EP1597278A4

EP1597278A4 - Polyethylene glycol modified interferon compositions and methods of use thereof

Info

Publication number: EP1597278A4
Application number: EP04714217A
Authority: EP
Inventors: Vlasselaer Peter Van; Michael J Roberts; Gary Visor; Stephen A Charles
Original assignee: Intermune Inc
Current assignee: Intermune Inc
Priority date: 2003-02-26
Filing date: 2004-02-24
Publication date: 2006-03-22
Also published as: WO2004076474A3; JP2006519235A; EP1597278A2; CN1997666A; WO2004076474A2; CA2516552A1

Abstract

Interferon alpha (IFN-α) linked to a linear polyethylene glycol (PEG) molecule having a nominal molecular weight of about 30 kD, and compositions comprising the same, are provided. The invention further provides methods of treating a viral infection with the subject PEG-modified IFN-α.

Description

POLYETHYLENE GLYCOL MODIFIED INTERFERON COMPOSITIONS AND METHODS OF USE THEREOF

FIELD OF THE INVENTION

[0001] The present invention is in the field of antiviral agents for the treatment of hepatitis virus infection.

BACKGROUND OF THE INVENTION

[0002] Hepatitis C virus (HCN) infection is the most common chronic blood borne infection in the United States. Although the numbers of new infections have declined, the burden of chronic infection is substantial, with Centers for Disease Control estimates of 3.9 million (1.8%) infected persons in the United States. Clironic liver disease is the tenth leading cause of death among adults in the United States, and accounts for approximately 25,000 deaths annually, or approximately 1% of all deaths. Studies indicate that 40% of chronic liver disease is HCN-related, resulting in an estimated 8,000-10,000 deaths each year. HCN-associated end- stage liver disease is the most frequent indication for liver transplantation among adults.

[0003] Interferon-alpha (IFΝ-α) treatment is the therapy of choice for treating hepatitis C virus infection. However, approximately 50% of patients fail to achieve a sustained virological response. Because the serum half-life of IFΝ-α is in the range of 8 to 8.5 hours, repeat doses of IFΝ-α are administered in order to maintain a higher serum level. For example, an accepted dosage regimen is administration of IFΝ-α three times in week (TIW) for a period of 24 - 48 weeks. The peaks and troughs of drug levels associated with such repeat drug dosing has been assumed to result in the severe side effects of the interferons during therapy.

[0004] There is a need in the art for a pharmaceutical agent having superior antiviral activity of

IFΝ-α, and which has desired pharmacokinetic properties that will allow for a reduced frequency dosing regimen with sustained high enough concentrations of the drug in blood while providing for a reduction in viral load. Literature

[0005] U.S. Patent Νos. 5,252,714; 5,382,657; 5,539,063; 5,559,213; 5,672,662; 5,747,646;

5,766,581; 5,792,834; 5,795,569; 5,798,232; 5,824,784; 5,834,594; 5,849,860; 5,928,636; 5,951,974; 5,595,732; 5,981,709; 5,985,265; 6,005,075; 6,180,096; 6,250,469; 6,277,830. PCT Publication No. WO 99/37779. Chamov et al. (1994) Bioconj. Chem. 5:133-140; Harris et al. (2001) Clin. Pharmacokinet. 40:539-551; Reddy (2000) Ann. Pharmacother. 34:915-923; Ballon et al. (2001) Bioconj. Chem. 12:195-202; Reddy et al. (2002) Adv. Dr gDeliv. Rev. 54:571-586.

SUMMARY OF THE INVENTION [0006] Interferon alpha (IFN-α) linked to a linear polyethylene glycol (PEG) molecule having an average molecular weight of about 30 kD, and compositions comprising the same, are provided. The invention further provides methods of treating a viral infection with the subject PEG-modified IFN-α.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 depicts the amino acid sequence of the consensus interferon IFN-alpha conl

(SEQ ID NO:l).

[0008] Figure 2 is a chromatogram depicting the size exclusion chromatographic profile of a pegylated consensus interferon alpha designated PEG-Alfacon.

[0009] Figure 3 is a photograph depicting the results of SDS-PAGE electrophoretic (reducing and non-reducing) analyses of a pegylated consensus interferon alpha designated PEG- Alfacon.

[0010] Figure 4 is a chromatogram depicting reversed phase HPLC chromatographic profiles of PEG-Alfacon, CIFN (Infergen) and a mixture of the two compounds.

[0011] Figure 5 is a graph depicting the serum concentration-time profile of pegylated interferon alpha analogs (IM-001, IM-003, IM-005, and IM-006), Pegasys, and PEG-Intron administered by subcutaneous injection in rats.

[0012] Figure 6 is a semilog plot depicting the serum concentration-time profile for PEG-

Alfacon (preparation no. IM-006) administered by subcutaneous injection in rats.

[0013] Figure 7 is a semilog plot depicting the pharmacokinetic profile of subcutaneously

(Sub-Q) administered PEG-Alfacon in cynomolgus monkeys.

[0014] Figure 8 is a log plot depicting the antiviral activities of Infergen, PEG-Alfacon- 1,

PEG-Intron, and Pegasys, expressed as International Units of activity per milligram normalized to the reference standard of human leukocyte-derived interferon-alpha supplied by the World Health Organization (WHO), measured in A549 cell/EMCN assays. Figure 8 depicts an analysis of the results with A549 cells.

[0015] Figure 9 is a log plot depicting the antiviral activities of Infergen, PEG-Alfacon- 1,

PEG-Intron, and Pegasys, expressed as International Units of activity per milligram normalized to the reference standard of human leukocyte-derived interferon-alpha supplied by the World Health Organization (WHO), measured in HeLa cell/ECMN assays. Figure 9 depicts an analysis of the results with HeLa cells.

[0016] Figure 10 is a log plot depicting the antiviral activities of Infergen, PEG-Alfacon- 1,

PEG-Intron, and Pegasys, expressed as International Units of activity per milligram normalized to the reference standard of human leukocyte-derived interferon-alpha supplied by the World Health Organization (WHO), measured in ME 180 cell/ECMN assays. Figure 10 depicts an analysis of the results with Me 180 cells.

[0017] Figure 11 is a graph depicting mean PEG-alfacon serum pharmacokinetic profiles from six subjects/group.

[0018] Figures 12A and B depict dose corrected C_max (pg/mL; Figure 12A) and AUCo-i_ast

(pg*h/mL; Figure 12B) vs. body mass index (BMI) values by dose group.

[0019] Figure 13 depicts mean serum pharmacokinetic profiles for 4-6 subjects and corresponding fitted curves using a 1 -compartment model.

[0020] Figures 14A-14G depict simulated serum pharmacokinetic profiles for various dosing regimens. Each panel in Figures 14A-14G uses different datasets that are fitted to a 1- compartment model. Figure 14A depicts simulated serum pharmacokinetic profiles for a dosing regimen with dosing every 10 days with 60 μg. Figure 14B depicts simulated serum pharmacokinetic profiles for a dosing regimen with dosing every 10 days with 100 μg. Figure 14C depicts simulated serum pharmacokinetic profiles for a dosing regimen with dosing every 10 days with 150 μg. Figure 14D depicts simulated serum pharmacokinetic profiles for a dosing regimen with dosing every 10 days with 200 μg. Figure 14E depicts simulated serum pharmacokinetic profiles for a dosing regimen with dosing every 7 days with 100 μg. Figure 14F depicts simulated serum pharmacokinetic profiles for a dosing regimen with dosing every 7 days with 150 μg. Figure 14G depicts simulated serum pharmacokinetic profiles for a dosing regimen with dosing every 7 days with 200 μg.

[0021] Figure 15 depicts mean percent (%) change in serum 2',5'-oligoadenylate synthetase

(OAS) for 6 subjects/dose group. The dosage of subcutaneous PEG-alfacon received in each dosage group is identified in the figure legend. The group treated with 15 μg subcutaneous Infergen® interferon alfacon-1 is identified as "Control" in the figure legend.

[0022] Figure 16 depicts measured and predicted percent (%) change in OAS serum values expressed as percent of baseline (pretreatment) values, from pharmacokinetic/pharmacodynamic (PK-PD) modeling of mean serum profiles using an Emax model with minimum response fixed at 0. FEATURES OF THE INVENTION

[0023] The present invention features a monopegylated consensus interferon (CIFN) molecule comprised of a single CIFN polypeptide and a single polyethylene glycol (PEG) moiety, where the PEG moiety is linear and about 30 kD in molecular weight and is directly or indirectly linked through a stable covalent linkage to either the N-terminal residue in the CIFN polypeptide or a lysine residue in the CIFN polypeptide.

[0024] In some embodiments, the PEG moiety is linked to either the alpha-amino group of the

N-terminal residue in the CIFN polypeptide or the epsilon-amino group of a lysine residue in the CIFN polypeptide. In further embodiments, the linkage comprises an amide bond between the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon- amino group of the lysine residue in the CIFN polypeptide. In still further embodiments, the linkage comprises an amide bond between a propionyl group of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide. In additional embodiments, the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide, thereby forming a hydrolytically stable linkage between the PEG moiety and the CIFN polypeptide.

[0025] In some embodiments, the PEG moiety is linked to the N-terminal residue in the CIFN polypeptide. In other embodiments, the PEG moiety is linked to the alpha-amino group of the N-terminal residue in the CIFN polypeptide. In further embodiments, the linkage comprises an amide bond between the PEG moiety and the alpha-amino group of the N-terminal residue in the CIFN polypeptide. In still further embodiments, the linkage comprises an amide bond between a propionyl group of the PEG moiety and the alpha-amino group of the N-terminal residue in the CIFN polypeptide. In additional embodiments, the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the alpha-amino group of the N-terminal residue of the CIFN polypeptide.

[0026] In some embodiments, the PEG moiety is linked to a lysine residue in the CIFN polypeptide. In other embodiments, the PEG moiety is linked to the epsilon-amino group of a lysine residue in the CIFN polypeptide. In further embodiments, the linkage comprises an amide bond between the PEG moiety and the epsilon-amino group of the lysine group in the CIFN polypeptide. In still further embodiments, the linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the lysine group in the CIFN polypeptide. In additional embodiments, the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon- amino group of the lysine residue in the CIFN polypeptide.

[0027] In some embodiments, the PEG moiety is linked to a surface-exposed lysine residue in the CIFN polypeptide. In other embodiments, the PEG moiety is linked to the epsilon-amino group of a surface-exposed lysine residue in the CIFN polypeptide. In further embodiments, the linkage comprises an amide bond between the PEG moiety and the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide. In still further embodiments, the linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide. In additional embodiments, the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide.

[0028] In some embodiments, the PEG moiety is linked to a lysine chosen from lys³¹, lys⁵ , lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide. In other embodiments, the PEG moiety is linked to the epsilon-amino group of a lysine chosen from lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide. In further embodiments, the linkage comprises an amide bond between the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide. In still further embodiments, the linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide. In additional embodiments, the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.

[0029] In some embodiments, the PEG moiety is linked to a lysine chosen from lys⁵⁰, lys⁷¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide. In other embodiments, the PEG moiety is linked to the epsilon-amino group of a lysine chosen from lys⁵⁰, lys⁷¹, lys¹³⁴, lys¹³⁵, and lys^{1 5} of the CIFN polypeptide. In further embodiments, the linkage comprises an amide bond between the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide. In still further embodiments, the linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide. In additional embodiments, the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide. [0030] In some embodiments, the PEG moiety is linked to a lysine chosen from lys¹²¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide. In other embodiments, the PEG moiety is linked to the epsilon-amino group of a lysine chosen from lys¹²¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide. In further embodiments, the linkage comprises an amide bond between the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide. In still further embodiments, the linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide. In additional embodiments, the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon- amino group of the chosen lysine residue in the CIFN polypeptide.

[0031] In connection with the above-described monopegylated CIFN molecules, the invention contemplates embodiments of each such molecule where the CIFN polypeptide is chosen from interferon alpha-co , interferon alpha-con₂, and interferon alpha-con₃, the amino acid sequences of which CIFN polypeptides are disclosed in U.S. Pat. No. 4,695,623.

[0032] The invention also features a composition comprising a population of monopegylated consensus interferon (CIFN) molecules, where the population consists of one or more species of molecules, where each species is comprised of a single CIFN polypeptide and a single PEG moiety, where the PEG moiety is linear and about 30 kD in molecular weight and is directly or indirectly attached through a covalent linlcage to either the N-terminal residue or a lysine residue in the CIFN polypeptide. In further embodiments, in each such species in the population the linkage comprises an amide bond between the PEG moiety and either the alpha- amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide. In still further embodiments, in each such species in the population the linkage comprises an amide bond between a propionyl group of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide. In additional embodiments, in each such species in the population the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide.

[0033] In some embodiments, the population consists of one or more species of molecules, where each species is comprised of a single CIFN polypeptide and a single PEG moiety, where the PEG moiety is linear and about 30 kD in molecular weight and is directly or indirectly attached tlirough a covalent linlcage to either the N-terminal residue or a surface-exposed lysine residue in the CIFN polypeptide. In further embodiments, in each such species in the population the linlcage comprises an amide bond between the PEG moiety and either the alpha- amino group of the N-terminal residue or the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide. In still further embodiments, in each such species in the population the linlcage comprises an amide bond between a propionyl group of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide. In additional embodiments, in each such species in the population the amide bond is formed by condensation of an alpha- methoxy, omega-propanoic acid activated ester of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide.

[0034] In some embodiments, the population consists of one or more species of molecules, where each species is comprised of a single CIFN polypeptide and a single PEG moiety, where the PEG moiety is linear and about 30 kD in molecular weight and is directly or indirectly attached through a covalent linlcage to either the N-terminal residue or a lysine residue chosen from lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide. In further embodiments, in each such species in the population the linlcage comprises an amide bond between the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide. In still further embodiments, in each such species in the population the linkage comprises an amide bond between a propionyl group of the PEG moiety and either the alpha-amino group of the N- terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide. In additional embodiments, in each such species in the population the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide.

[0035] In some embodiments, the population consists of one or more species of molecules, where each species is comprised of a single CIFN polypeptide and a single PEG moiety, where the PEG moiety is linear and about 30 kD in molecular weight and is directly or indirectly attached through a covalent linkage to either the N-terminal residue or a lysine residue chosen from lys⁵⁰, lys⁷¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide. In further embodiments, in each such species in the population the linkage comprises an amide bond between the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide. In still further embodiments, in each such species in the population the linkage comprises an amide bond between a propionyl group of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon- amino group of the lysine residue in the CIFN polypeptide. In additional embodiments, in each such species in the population the amide bond is formed by condensation of an alpha- methoxy, omega-propanoic acid activated ester of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide.

[0036] In some embodiments, the population consists of one or more species of molecules, where each species is comprised of a single CIFN polypeptide and a single PEG moiety, where the PEG moiety is linear and about 30 kD in molecular weight and is directly or indirectly attached through a covalent linlcage to either the N-terminal residue or a lysine residue chosen from lys , lys , lys , and lys of the CIFN polypeptide. In further embodiments, in each such species in the population the linkage comprises an amide bond between the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide. In still further embodiments, in each such species in the population the linlcage comprises an amide bond between a propionyl group of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide. In additional embodiments, in each such species in the population the amide bond is formed by condensation of an alpha-methoxy, omega- propanoic acid activated ester of the PEG moiety and either the alpha-amino group of the N- terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide.

[0037] In some embodiments, the population consists of one or more species of monopegylated CIFN in which the PEG moiety is linked to the N-terminal residue of the CIFN polypeptide. In other embodiments, in each such species in the population the PEG moiety is linked to the alpha-amino group of the N-terminal residue in the CIFN polypeptide. In further embodiments, in each such species in the population the linkage comprises an amide bond between the PEG moiety and the alpha-amino group of the N-terminal residue in the CIFN polypeptide. In still further embodiments, in each such species in the population the linlcage comprises an amide bond between a propionyl group of the PEG moiety and the alpha-amino group of the N-terminal residue in the CIFN polypeptide. In additional embodiments, in each such species in the population the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the alpha-amino group of the N- terminal residue of the CIFN polypeptide.

[0038] In some embodiments, the population consists of one or more species of monopegylated CIFN in which the PEG moiety is linked to a lysine residue in the CIFN polypeptide. In other embodiments, in each such species in the population the PEG moiety is linked to the epsilon-amino group of a lysine residue in the CIFN polypeptide. In further embodiments, in each such species in the population the linkage comprises an amide bond between the PEG moiety and the epsilon-amino group of the lysine group in the CIFN polypeptide. In still further embodiments, in each such species in the population the linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the lysine group in the CIFN polypeptide. In additional embodiments, in each such species in the population the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the lysine residue in the CIFN polypeptide.

[0039] In some embodiments, the population consists of one or more species of monopegylated CIFN in which the PEG moiety is linked to a surface-exposed lysine residue in the CIFN polypeptide. In further embodiments, in each such species in the population the linkage comprises an amide bond between the PEG moiety and the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide. In still further embodiments, in each such species in the population the linlcage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide. In additional embodiments, in each such species in the population the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide.

[0040] In some embodiments, the population consists of one or more species of monopegylated CIFN in which the PEG moiety is linked to a lysine residue chosen from lys , lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide. In other embodiments, in each such species in the population the PEG moiety is linked to the epsilon- amino group of a lysine chosen from lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide. In further embodiments, in each such species in the population the linlcage comprises an amide bond between the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide. In still further embodiments, in each such species in the population the linlcage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide. In additional embodiments, in each such species in the population the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.

[0041] In some embodiments, the population consists of one or more species of monopegylated CIFN in which the PEG moiety is linked to a lysine residue chosen from lys⁵⁰, lys⁷¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide. In other embodiments, in each such species in the population the PEG moiety is linked to the epsilon-amino group of a lysine chosen from lys⁵⁰, lys⁷¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide. In further embodiments, in each such species in the population the linlcage comprises an amide bond between the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide. In still further embodiments, in each such species in the population the linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide. In additional embodiments, in each such species in the population the amide bond is formed by condensation of an alpha- methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.

[0042] In some embodiments, the population consists of one or more species of monopegylated CIFN in which the PEG moiety is linked to a lysine residue chosen from lys , lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide. In other embodiments, in each such species in the population the PEG moiety is linked to the epsilon-amino group of a lysine chosen from lys¹²¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide. In further embodiments, in each such species in the population the linkage comprises an amide bond between the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide. In still further embodiments, in each such species in the population the linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide. In additional embodiments, in each such species in the population the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.

[0043] In connection with each of the above-described populations consisting of monopegylated CIFN molecules where each such species comprises a PEG moiety linked to the N-terminal residue or a lysine residue of a CIFN polypeptide, the invention contemplates embodiments where the population consists of a first monopegylated CIFN molecule comprising a PEG moiety linked to the N-terminal residue of a first CIFN polypeptide and a second monopegylated CIFN molecule comprising a PEG moiety linked to a lysine residue in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different.

[0044] In connection with each of the above-described populations consisting of monopegylated CIFN molecules where each such species comprises a PEG moiety linked to the N-terminal residue or a lysine residue of a CIFN polypeptide, the invention contemplates embodiments where the population consists of a first monopegylated CIFN molecule comprising a PEG moiety linked to the N-terminal residue of a first CIFN polypeptide, a second monopegylated CIFN molecule comprising a PEG moiety linked to a first lysine residue in a second CIFN polypeptide, and a third monopegylated CIFN molecule comprising a PEG moiety linked to a second lysine residue in a third CIFN polypeptide, where each of the first, second and third CIFN polypeptides can be the same or different from any of the other CIFN polypeptides, and where the location of the linkage site in the second CIFN polypeptide is not the same as the location of the linkage site in the third CIFN polypeptide.

[0045] In connection with each of the above-described populations consisting of monopegylated CIFN molecules where each such species comprises a PEG moiety linked to the N-terminal residue or a lysine residue of a CIFN polypeptide, the invention contemplates embodiments where the population consists of a first monopegylated CIFN molecule comprising a PEG moiety linked to the N-terminal residue of a first CIFN polypeptide, a second monopegylated CIFN molecule comprising a PEG moiety linked to a first lysine residue in a second CIFN polypeptide, a third monopegylated CIFN molecule comprising a PEG moiety linked to a second lysine residue in a third CIFN polypeptide, and a fourth monopegylated CIFN molecule comprising a PEG moiety linked to a third lysine residue in a fourth CIFN polypeptide, where each of the first, second, third and fourth CIFN polypeptides can be the same or different from any of the other CIFN polypeptides, and where the location of the linkage site in each of the second, third and fourth CIFN polypeptides is not the same as the location of the linlcage site in any other CIFN polypeptide.

[0046] In connection with each of the above-described populations consisting of monopegylated CIFN molecules where each such species comprises a PEG moiety linked to the N-terminal residue or a lysine residue of a CIFN polypeptide, the invention contemplates embodiments where the population consists of a first monopegylated CIFN molecule comprising a PEG moiety linked to the N-terminal residue of a first CIFN polypeptide, a second monopegylated CIFN molecule comprising a PEG moiety linked to a first lysine residue in a second CIFN polypeptide, a third monopegylated CIFN molecule comprising a PEG moiety linked to a second lysine residue in a third CIFN polypeptide, a fourth monopegylated CIFN molecule comprising a PEG moiety linked to a third lysine residue in a fourth CIFN polypeptide, and a fifth monopegylated CIFN molecule comprising a PEG moiety linked to a fourth lysine residue in a fifth CIFN polypeptide, where each of the first, second, third, fourth and fifth CIFN polypeptides can be the same or different from any of the other CIFN polypeptides, and where the location of the linkage site in each of the second, third, fourth and fifth CIFN polypeptides is not the same as the location of the linkage site in any other CIFN polypeptide.

[0047] In connection with each of the above-described populations consisting of monopegylated CIFN molecules where each such species comprises a PEG moiety linked to the N-terminal residue or a lysine residue of a CIFN polypeptide, the invention contemplates embodiments where the population consists of a first monopegylated CIFN molecule comprising a PEG moiety linked to the N-terminal residue of a first CIFN polypeptide, a second monopegylated CIFN molecule comprising a PEG moiety linked to a first lysine residue in a second CIFN polypeptide, a third monopegylated CIFN molecule comprising a PEG moiety linked to a second lysine residue in a third CIFN polypeptide, a fourth monopegylated CIFN molecule comprising a PEG moiety linked to a third lysine residue i a fourth CIFN polypeptide, a fifth monopegylated CIFN molecule comprising a PEG moiety linked to a fourth lysine residue in a fifth CIFN polypeptide, and a sixth monopegylated CIFN molecule comprising a PEG moiety linked to a fifth lysine residue in a sixth CIFN polypeptide, where each of the first, second, third, fourth, fifth and sixth CIFN polypeptides can be the same or different from any of the other CIFN polypeptides, and where the location of the linlcage site in each of the second, third, fourth, fifth and sixth CIFN polypeptides is not the same as the location of the linkage site in any other CIFN polypeptide. Optionally, the population can include at least one additional monopegylated CIFN molecule comprising a PEG moiety linked to a lysine residue of an additional CIFN polypeptide, where the CIFN polypeptide is the same or different as any of the first, second, third, fourth, fifth and sixth CIFN polypeptides, and where the location of the linlcage site in the additional CIFN polypeptide is not the same as the location of the linlcage site in any other CIFN polypeptide.

[0048] In connection with each of the above-described populations of monopegylated CIFN molecules where each such species comprises a PEG moiety linked to a lysine residue of a CIFN polypeptide, it will be understood that the invention contemplates embodiments characterized by a plurality of species of monopegylated, lysine-derivatized CIFN molecules, where each such species is characterized by a site of linkage that is not the same as the site of linlcage in any other species, in a manner analogous to the description of embodiments relating to populations including monopeyglated, N-terminally derivatized CIFN molecules above. In particular, the embodiments of the invention characterized by a plurality of species of monopegylated, lysine-derivatized CIFN molecules can be obtained by modifying the description of embodiments relating to populations including monopeyglated, N-terminally derivatized CIFN molecules above so as to remove the N-terminally derivatized species contained therein.

[0049] In connection with each of the above-described populations of monopegylated CIFN molecules, the invention contemplates embodiments where the molecules in each such population comprise a CIFN polypeptide chosen from interferon alpha-co , interferon alpha- con₂, and interferon alpha-con₃.

[0050] The invention further features a product that is produced by the process of reacting

CIFN polypeptide with a succinimidyl ester of alpha-methoxy, omega-propionylpoly(ethylene glycol) (mPEGspa) that is linear and about 30 kD in molecular weight, where the reactants are initially present at a molar ratio of about 1 : 1 to about 1 :5 CIFN:mPEGspa, and where the reaction is conducted at a pH of about 7 to about 9, followed by recovery of the monopegylated CIFN product of the reaction. In one embodiment, the reactants are initially present at a molar ratio of about 1 :3 CIFN:mPEGspa and the reaction is conducted at a pH of about 8. In another embodiment where the product of the invention is generated by a scaled-up procedure needed for toxicological and clinical investigations, the reactants are initially present in a molar ratio of 1 :2 CIFN:mPEGspa and the reaction is conducted at a pH of about 8.0.

[0051] In connection with the above-described product-by-process, the invention contemplates embodiments where the CIFN reactant is chosen from interferon alpha-con_! , interferon alpha- con , and interferon alpha-con₃.

[0052] In some aspects, the monopegylated CIFN molecule(s) or population(s) of the invention exhibit an antiviral activity that is at least 10-fold greater than that of PEGASYS® Peg- interferon-alfa2a, an approved marketed product to treat chronic hepatitis C. In other aspects, the monopegylated CIFN molecule(s) or population(s) of the invention exhibit an antiviral activity that is approximately the same as that of INFERGEN® Alfacon-1.

[0053] The invention additionally features a pharmaceutical composition comprising any monopegylated CIFN molecule or population of such molecules described above and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises the monopegylated CIFN molecule or population of the invention in an amount that is therapeutically effective in the treatment of a viral disease in a patient. In other embodiments, the pharmaceutical composition comprises the monopegylated CIFN molecule or population of the invention in an amount that is therapeutically effective in the treatment of a hepatitis viral disease in a patient. In further embodiments, the pharmaceutical composition comprises the monopegylated CIFN molecule or population of the invention in an amount that is therapeutically effective in the treatment of a hepatitis C viral (HCN) disease in a patient.

DEFINITIONS

[0054] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease (as in liver fibrosis that can result in the context of chronic HCN infection); (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0055] The terms "individual," "host," "subject," and "patient" are used interchangeably herein, and refer to a mammal, including, but not limited to, primates, including simians and humans.

[0056] The term "pharmacokinetic profile," as used herein, refers to the profile of the curve that results from plotting serum concentration of IFΝ-α over time, following administration of IFΝ-α to a subject. "Area under the curve," or "AUC," refers to the integrated area under the curve generated by plotting serum concentration of IFΝ-α over time following administration of IFΝ-α.

[0057] The term "hepatitis virus infection" refers to infection with one or more of hepatitis A,

B, C, D, or E virus, with blood-borne hepatitis viral infection being of particular interest, particularly hepatitis C virus infection.

[0058] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. [0059] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed witliin the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0061] It must be noted that as used herein and in the appended claims, the singular forms "a",

"and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a modified IFN-α polypeptide" includes a plurality of such polypeptides and reference to "the PEG molecule" includes reference to one or more PEG molecules and equivalents thereof known to those skilled in the art, and so forth.

[0062] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION [0063] The present invention provides IFN-α that is modified with linear polyethylene glycol

(PEG). In particular, the invention provides an IFN-α polypeptide that is linked to a single molecule of PEG (i.e., the IFN-α polypeptide is "monopegylated"). The pharmacokinetic profile of the PEG-modified IFN-α of the invention is such that the PEG-modified IFN-α is effective for treating viral infections, particularly hepatitis virus infections. Accordingly, the invention further provides methods of treating a hepatitis virus infection. The methods generally involve administering an effective amount of a PEG-modified IFN-α of the invention to an individual having or susceptible to a viral hepatitis infection. PEG-MODIFIED IFN-α

[0064] The present invention provides IFN-α that is modified with a polyethylene glycol

(PEG) molecule of less than about 40 kDa average molecular weight. In particular, the invention provides an IFN-α polypeptide that is linked to a single molecule of PEG of less than about 40 lcDa, with a PEG molecule of about 30 kDa being of particular interest, more particularly a linear 30 kDa PEG molecule attached to IFN-α, with CIFN being of particular interest. IFN-α

[0065] The term "interferon-alpha" as used herein refers to a family of related polypeptides that inhibit viral replication and cellular proliferation and modulate immune response. The term "IFN-α" includes IFN-α polypeptides that are naturally occurring; non-naturally- occurring IFN-α polypeptides; and analogs of naturally occurring or non-naturally occurring IFN-α that retain antiviral activity of a parent naturally-occurring or non-naturally occurring IFN-α.

[0066] Suitable alpha interferons include, but are not limited to, naturally-occurring IFN-α

(including, but not limited to, naturally occurring IFN-α2a, IFN-α2b); recombinant interferon alpha-2b such as Intron®A interferon available from Schering Corporation, Kenilworth, N.J.; recombinant interferon alpha-2a such as Roferon® interferon available from Hoffmann-La Roche, Nutley, N. ; recombinant interferon alpha-2C such as Berofor® alpha 2 interferon available from Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, Conn.; interferon alpha- nl, a purified blend of natural alpha interferons such as Sumiferon available from Sumitomo, Japan or as Wellferon® interferon alpha-nl (INS) available from the Glaxo- Wellcome Ltd., London, Great Britain; and interferon alpha-n3 a mixture of natural alpha interferons made by Interferon Sciences and available from the Purdue Frederick Co., Norwalk, Conn., under the Alferon® Tradename.

[0067] The term "IFN-α," as used herein, also encompasses consensus IFN-α. As used herein, the term "consensus IFN-α" refers to a non-naturally-occurring polypeptide, which includes those amino acid residues that are common to all naturally-occurring human leukocyte IFN-α subtype sequences and which includes, at one or more of those positions where there is no amino acid common to all subtypes, an amino acid which predominantly occurs at that position, provided that at any such position where there is no amino acid common to all subtypes, the polypeptide excludes any amino acid residue which is not present in at least one naturally-occurring subtype. Amino acid residues that are common to all naturally-occurring human leukocyte IFN-α subtype sequences ("common amino acid residues"), and amino acid residues that occur predominantly at non-common residues ("consensus amino acid residues") are known in the art. See Figure 1 for the amino acid sequence of IFN-alpha conl. Thus, a consensus interferon is a wholly synthetic Type I interferon developed by scanning several interferon-alpha non-allelic subtypes and assigning the most frequently observed amino acids in each position.

[0068] Consensus IFN-α (also referred to as "CIFN" and "IFN-con" and "IFN-alpha con") encompasses but is not limited to the amino acid sequences designated IFN-coni (sometimes referred to as "CIFN-alpha conl," "IFN-alpha conl," or "IFN-conl," or "alphacon"), IFN-con₂ and IFN-con₃ which are disclosed in U.S. Pat. Nos. 4,695,623 and 4,897,471; and Infergen® (Amgen, Thousand Oaks, Calif). Consensus interferons are generally defined by determination of a consensus sequence of naturally occurring interferon alphas. PEG-modified CIFN, especially Infergen®, is of particular interest in some embodiments.

[0069] IFN-α polypeptides can be produced by any known method. DNA sequences encoding

IFN-con may be synthesized as described in the above-mentioned patents or other standard methods. In many embodiments, IFN-α polypeptides are the products of expression of manufactured DNA sequences transformed or transfected into bacterial hosts, e.g., E. coli, or in eukaryotic host cells (e.g., yeast; mammalian cells, such as CHO cells; and the like). In these embodiments, the IFN-α is "recombinant IFN-α." Where the host cell is a bacterial host cell, the IFN-α is modified to comprise an N-terminal methioiiine. IFN-α produced in E. coli is generally purified by procedures known to those skilled in the art and generally described in Klein et al. ((1988) J Chromatog. 454:205-215) for IFN-con_L

[0070] Bacterially produced IFN-α may comprise a mixture of isoforms with respect to the N- terminal amino acid residue. For example, purified IFN-con may comprise a mixture of isoforms with respect to the N-terminal methionine status. For example, in some embodiments, an IFN-con comprises a mixture of N-terminal methionyl IFN-con, des- methionyl IFN-con with an unblocked N-terminus, and des-methionyl IFN-con with a blocked N-terminus. As one non-limiting example, purified IFN-coni comprises a mixture of methionyl IFN-con^ des-methionyl IFN-coni and des-methionyl IFN-coni with a blocked N- terminus. Klein et al. ((1990) Arch. Biochemistry & Biophys. 276:531-537). Alternatively, IFN-con may comprise a specific, isolated isoform. Isoforms of IFN-con are separated from each other by techniques such as isoelectric focusing which are known to those skilled in the art. [0071] It is to be understood that IFN-α as described herein may comprise one or more modified amino acid residues, e.g., glycosylations, chemical modifications, and the like. Site of Linkage

[0072] PEG is coupled either directly (i.e., without a linking group), or via a linker (as described in detail below), to an amino group on the IFN-α polypeptide.

[0073] In some embodiments, the PEGylated IFN-α is PEGylated at or near the amino terminus (N-terminus) of the IFN-α polypeptide, e.g., the PEG moiety is conjugated to the IFN-α polypeptide at an amino acid residue from amino acid 1 through amino acid 4, or from amino acid 5 through about 10.

[0074] In other embodiments, the PEGylated IFN-α is PEGylated at an amino acid residues from about 10 to about 28.

[0075] In other embodiments, the PEGylated IFN-α is PEGylated at an amino acid residue from amino acids 100-114.

[0076] In particular embodiments of interest, where the IFN-α is CIFN, the PEG molecule is linked to the NH₂ terminal amino acid residue of the CIFN polypeptide. In these embodiments, the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.

[0077] In some embodiments, the PEGylated IFN-α comprises CIFN PEGylated at the epsilon amino group of a lysine residue. See Figure 1 for the amino acid sequence of an exemplary IFN-α, showing the locations of the lysine residues.

[0078] Generally, the PEG moiety is linked to a surface-exposed lysine ("lys") residue.

Whether a lysine is surface exposed can be determined using any known method. Generally, analysis of hydrophilicity (e.g., Kyte-Doolittle and Hoppe- Woods analysis) and/or predicted surface-forming regions (e.g., Emini surface-forming probability analysis) is carried out using appropriate computer programs, which are well known to those skilled in the art. Suitable computer programs include PeptideStructure, and the like. Alternatively, NMR investigations can identify the surface accessible residues by virtue of the chemical shift of the protons of a specific functional group in the spectrum and how they are affected by the inclusion of "shift reagents". In other cases, the inaccessibility or accessibility of residues to solvents or environment can be assessed by fluorescence. In yet other cases, the surface exposure of accessible lysines can be ascertained by the chemical reactivity to water soluble reagents e.g., Trinitrobenzene sulfonate or TNBS, and like measurements.

[0079] In some embodiments, the invention provides PEG-modified CIFN, where the PEG moiety is attached to a lysine residue chosen from lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵. In these embodiments, the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.

[0080] In other embodiments, the invention provides PEG-modified CIFN, where the PEG moiety is attached to a lysine residue chosen from lys⁵⁰, lys⁷¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵. hi these embodiments, the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 lcD

[0081] In other embodiments, the invention provides PEG-modified CIFN, where the PEG moiety is attached to a lysine residue chosen from lys¹²¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵. In these embodiments, the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD. Populations of IFN-α

[0082] The invention further provides a composition comprising a population of monopegylated IFNα molecules, where the population consists of one or more species of monopegylated IFNα molecules as described above. As discussed above, the PEG-modified IFN-α of the invention comprises a single PEG molecule per IFN-α polypeptide molecule. In some embodiments, a subject composition comprises a population of modified IFN-α polypeptides, each with a single PEG molecule linked to a single amino acid residue of the polypeptide.

[0083] In some of these embodiments, the population comprises a mixture of a first IFN-α polypeptide linked to a PEG molecule at a first amino acid residue; and at least a second IFN-α polypeptide linked to a PEG molecule at a second amino acid residue, wherein the first and second IFN-α polypeptides are the same or different, and wherein the location of the first amino acid residue in the amino acid sequence of the first IFN-α polypeptide is not the same as the location of the second amino acid residue in the second IFN-α polypeptide. As one non- limiting example, a subject composition comprises a population of PEG-modified IFN-α polypeptides, the population comprising an IFN-α polypeptide linked at its amino terminus to a linear PEG molecule; and an IFN-α polypeptide linked to a linear PEG molecule at a lysine residue.

[0084] Generally, a given modified IFN-α species represents from about 0.5% to about 99.5% of the total population of monopegylated IFNα polypeptide molecules in a population, e.g, a given modified IFN-α species represents about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 99.5% of the total population of monopegylated IFN-α polypeptide molecules in a population. In some embodiments, a subject composition comprises a population of monopegylated IFN-α polypeptides, which population comprises at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, IFN-α polypeptides linked to PEG at the same site, e.g., at the N-terminal amino acid.

[0085] In particular embodiments of interest, a subject composition comprises a population of monopegylated CIFN molecules, the population consisting of one or more species of molecules, where each species is a single CIFN polypeptide linked, directly or indirectly in a covalent linkage, to a single linear PEG moiety of about 30 kD in molecular weight, and where the linkage is to either a lysine residue in the CIFN polypeptide, or the N-terminal amino acid residue of the CIFN polypeptide.

[0086] The amino acid residue to which the PEG is attached is in many embodiments the N- terminal amino acid residue. In other embodiments, the PEG moiety is attached (directly or via a linker) to a surface-exposed lysine residue. In additional embodiments, the PEG moiety is attached (directly or via a linker) to a lysine residue chosen from lys , lys , lys , lys , lys , lys , lys , lys , and lys of the CIFN polypeptide. In further embodiments, the PEG moiety is attached (directly or via a linker) to a lysine residue chosen from lys , lys , lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide. In additional embodiments, the PEG moiety is attached (directly or via a linker) to a lysine residue chosen from lys¹²¹, lys¹³ , lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide.

[0087] As an example, a subject composition comprises a population of monopegylated CIFN molecules, consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked at the N-terminal amino acid residue of a first CIFN polypeptide, and a second monopegylated CIFN polypeptide species having a PEG moiety linked to a first lysine residue of a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different. A subject composition can further comprise at least one additional monopegylated CIFN polypeptide species having a PEG moiety linked to a lysine residue in the CIFN polypeptide, where the location of the linlcage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linkage site in any other species. In all species in this example, the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.

[0088] As another example, a subject composition comprises a population of monopegylated

CIFN molecules, consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked at the N-terminal amino acid residue of a first CIFN polypeptide, and a second monopegylated CIFN polypeptide species having a PEG moiety linked to a first surface- exposed lysine residue of a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different. A subject composition can further comprise at least one additional monopegylated CIFN polypeptide species having a PEG moiety linked to a surface- exposed lysine residue in the CIFN polypeptide, where the location of the linkage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linkage site in any other species. In all species in this example, the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.

[0089] As another example, a subject composition comprises a population of monopegylated

CIFN molecules, consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked at the N-terminal amino acid residue of a first CIFN polypeptide, and a second monopegylated CIFN polypeptide species having a PEG moiety linked to a first lysine residue selected from one of lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different. A subject composition can further comprise a third monopegylated CIFN polypeptide species having a PEG moiety linked to a second lysine residue selected from one of lys , lys , lys , lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ in a third CIFN polypeptide, where the third CIFN polypeptide is the same or different from either of the first and second CIFN polypeptides, where the second lysine residue is located in a position in the amino acid sequence of the third CIFN polypeptide that is not the same as the position of the first lysine residue in the amino acid sequence of the second CIFN polypeptide. A subject composition may further comprise at least one additional monopegylated CIFN polypeptide species having a PEG moiety linked to one of lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵, where the location of the linlcage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linlcage site in any other species. In all species in this example, the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.

[0090] As another example, a subject composition comprises a population of monopegylated

CIFN molecules, consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked at the N-terminal amino acid residue of a first CIFN polypeptide, and a second monopegylated CIFN polypeptide species having a PEG moiety linked to a first lysine residue selected from one of lys⁵⁰, lys⁷¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different. A subject composition can further comprise a third monopegylated CIFN polypeptide species having a PEG moiety linked to a second lysine residue selected from one of lys⁵⁰, lys⁷¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ in a third CIFN polypeptide, where the third CIFN polypeptide is the same or different from either of the first and second CIFN polypeptides, where the second lysine residue is located in a position in the amino acid sequence of the third CIFN polypeptide that is not the same as the position of the first lysine residue in the amino acid sequence of the second CIFN polypeptide. A subject composition may further comprise at least one additional monopegylated CIFN polypeptide species having a PEG moiety linked to one of lys⁵⁰, lys⁷¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵, where the location of the linkage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linlcage site in any other species. In all species in this example, the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.

[0091] As another example, a subject composition comprises a population of monopegylated

CIFN molecules, consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked at the N-terminal amino acid residue of a first CIFN polypeptide, and a second monopegylated CIFN polypeptide species having a PEG moiety linked to a first lysine residue selected from one of lys¹²¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different. A subject composition can further comprise a third monopegylated CIFN polypeptide species having a PEG moiety linked to a second lysine residue selected from one of lys¹²¹, lys ⁴, lys¹³⁵, and lys^{1 5} in a third CIFN polypeptide, where the third CIFN polypeptide is the same or different from either of the first and second CIFN polypeptides, where the second lysine residue is located in a position in the amino acid sequence of the third CIFN polypeptide that is not the same as the position of the first lysine residue in the amino acid sequence of the second CIFN polypeptide. A subject composition may further comprise at least one additional monopegylated CIFN polypeptide species having a PEG moiety linked to one of lys¹²¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵, where the location of the linlcage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linlcage site in any other species. In all species in this example, the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.

[0092] As another non-limiting example, a subject composition comprises a population of monopegylated CIFN molecules, consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked to a first lysine residue in a first CIFN polypeptide; and a second monopegylated CIFN polypeptide species having a PEG moiety linked at a second lysine residue in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different, and where the first lysine is located in a position in the amino acid sequence of the first CIFN polypeptide that is not the same as the position of the second lysine residue in the amino acid sequence of the second CIFN polypeptide. A subject composition may further comprise at least one additional monopegylated CIFN species having a PEG moiety linked to a lysine residue in the CIFN polypeptide, where the location of the linlcage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linkage site in any other species. In all species in this example, the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.

[0093] As another non-limiting example, a subject composition comprises a population of monopegylated CIFN molecules, consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked at a first lysine residue chosen from lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ in a first CIFN polypeptide; and a second monopegylated CIFN polypeptide species having a PEG moiety linked at a second lysine residue chosen from lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different, and where the second lysine residue is located in a position in the amino acid sequence of in the second CIFN polypeptide that is not the same as the position of the first lysine residue in the first CIFN polypeptide. The composition may further comprise at least one additional monopegylated CIFN polypeptide species having a PEG moiety linked to one of lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys ⁵, and lys^{1 5}, where the location of the linkage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linkage site in any other species. In all species in this example, the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.

[0094] As another non-limiting example, a subject composition comprises a population of monopegylated CIFN molecules, consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked at a first lysine residue chosen from lys⁵⁰, lys⁷¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ in a first CIFN polypeptide; and a second monopegylated CIFN polypeptide species having a PEG moiety linked at a second lysine residue chosen from lys⁵⁰, lys⁷¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different, and where the second lysine residue is located in a position in the amino acid sequence of in the second CIFN polypeptide that is not the same as the position of the first lysine residue in the first CIFN polypeptide. The composition may further comprise at least one additional monopegylated CIFN polypeptide species having a PEG moiety linked to one of lys⁵⁰, lys⁷¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵, where the location of the linlcage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linlcage site in any other species. In all species in this example, the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD. [0095] As another non-limiting example, a subject composition comprises a population of monopegylated CIFN molecules, consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked at a first lysine residue chosen from lys¹²¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ in a first CIFN polypeptide; and a second monopegylated CIFN polypeptide species having a PEG moiety linked at a second lysine residue chosen from lys¹²¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different, and where the second lysine residue is located in a position in the amino acid sequence of in the second CIFN polypeptide that is not the same as the position of the first lysine residue in the first CIFN polypeptide. The composition may further comprise at least one additional monopegylated CIFN polypeptide species having a PEG moiety linked to one of lys¹²¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵, where the location of the linkage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linlcage site in any other species. In all species in this example, the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.

[0096] As another non-limiting example, a subject composition comprises a monopegylated population of CIFN molecules, consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked to a first surface-exposed lysine residue in a first CIFN polypeptide; and a second monopegylated CIFN polypeptide species having a PEG moiety linked at a second surface-exposed lysine residue in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different, and where the first surface- exposed lysine is located in a position in the amino acid sequence of the first CIFN polypeptide that is not the same as the position of the second surface-exposed lysine residue in the amino acid sequence of the second CIFN polypeptide. A subject composition may further comprise at least one additional monopegylated CIFN species having a PEG moiety linked to a surface- exposed lysine residue in the CIFN polypeptide, where the location of the linkage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linlcage site in any other species. In all species in this example, the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 lcD. Linking groups

[0097] In some embodiments, PEG is attached to IFN-α via a linking group. The linking group is any biocompatible linking group, where "biocompatible" indicates that the compound or group is essentially non-toxic and may be utilized in vivo without causing a significant adverse response in the subject, e.g., injury, sickness, disease, undesirable immune response, or death. PEG can be bonded to the linking group, for example, via an ether bond, an ester bond, a thio ether bond or an amide bond. Suitable biocompatible linking groups include, but are not limited to, an ester group, an amide group, an imide group, a carbamate group, a carboxyl group, a hydroxyl group, a carbohydrate, a succinimide group (including, for example, succinimidyl succinate (SS), succinimidyl propionate (SPA), succinimidyl butanoic acid (SBA), succinimidyl carboxymethylate (SCM), succinimidyl succinamide (SSA) or N-hydroxy succinimide (NHS)), an epoxide group, an oxycarbonylimidazole group (including, for example, carbonyldimidazole (CDI)), a nitro phenyl group (including, for example, nitrophenyl carbonate (NPC) or trichlorophenyl carbonate (TPC)), a trysylate group, an aldehyde group, an isocyanate group, a vinylsulfone group, a tyrosine group, a cysteine group, a histidine group or a primary amine.

[0098] In many embodiments, the PEG is a monomethoxyPEG molecule that reacts with primary amine groups on the IFN-α polypeptide. Methods of modifying polypeptides with monomethoxy PEG via reductive alkylation are known in the art. See, e.g., Chamow et al. (1994) Bioconj. Chem. 5:133-140.

[0099] In one non-limiting example, PEG is linked to IFN-α via an SPA linking group. SPA esters of PEG, and methods for making same, are described in U.S. Patent No. 5,672,662. SPA linkages provide for linkage to free amine groups on the IFN-α polypeptide.

[00100] For example, a PEG molecule is covalently attached via a linkage that comprises an amide bond between a propionyl group of the PEG moiety and the epsilon amino group of a surface-exposed lysine residue in the IFN-α polypeptide. Such a bond can be formed, e.g., by condensation of an α-methoxy, omega propanoic acid activated ester of PEG (mPEGspa).

[00101] As one non-limiting example, monopegylated CIFN has a linear PEG moiety of about

30 kD attached via a covalent linkage to the CIFN polypeptide, where the covalent linkage is an amide bond between a propionyl group of the PEG moiety and the epsilon amino group of a surface-exposed lysine residue in the CIFN polypeptide, where the surface-exposed lysine residue is chosen from lys⁵⁰, lys⁷¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵, and the amide bond is formed by condensation of an α-methoxy, omega propanoic acid activated ester of PEG.

[00102] As another non-limiting example, monopegylated CIFN has a linear PEG moiety of about 30 kD attached via a covalent linlcage to the CIFN polypeptide, where the covalent linlcage is an amide bond between a propionyl group of the PEG moiety and the epsilon amino group of a surface-exposed lysine residue in the CIFN polypeptide, where the surface-exposed lysine residue is chosen from lys¹²¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵, and the amide bond is formed by condensation of an α-methoxy, omega propanoic acid activated ester of PEG. [00103] Methods for attaching a PEG molecule to an IFN-α polypeptide are known in the art, and any known method can be used. See, for example, by Park et al, Anticancer Res., 1 :373- 376 (1981); Zaplipsky and Lee, Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, ed., Plenum Press, NY, Chapter 21 (1992); and U.S. Patent No. 5,985,265. Polyethylene glycol

[00104] Polyethylene glycol is soluble in water at room temperature, and has the general formula R-O-(CH₂-CH₂O)_n-R, where R is hydrogen or a protective group such as an allcyl or an alkanol group, and where n is an integer from 1 to 1000. Where R is a protective group, it generally has from 1 to 8 carbons.

[00105] In many embodiments, PEG has at least one hydroxyl group, e.g., a terminal hydroxyl group, which hydroxyl group is modified to generate a functional group that is reactive with an amino group, e.g., an epsilon amino group of a lysine residue, a free amino group at the N- terminus of a polypeptide, or any other amino group such as an amino group of asparagine, glutamine, arginine, or histidine.

[00106] In other embodiments, PEG is derivatized so that it is reactive with free carboxyl groups in the IFN-α polypeptide, e.g., the free carboxyl group at the carboxyl terminus of the IFN-α polypeptide. Suitable derivatives of PEG that are reactive with the free carboxyl group at the carboxyl-terminus of IFN-α include, but are not limited to PEG-amine, and hydrazine derivatives of PEG (e.g., PEG-NH-NH₂).

[00107] In other embodiments, PEG is derivatized such that it comprises a terminal thiocarboxylic acid group, -COSH, which selectively reacts with amino groups to generate amide derivatives. Because of the reactive nature of the thio acid, selectivity of certain amino groups over others is achieved. For example, -SH exhibits sufficient leaving group ability in reaction with N-terminal amino group at appropriate pH conditions such that the ε-amino groups in lysine residues are protonated and remain non-nucleophilic. On the other hand, reactions under suitable pH conditions may make some of the accessible lysine residues to react with selectivity.

[00108] In other embodiments, the PEG comprises a reactive ester such as an N-hydroxy succinimidate at the end of the PEG chain. Such an N-hydroxysuccinimidate-containing PEG molecule reacts with select amino groups at particular pH conditions such as neutral pH 6.5- 7.5. For example, the N-terminal amino groups may be selectively modified under neutral pH conditions. However, if the reactivity of the reagent were extreme, accessible-NH₂ groups of lysine may also react. [00109] In other embodiments, the PEG comprises a sufficiently reactive N-hydroxy succinimidyl ester at the end of the PEG chain by virtue of having a suitable spacer e.g., a propionyl group, between the end of the PEG chain and the ester such that the ester does not hydrolyze rapidly and reacts more selectively at particular pH conditions ranging from neutral to alkaline i.e., pH 7.0-9.0. For example, the ε- amino group of certain lysines in the polypeptide chain may be selectively modified with a N-hydroxysuccinimdyl propionate ester- activated PEG. The specific process conditions used are selected to yield products of definite compositions and activities.

[00110] The PEG can be conjugated directly to the IFN-α polypeptide, or through a linker. In some embodiments, a linker is added to the IFN-α polypeptide, forming a linker-modified IFN- α polypeptide. Such linkers provide various functionalities, e.g., reactive groups such as sulfl ydryl, amino, or carboxyl groups to couple a PEG reagent to the linker-modified IFN-α polypeptide.

[00111] In some embodiments, the PEG conjugated to the IFN-α polypeptide is linear. In other embodiments, the PEG conjugated to the IFN-α polypeptide is branched. Branched PEG derivatives such as those described in U.S. Pat. No. 5,643,575, "star-PEG's" and multi-armed PEG's such as those described in Shearwater Polymers, Inc. catalog "Polyethylene Glycol Derivatives 1997-1998." Star PEGs are described in the art including, e.g., in U.S. Patent No. 6,046,305.

[00112] In embodiments of particular interest, the PEG conjugated to the IFN-α polypeptide is linear.

[00113] The PEG-modified IFN-α polypeptides of the invention comprise a single molecule of

PEG, having a molecular weight of less than 40 kDa. As used herein, and as is well known in the art, "30 kDa" PEG has an average molecular weight of 30 kDa. PEG having an average molecular weight in a range of from about 2 kDa to about 30 kDa, is generally used. For example, the molecular weight of the linear PEG molecule is in the range of from about 20 kD to about 40 lcD, from about 22 lcD to about 38 kD, from about 24 lcD to about 36 kD, from about 26 lcD to about 34 lcD, or from about 28 lcD to about 32 lcD. In particular embodiments, the PEG has a molecular weight of about 30 lcD and is linear.

[00114] The molecular weight of PEG molecules is ascertained by gel filtration column chromatography with suitable molecular weight markers, or by MALDI-TOF mass spectrometry. PEG-modified IFN-α

[00115] PEG-modified IFN-α of the invention has a molecular weight that is less than that of

IFN-α2a linked to a single molecule of branched 40 kDa PEG. An exemplary IFN-α2a conjugated to a single molecule of branched 40kDa PEG is referred to as Pegasys® (Reddy et al. ((2002) Adv. Drug Deliv. Rev. 54:571-586). The molecular weight of a subject PEG- modified IFN-α polypeptide is from about 5 kDa to about 20 kDa, from about 6 kDa to about 15 IcDa, from about 8 kDa to about 12 kDa, or from about 7 kDa to about 10 kDa less than the molecular weight of Pegasys®. In many embodiments, a subject PEG-modified IFN-α polypeptide has a molecular weight that is about 8 kDa to about 12 kDa less than that of Pegasys®.

[00116] Whether a subject PEG-modified IFN-α has a molecular weight less than that of

Pegasys® can be readily determined using standard methods of determining the molecular weight of a protein. Such methods include, but are not limited to, high performance liquid chromatography (HPLC); reverse phase HPLC; size exclusion chromatography; sodium dodecyl sulfate polyacrylamide gel electrophoresis; HPLC size exclusion chromatography (SEC); HPLC/SEC/laser light scattering; and matrix-assisted laser desorption ionization Stime- of-flight mass spectroscopy (MALDI-TOF MS). Preferably, to determine whether a subject PEG-modified IFN-α has a lower molecular weight than that of Pegasys®, the subject PEG- modified IFN-α and the Pegasys® are subjected to size exclusion HPLC under the same conditions.

[00117] A PEG-modified IFN-α polypeptide of the invention has a molecular weight of less than about 60 IcDa, and generally is from about 40 kDa to about 55 kDa, or from about 45 kDa to about 50 IcDa. In some embodiments, a subject PEG-modified IFN-α polypeptide has a molecular weight of about 50 IcDa, e.g., from about 48 kDa to about 52 kDa. Preparing PEG-IFN-α conjugates

[00118] As discussed above, the PEG moiety can be attached, directly or via a linker, to an amino acid residue at or near the N-terminus, or internally (e.g., at a surface-exposed lysine residue). Conjugation can be carried out in solution or in the solid phase.

[00119] Methods for attaching a PEG moiety to an amino acid residue at or near the N-terminus of an IFN-α polypeptide are known in the art. See, e.g., U.S. Patent No. 5,985,265.

[00120] In some embodiments, known methods for selectively obtaining an N-terminally chemically modified IFN-α are used. For example, a method of protein modification by reductive all viation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminus) available for derivatization in a particular protein can be used. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved. The reaction is performed at pH which allows one to take advantage of the pK_a differences between the ε- amino groups of the lysine residues and that of the α-amino group of the N-terminal residue of the protein. By such selective derivatization attachment of a PEG moiety to the IFN-α is controlled: the conjugation with the polymer takes place predominantly at the N-terminus of the IFN-α and no significant modification of other reactive groups, such as the lysine side chain amino groups, occurs.

[00121] If desired, PEGylated IFN-α is separated from unPEGylated IFN-α using any known method, including, but not limited to, ion exchange chromatography, size exclusion chromatography, and combinations thereof. For example, where the PEG-IFN-α conjugate is a monoPEGylated IFN-α, the products are first separated by ion exchange chromatography to obtain material having a charge characteristic of monoPEGylated material (other multi- PEGylated material having the same apparent charge may be present), and then the monoPEGylated materials are separated using size exclusion chromatography.

[00122] In some embodiments, a modified IFN-α is prepared by reacting an IFN-α polypeptide with a succinimidyl ester of alpha-methoxy, omega-propionylpoly(ethylene glycol) (mPEGspa). In general, the reaction is carried out with IFN-α and mPEGspa in a molar ratio of from about 1 : 1 to about 1 :5. Typically, the reaction is carried out in a solution having a pH of from about 7 to about 9.

[00123] In particular embodiments, CIFN is reacted with mPEGspa that is linear and has a molecular weight of about 30 kD, where the reaction is carried out with a CIFNmiPEGspa molar ratio of from about 1 : 1 to about 1:5, and where the pH of the reaction is from about 7 to about 9. In a particular embodiment, the CIFN:mPEGspa ratio is about 1 :2 and the pH of the reaction is about 8.

[00124] In some embodiments, the invention provides a modified CIFN that is produced by the process of reacting CIFN and a succinimidyl ester of alpha-methoxy, omega- propionylpoly(ethylene glycol) (mPEGspa) that is linear and has a molecular weight of about 30 lcD, where the reaction is carried out with a CIFN:mPEGspa molar ratio of from about 1 : 1 to about 1:5, and where the pH of the reaction is from about 7 to about 9. In a particular embodiment, the invention provides a modified CIFN that is produced by the process of reacting CIFN and a succinimidyl ester of alpha-methoxy, omega-propionylpoly(ethylene glycol) (mPEGspa) that is linear and has a molecular weight of about 30 kD, where the reaction is carried out with a CIFN:mPEGspa ratio of about 1:3, and at apH of about 8. Pharmacokinetic properties of PEG-modified IFN-α

[00125] A PEG-modified IFN-α polypeptide of the invention has a serum half-life (e.g., mean plasma residence time) of at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 15 hours, at least about 17 hours, at least about 20 hours, or at least about 25 hours, or longer.

[00126] Serum clearance of the PEG-modified IFN-α is reduced compared to serum clearance of unmodified IFN-α, e.g., the serum clearance of PEG-modified IFN-α is at least about 25%, at least about 50%, at least about 75%, or at least about 90%) less than the serum clearance of unmodified IFN-α of the same amino acid sequence.

[00127] The area under the curve (AUC; expressed as hr x mg/niL) for PEG-modified IFN-α of the invention is as shown in Figure 5, 6 or 7

[00128] In many embodiments, a PEG-modified IFN-α polypeptide of the invention has substantially similar pharmacokinetic profile (e.g., as described by the AUC) as interferon-α2a linked to a branched PEG molecule having a molecular weight of 40 kDa. Thus, e.g., a once- weekly dose of 180 μg of a PEG-modified IFN-α of the invention for a period of 48 weeks produces the same pharmacokinetic profile as a once- weekly dose of 180 μg of Pegasys® for a period of 48 weeks. Antiviral and anti-proliferative properties of PEG-modified IFN-α

[00129] In some embodiments, a PEG-modified IFN-α polypeptide of the invention (or a subject population of IFN polypeptides comprising PEGylated IFN-α polypeptides) exhibit antiviral activity that is at least 5-fold greater than that of PEGASYS® Peg-interferon-α2a. In some embodiments, a monopegylated IFN-α molecule(s) or population(s) of the invention exhibits antiviral activity that is at least 5-fold greater, at least 10-fold greater, at least 15-fold greater, or at least 20-fold greater, than that of PEGASYS® Peg-interferon-α2a. In some embodiments, a subject monopegylated CIFN molecule(s) or population(s) exhibits antiviral activity that is at least 5-fold greater, at least 10-fold greater, at least 15-fold greater, or at least 20-fold greater, than that of PEGASYS® Peg-interferon-α2a. The basis for comparison of antiviral activities can be any known assay, including, but not limited to, the MDBK/VSV assay as described in Example 1.

[00130] In some embodiments, a PEG-modified IFN-α polypeptide of the invention (or a subject population of IFN polypeptides comprising PEGylated IFN-α polypeptides) retains at least about 5%, at least about 7%, at least about 10%, at least about 12%, at least about 15%), or at least about 20%, or more, of the antiviral activity of the parent (non-PEGylated) IFN-α polypeptide. In some embodiments, a subject PEG-Alfacon- 1 molecule retains at least about 5%, at least about 7%, at least about 10%, at least about 12%, at least about 15%, or at least about 20%, or more, of the antiviral activity of INFERGEN® Alfacon-1. The basis for comparison of antiviral activities can be any known assay, including, but not limited to, the MDBK/VSV assay as described in Example 1.

[00131] In some embodiments, a PEG-modified IFN-α polypeptide of the invention (or a subject population of IFN polypeptides comprising PEGylated IFN-α polypeptides) exhibit anti-proliferative activity that is at least 5-fold greater than that of PEGASYS® Peg-interferon- α2a. In some embodiments, a monopegylated IFN-α molecule(s) or population(s) of the invention exhibits anti-proliferative activity that is at least 5-fold greater, at least 10-fold greater, at least 15-fold greater, or at least 20-fold greater, than that of PEGASYS® Peg- interferon-α2a. In some embodiments, a subject monopegylated CIFN molecule(s) or population(s) exhibits anti-proliferative activity that is at least 5-fold greater, at least 10-fold greater, at least 15-fold greater, or at least 20-fold greater, than that of PEGASYS® Peg- interferon-α2a. The basis for comparison of anti-proliferative activities can be any known assay, including, but not limited to, the Daudi cell assay as described in Example 1. Formulations

[00132] The above-discussed compositions can be formulated using well-known reagents and methods. Compositions are provided in formulation with a pharmaceutically acceptable excipient(s). A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7^th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al, eds., 3^rd ed. Amer. Pharmaceutical Assoc.

[00133] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

[00134] In some embodiments, a PEGylated IFN-α is formulated in an aqueous buffer. Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers varying in strengths from 5mM to lOOmM. In some embodiments, the aqueous buffer includes reagents that provide for an isotonic solution. Such reagents include, but are not limited to, sodium chloride; and sugars e.g., mannitol, dextrose, sucrose, and the like. In some embodiments, the aqueous buffer further includes a non-ionic surfactant such as polysorbate 20 or 80. Optionally the formulations may further include a preservative. Suitable preservatives include, but are not limited to, a benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. In many cases, the formulation is stored at 2-8°C. Formulations may also be lyophilized, in which case they generally include cryoprotectants such as sucrose, trehalose, lactose, maltose, mannitol, and the like. Lyophilized formulations can be stored over extended periods of time, even at ambient temperatures.

[00135] In the subject methods, the active agents may be administered to the host using any convenient means capable of resulting in the desired therapeutic effect. Thus, the agents can be incorporated into a variety of formulations for therapeutic administration. More particularly, the agents of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.

[00136] As such, administration of the agents can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc.

[00137] In pharmaceutical dosage forms, the agents may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.

[00138] For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[00139] The agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. [00140] Furthermore, the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

[00141] Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

[00142] The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[00143] Effective dosages of the subject PEG-modified IFN-α range from about 50 μg to about

300 μg, or from about 90 to about 180 μg per dose. Effective dosages the subject PEG- modified IFN-α range from 0.5 μg/kg body weight to 3.5 μg/kg body weight per dose. METHODS OF TREATING A HEPATITIS VIRUS INFECTION

[00144] The instant invention provides method of treating a hepatitis virus infection. The methods generally involve administering an effective amount of a PEG-modified IFN-α polypeptide of the invention to an individual.

[00145] In some embodiments, an "effective amount" of the subject PEG-modified IFN-α is an amount that is effective to achieve a 1.5-log, a 2-log, a 2.5-log, a 3-log, a 3.5-log, a 4-log, a 4.5-log, or a 5-log reduction in viral titer in the serum of the individual within a period of from about 12 hours to about 48 hours, from about 48 hours to about 3 days, from about 3 days to about 7 days, from about 7 days to about 2 weeks, from about 2 weeks to about 4 weeks, or from about 4 weeks to about 8 weeks, from about 8 weeks to about 12 weeks, from about 12 weeks to about 16 weeks, from about 16 weeks to about 24 weeks, or from about 24 weeks to about 48 weeks after the beginning of the dosing regimen. [00146] Patients with chronic hepatitis C generally have circulating virus at levels of 10⁵-10⁷ genome copies/ml. An effective amount of a subject PEG-modified IFN-α is an amount that is effective to reduce HCV titer down to about 5 x 10⁴ to about 10⁵, to about 10⁴ to about 5 x 10⁴, or to about 5 x 10³ to about 10⁴ genome copies per milliliter serum.

[00147] In some embodiments, an effective amount of the subject PEG-modified IFN-α is an amount that is effective to reduce HCV titer down to about 5 x 10⁴ to about 10⁵, to about 10⁴ to about 5 x 10⁴, or to about 5 x 10³ to about 10 genome copies per milliliter serum within a period of from about 12 hours to about 48 hours, or from about 16 hours to about 24 hours after the beginning of the dosing regimen.

[00148] In some embodiments, an effective amount of a PEG-modified IFN-α of the invention is an amount that is effective to reduce viral titers to undetectable levels, e.g., to about 1000 to about 5000, to about 500 to about 1000, or to about 100 to about 500 genome copies/mL serum. In some embodiments, an effective amount of a subject PEG-modified IFN-α polypeptide is an amount that is effective to reduce viral load to lower than 100 genome copies/mL serum.

[00149] In some embodiments, an effective amount of a PEG-modified IFN-α of the invention is an amount that is effective to achieve a sustained viral response, e.g., no detectable HCV RNA (e.g., less than about 500, less than about 400, less than about 200, or less than about 100 genome copies per milliliter serum) is found in the patient's serum for a period of at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, or more preferably to at least about six months following cessation of therapy.

[00150] A PEG-modified IFN-α of the invention provides for a serum concentration of PEG- modified IFN-α in the serum. The serum concentration of PEG-modified IFN-α of the invention is maintained for a period of from about 24 hours to about 48 hours, from about 2 days to about 4 days, from about 4 days to about 7 days, from about 1 week to about 2 weeks, from about 2 weeks to about 4 weeks, from about 4 weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about 8 weeks to about 12 weeks, from about 12 weeks to about 16 weeks, from about 16 weeks to about 24 weeks, or from about 24 weeks to about 48 weeks.

[00151] In some embodiments, a subject PEG-modified IFN-α provides for a serum concentration of PEG-modified IFN-α that is at or near the maximum level that is tolerable by the patient. The serum concentration that is achieved is in a range of from about 10 to about 1000, from about 10 to about 500, from about 20 to about 250, from about 30 to about 100, or from about 50 to about 75 International Units (IU)/ml. The serum concentration is maintained for a period of from about 24 hours to about 48 hours, from about 2 days to about 4 days, from about 4 days to about 7 days, from about 1 week to about 2 weeks, from about 2 weeks to about 4 weeks, from about 4 weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about 8 weeks to about 12 weeks, from about 12 weeks to about 16 weeks, from about 16 weeks to about 24 weeks, or from about 24 weeks to about 48 weeks.

[00152] In some embodiments, PEG-modified IFN-α of the invention is administered in an amount that is effective to achieve and maintain a serum concentration of the subject PEG- modified IFN-α that is from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 100% of the maximum tolerated dose (MTD). Thus, within a period of from about 6 hours to about 12 hours, from about 12 hours to about 24 hours, or from about 24 hours to about 48 hours from the beginning of the dosing regimen, a serum concentration of the subject PEG-modified IFN-α is achieved that is from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80%) to about 85%, from about 85%> to about 90%, from about 90% to about 95%), or from about 95% to about 100%ι of the maximum tolerated dose (MTD). The achieved serum concentration can be maintained for a period of about 7 days to about 2 weeks, from about 2 weeks to about 4 weeks, from about 4 weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about 8 weeks to about 12 weeks, from about 12 weeks to about 16 weeks, from about 16 weeks to about 24 weeks, or from about 24 weeks to about 48 weeks.

[00153] The administered dose of PEG-modified IFN-α is in a range of from about 50 μg to about 300 μg, e.g., from about 50 μg to about 70 μg, from about 70 μg to about 90 μg, from about 90 μg to about 100 μg, from about 100 μg to about 120 μg, from about 120 μg to about 150 μg, from about 150 μg to about 170 μg, from about 170 μg to about 200 μg, from about 200 μg to about 230 μg, from about 230 μg to about 270 μg, or from about 270 μg to about 300 μg-

[00154] Amounts of PEG-modified IFN-α to be administered are expressed in micrograms, as described above. Alternatively, the doses are also expressed as Units or International Units (IU) of activity. Units or IU are measured in vitro as the ability of the interferon to inhibit the cytopathic effect of a suitable virus (e.g. endomyocarditis virus (EMC), vesicular stomatitis virus, Semliki forest virus) after infection of an appropriate cell line (e.g., the human lung carcinoma cell lines, A549; HEP2/C; and the like). The antiviral activity is measured against a reference standard such as human interferon alpha supplied by WHO. Such methods are detailed in numerous references, including the following: Familletti, P.C., Rubinstein, S and Pestlca, S.(1981)"A convenient and rapid cytopathic effect inhibition assay for interferon", Methods in Enzymol, Vol 78(S.Pestka, ed), Academic Press, New York pages 387-394.

[00155] In some embodiments, the invention provides a method of treating a hepatitis virus infection, the method involving administering the subject PEG-modified IFN-α in an amount effective to reduce viral load.

[00156] PEG-modified IFN-α of the invention is administered daily, twice a week, once a week, once every two weeks, or three, times a week for a period of from about 24 hours to about 48 hours, from about 2 days to about 4 days, from about 4 days to about 7 days, from about 1 week to about 2 weeks, from about 2 weeks to about 4 weeks, from about 4 weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about 8 weeks to about 12 weeks, from about 12 weeks to about 16 weeks, from about 16 weeks to about 24 weeks, or from about 24 weeks to about 48 weeks.

[00157] In particular embodiments, a PEG-modified IFN-α of the invention is administered once per week for a period of from about 2 weeks to about 4 weeks, from about 4 weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about 8 weeks to about 12 weeks, from about 12 weeks to about 16 weeks, from about 16 weeks to about 24 weeks, or from about 24 weeks to about 48 weeks.

[00158] In some embodiments, the subject PEG-modified IFN-α is administered at a dosage of about 45 μg to about 270 μg, or about 180 μg, or about 120 μg per week subcutaneously for a period of from about 2 weeks to about 4 weeks, from about 4 weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about 8 weeks to about 12 weeks, from about 12 weeks to about 16 weeks, from about 16 weeks to about 24 weeks, or from about 24 weeks to about 48 weeks. The weekly dosage may be delivered by a single bolus injection or by a pump- controlled continuous infusion system

[00159] In some embodiments, the subject PEG-modified IFN-α is administered in a combination therapy, e.g., another anti-viral agent or other therapeutic agent is administered: (1) substantially simultaneously and in a separate formulation; (2) substantially simultaneously and in the same formulation; or (3) in separate formulations, and at separate times. Combination therapies are discussed in detail below. Combination therapies

[00160] In some embodiments, the methods provide for combination therapy comprising administering a composition of the invention and an additional therapeutic agent such as IFN-γ and/or ribavirin. [00161] In some embodiments, the additional therapeutic agent(s) is administered during the entire course of PEG-modified IFN-α treatment, and the beginning and end of the treatment periods coincide. In other embodiments, the additional therapeutic agent(s) is administered for a period of time that is overlapping with that of the PEG-modified IFN-α treatment, e.g., treatment with the additional therapeutic agent(s) begins before the PEG-modified IFN-α treatment begins and ends before the PEG-modified IFN-α treatment ends; treatment with the additional therapeutic agent(s) begins after the PEG-modified IFN-α treatment begins and ends after the IFN-α treatment ends; treatment with the additional therapeutic agent(s) begins after the PEG-modified IFN-α treatment begins and ends before the PEG-modified IFN-α treatment ends; or treatment with the additional therapeutic agent(s) begins before the PEG-modified IFN-α treatment begins and ends after the PEG-modified IFN-α treatment ends.

[00162] In still other embodiments, the additional therapeutic agent(s) is administered before the PEG-modified IFN-α treatment begins, and ends once PEG-modified IFN-α treatment begins, e.g., the additional therapeutic agent is used in a "priming" dosing regimen. Ribavirin and other antiviral agents

[00163] Ribavirin, l-β-D-ribofuranosyl-lH-l,2,4-triazole-3-carboxamide, available from ICN

Pharmaceuticals, Inc., Costa Mesa, Calif, is described in the Merck Index, compound No. 8199, Eleventh Edition. Its manufacture and formulation is described in U.S. Pat. No. 4,211,771. The invention also contemplates use of derivatives of ribavirin (see, e.g., U.S. Pat. No. 6,277,830). Ribavirin is administered orally in dosages of about 400, about 800, or about 1200 mg per day.

[00164] Other antiviral agents can be delivered in the treatment methods of the invention. For example, compounds that inhibit inosine monophosphate dehydrogenase (IMPDH) may have the potential to exert direct anti viral activity, and such compounds can be administered in combination with an IFN-α composition, as described herein. Drugs that are effective inhibitors of hepatitis C NS3 protease may be administered in combination with an IFN-α composition, as described herein. Hepatitis C NS3 protease inhibitors inhibit viral replication. Other agents such as inhibitors of HCV NS3 helicase are also attractive drugs for combinational therapy, and are contemplated for use in combination therapies described herein. Ribozymes such as Heptazyme™ and phosphorothioate oligonucleotides which are complementary to HCV protein sequences and which inhibit the expression of viral core proteins are also suitable for use in combination therapies described herein. In addition, suitable analogs of ribavirin including enantiomers and immune enhancers such as Zadaxin® are also suitable for use in combination therapies described herein. Determining effectiveness of treatment

[00165] Whether a subject method is effective in treating a hepatitis virus infection, particularly an HCV infection, can be determined by measuring viral load, or by measuring a parameter associated with HCV infection, including, but not limited to, liver fibrosis.

[00166] Viral load can be measured by measuring the titer or level of virus in serum. These methods include, but are not limited to, a quantitative polymerase chain reaction (PCR) and a branched DNA (bDNA) test. For example, quantitative assays for measuring the viral load (titer) of HCV RNA have been developed. Many such assays are available commercially, including a quantitative reverse transcription PCR (RT-PCR) (Amplicor HCV Monitor™, Roche Molecular Systems, New Jersey); and a branched DNA (deoxyribonucleic acid) signal amplification assay (Quantiplex™ HCV RNA Assay (bDNA), Chiron Corp., Emeryville, California). See, e.g., Gretch et al. (1995) Ann. Intern. Med. 123:321-329.

[00167] As noted above, whether a subject method is effective in treating a hepatitis virus infection, e.g., an HCV infection, can be determined by measuring a parameter associated with hepatitis virus infection, such as liver fibrosis. Liver fibrosis reduction is determined by analyzing a liver biopsy sample. An analysis of a liver biopsy comprises assessments of two major components: necro inflammation assessed by "grade" as a measure of the severity and ongoing disease activity, and the lesions of fibrosis and parenchymal or vascular remodeling as assessed by "stage" as being reflective of long-term disease progression. See, e.g., Brant (2000) Hepatol 31:241-246; and METAVIR (1994) Hepatology 20:15-20. Based on analysis of the liver biopsy, a score is assigned. A number of standardized scoring systems exist which provide a quantitative assessment of the degree and severity of fibrosis. These include the METAVIR, Knodell, Scheuer, Ludwig, and Ishalc scoring systems.

[00168] Serum markers of liver fibrosis can also be measured as an indication of the efficacy of a subject treatment method. Serum markers of liver fibrosis include, but are not limited to, hyaluronate, N-terminal procollagen III peptide, 7S domain of type IV collagen, C-terminal procollagen I peptide, and laminin. Additional biochemical markers of liver fibrosis include α- 2-macroglobulin, haptoglobin, gamma globulin, apolipoprotein A, and gamma glutamyl transpeptidase.

[00169] As one non-limiting example, levels of serum alanine aminotransferase (ALT) are measured, using standard assays. In general, an ALT level of less than about 45 international units per milliliter serum is considered normal. In some embodiments, an effective amount of IFNα is an amount effective to reduce ALT levels to less than about 45 IU/ml serum. SUBJECTS SUITABLE FOR TREATMENT

[00170] Individuals who have been clinically diagnosed as infected with a hepatitis virus (e.g.,

HAV, HBV, HCV, delta, etc.), particularly HCV, are suitable for treatment with the methods of the instant invention. Individuals who are infected with HCV are identified as having HCV RNA in their blood, and/or having anti-HCV antibody in their serum. Such individuals include naive individuals (e.g., individuals not previously treated for HCV, particularly those who have not previously received IFN-α-based or ribavirin-based therapy) and individuals who have failed prior treatment for HCV ("treatment failure" patients). Treatment failure patients include non-responders (e.g., individuals in whom the HCV titer was not significantly or sufficiently reduced by a previous treatment for HCV, particularly a previous IFN-α monotherapy using a single form of IFN-α); and relapsers (e.g., individuals who were previously treated for HCV (particularly a previous IFN-α monotherapy using a single form of IFN-α), whose HCV titer decreased significantly, and subsequently increased).

[00171] In particular embodiments of interest, individuals have an HCV titer of at least about

10⁵, at least about 5 x 10⁵, or at least about 10 , genome copies of HCV per milliliter of serum. The patient may be infected with any HCV genotype (genotype 1, including la and lb, 2, 3, 4, 6, 7, 8, 9, etc. and subtypes (e.g., 2a, 2b, 3a, etc.)), particularly a difficult to treat genotype such as HCV genotype 1 and particular HCV subtypes and quasispecies.

[00172] Also of interest are HCV-positive individuals (as described above) who exhibit severe fibrosis or early cirrhosis (non-decompensated, Child' s-Pugh class A or less), or more advanced cirrhosis (decompensated, Child' s-Pugh class B or C) due to chronic HCV infection and who are viremic despite prior anti- viral treatment with IFN-α-based therapies or who cannot tolerate IFN-α-based therapies, or who have a contraindication to such therapies. In particular embodiments of interest, HCV-positive individuals with stage 3 or 4 liver fibrosis according to the METAVIR scoring system are suitable for treatment with the methods of the present invention. In other embodiments, individuals suitable for treatment with the methods of the instant invention are patients with decompensated cirrhosis with clinical manifestations, including patients with far-advanced liver cirrhosis, including those awaiting liver transplantation. In still other embodiments, individuals suitable for treatment with the methods of the instant invention include patients with milder degrees of fibrosis including those with early fibrosis (stages 1 and 2 in the METAVIR, Ludwig, and Scheuer scoring systems; or stages 1, 2, or 3 in the Ishalc scoring system.)

[00173] In certain embodiments, the specific regimen of drug therapy used in treatment of the

HCV patient is selected according to certain disease parameters exhibited by the patient, such as the initial viral load, genotype of the HCV infection in the patient, antiviral treatment history of the patient, liver histology and/or stage of liver fibrosis in the patient. In one embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having advanced or severe stage liver fibrosis as measured by a Knodell score of 3 or 4 and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 24 weeks to about 60 weeks, or about 30 weeks to about one year, or about 36 weeks to about 50 weeks, or about 40 weeks to about 48 weeks, or at least about 24 weeks, or at least about 30 weeks, or at least about 36 weeks, or at least about 40 weeks, or at least about 48 weeks, or at least about 60 weeks.

[00174] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having advanced or severe stage liver fibrosis as measured by a Knodell score of 3 or 4 and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 40 weeks to about 50 weeks, or about 48 weeks.

[00175] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 1 infection and an initial viral load of greater than 2 million viral genome copies per ml of patient serum and then (2) administering to the patient a therapeutically effective amount of a subject PEG- modified IFN-α for a time period of about 24 weeks to about 60 weeks, or about 30 weeks to about one year, or about 36 weeks to about 50 weeks, or about 40 weeks to about 48 weeks, or at least about 24 weeks, or at least about 30 weeks, or at least about 36 weeks, or at least about 40 weeks, or at least about 48 weeks, or at least about 60 weeks.

[00176] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 1 infection and an initial viral load of greater than 2 million viral genome copies per ml of patient serum and then (2) administering to the patient a therapeutically effective amount of a subject PEG- modified IFN-α for a time period of about 40 weeks to about 50 weeks, or about 48 weeks.

[00177] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 1 infection and an initial viral load of greater than 2 million viral genome copies per ml of patient serum and no or early stage liver fibrosis as measured by a Knodell score of 0, 1, or 2 and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- α for a time period of about 24 weeks to about 60 weeks, or about 30 weeks to about one year, or about 36 weeks to about 50 weeks, or about 40 weeks to about 48 weeks, or at least about 24 weeks, or at least about 30 weeks, or at least about 36 weeks, or at least about 40 weeks, or at least about 48 weeks, or at least about 60 weeks.

[00178] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 1 infection and an initial viral load of greater than 2 million viral genome copies per ml of patient serum and no or early stage liver fibrosis as measured by a Knodell score of 0, 1, or 2 and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- α for a time period of about 40 weeks to about 50 weeks, or about 48 weeks.

[00179] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 1 infection and an initial viral load of less than or equal to 2 million viral genome copies per ml of patient serum and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 20 weeks to about 50 weeks, or about 24 weeks to about 48 weeks, or about 30 weeks to about 40 weeks, or up to about 20 weeks, or up to about 24 weeks, or up to about 30 weeks, or up to about 36 weeks, or up to about 48 weeks.

[00180] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 1 infection and an initial viral load of less than or equal to 2 million viral genome copies per ml of patient serum and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 20 weeks to about 24 weeks.

[00181] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 1 infection and an initial viral load of less than or equal to 2 million viral genome copies per ml of patient serum and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 24 weeks to about 48 weeks.

[00182] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 2 or 3 infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 24 weeks to about 60 weeks, or about 30 weeks to about one year, or about 36 weeks to about 50 weeks, or about 40 weeks to about 48 weeks, or at least about 24 weeks, or at least about 30 weeks, or at least about 36 weeks, or at least about 40 weeks, or at least about 48 weeks, or at least about 60 weeks.

[00183] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 2 or 3 infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 20 weeks to about 50 weeks, or about 24 weeks to about 48 weeks, or about 30 weeks to about 40 weeks, or up to about 20 weeks, or up to about 24 weeks, or up to about 30 weeks, or up to about 36 weeks, or up to about 48 weeks.

[00184] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 2 or 3 infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α and for a time period of about 20 weeks to about 24 weeks.

[00185] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 2 or 3 infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of at least about 24 weeks.

[00186] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 4 infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG- modified IFN-α for a time period of about 24 weeks to about 60 weeks, or about 30 weeks to about one year, or about 36 weeks to about 50 weeks, or about 40 weeks to about 48 weeks, or at least about 24 weeks, or at least about 30 weeks, or at least about 36 weeks, or at least about 40 weeks, or at least about 48 weeks, or at least about 60 weeks.

[00187] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV infection characterized by any of HCV genotypes 5, 6, 7, 8 and 9 and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 20 weeks to about 50 weeks.

[00188] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV infection characterized by any of HCV genotypes 5, 6, 7, 8 and 9 and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of at least about 24 weeks and up to about 48 weeks.

[00189] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying an antiviral treatment naive patient having a genotype 1 HCV infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 24 weeks to about 48 weeks.

[00190] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying an antiviral treatment naive patient having a genotype 1 HCV infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 48 weeks.

[00191] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying an antiviral treatment naive patient having a genotype 1 HCV infection and an initial viral load greater than 2 million HCV RNA genomes/ml of serum and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 48 weeks.

[00192] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying an antiviral treatment naive patient having a genotype 1 HCV infection and an initial viral load less than or equal to 2 million HCV RNA genomes/ml of serum and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 24 weeks to about 48 weeks.

[00193] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying an antiviral treatment naive patient having a genotype 2 or 3 HCV infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 12 weeks to about 24 weeks.

[00194] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying an antiviral treatment naive patient having a genotype 4 HCV infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 48 weeks.

[00195] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying patient who has an HCV infection and who failed and earlier course of antiviral treatment and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 24 weeks to about 60 weeks.

[00196] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient who has an HCV infection and who failed an earlier course of IFN-α therapy and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 24 weeks to about 60 weeks.

[00197] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient who has an HCV infection and who failed an earlier course of IFN-α 2a or 2b therapy and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 24 weeks to about 60 weeks.

[00198] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient who has an HCV infection and who failed an earlier course of PEGASYS® peginterferon alfa-2a or PEG-INTRON® peginterferon alfa-2b therapy and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 24 weeks to about 60 weeks.

[00199] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient who has an HCV infection and who failed an earlier course of consensus interferon therapy and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 24 weeks to about 60 weeks.

[00200] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient who has a genotype 2 or 3 HCV infection and who relapsed after responding to an earlier course of IFN-α therapy and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- α for a time period of about 24 weeks to about 48 weeks.

[00201] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient who has a genotype 1 or 4 HCV infection and who relapsed after responding to an earlier course of IFN-α therapy and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- α for a time period of about 48 weeks.

[00202] In another embodiment, the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient who has an HCV infection and who did not respond to an earlier course of IFN-α therapy and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN-α for a time period of about 48 weeks to about 60 weeks.

[00203] In connection with each of the methods tailored to the disease parameter(s) and/or other characteristics of the patient described above, the invention also contemplates co-administering to the patient a therapeutically effective amount of ribavirin for the duration of the desired course of PEG-modified IFN-α therapy.. In one embodiment, the subject method includes co- administering to the patient about 800 to about 1200 mg ribavirin orally per day, the daily dosage optionally being divided into two doses per day, for the desired course of PEG- modified IFN-α therapy. In another embodiment, the subject method includes co- administering to the patient for the duration of the desired course of PEG-modified IFN-α therapy (a) 1000 mg ribavirin orally per day if the patient has a body weight less than 75 kg or (b) 1200 mg ribavirin orally per day if the patient has a body weight greater than or equal to 75 kg, where the daily dosage is optionally divided into two doses per day. KITS

[00204] Kits with unit doses of a subject PEG-modified IFN-α, e.g. in oral or injectable doses, are provided. In such kits, in addition to the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the drugs in treating hepatitis. Preferred agents and unit doses are those described herein above.

[00205] In some embodiments, a subject kit includes a container comprising a solution comprising a unit dose of subject PEG-modified IFN-α and a pharmaceutically acceptable excipient; and instructions to administer a unit dose once per week. The instructions can be printed on a label affixed to the container, or can be a package insert that accompanies the container.

EXAMPLES [00206] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to malce and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

Example 1: Preparation and pharmacological characterization of PEG-modified CIFN Summary

[00207] CIFN (having an approximate one third of the protein as N-blocked variant, 3 mg/ml) in 20 mM sodium phosphate buffer containing 100 mM sodium chloride was treated with a two-fold molar excess of succinimido propionyl derivative of linear 30 kDa MPEG (mSPA 30K PEG) for one hour at 20°C. The crude mixture, when analyzed by SEC, showed that the monopegylated products accounted for -52% of the mixture. The major fraction was purified on a cation exchange column to provide the monopegylated CIFN with a purity of -97% or greater. This material (3 OK MPEG-monopegylated CIFN) retained a significant amount of the antiviral activity of the parent CIFN.

[00208] CIFN was N-terminally modified with 30 kD (linear) methoxypolyethylene glycol aldehyde to form N-terminal monopegylated CIFN (3 OK MPEG-α-amino-monopegylated CIFN) according to the method described in Example 3 of U.S. Pat. No. 5,985,265, except that 30 lcD (linear) methoxypolyethylene glycol aldehyde was substituted for 12 kD (linear) methoxypolyethylene glycol aldehyde. This material (3 OK MPEG-α-amino-monopegylated CIFN) retained a significant amount of the antiviral activity of the parent CIFN. Unlike the acylation reactions described below, N-terminal derivatization requires eight fold molar excess of methoxy PEG-propionaldehyde and sodium cyanoborohydride as reductant to effect the alkylation reaction at pH 4.0.

[00209] When tested in an in vitro antiproliferative activity model using Daudi cells which abundantly expresses IFN receptors, the 3 OK MPEG-monopegylated CIFN and 3 OK MPEG-α- amino-monopegylated CIFN molecules showed a substantial retention of activity of the parent molecule (CIFN) and exhibited a 20-30 fold higher activity than Peg-interferon α-2a (Pegasys®). 30K MPEG-monopegylated CIFN and 30K MPEG-α-amino-monopegylated CIFN compounds were evaluated for the pharmacokinetic behavior in vivo in rats. Both monopegylated CIFN compounds unexpectedly exhibited a circulation profile similar to the pegylated interferon α-2a (Pegasys®). MATERIALS AND METHODS CIFN and PEG

[00210] CIFN was manufactured and purified by Amgen and the final product was received as drug substance with a Certificate of Analysis at a concentration of 0.2 mg/ml. Succinimido propionyl methoxy PEG (Linear 30 lcD) was obtained as either research grade or GMP material from Shearwater Corporation, Huntsville, AL.

[00211] A typical protocol used in the small scale manufacture of one of the pegylated CIFNs is provided below:

[00212] Bulk protein (-750 mg) at a concentration of 0.2 mg/ml was subjected to ultrafiltration using a membrane area of 0.038 cm². The product was then diafiltered into 25 mM sodium phosphate buffer containing 100 mM sodium chloride adjusted to pH 8.00. The concentrated material was adjusted to 3mg/ml in protein concentration. The solution of CIFN was then mixed with two molar excess of mSPA 3 OK PEG reagent in 2mM HC1 and incubated at 21°C for one hour. The reaction was quenched by the addition of three molar excess of glycine over the amount of reagent. The reaction mixture was then diluted ten fold with 50mM sodium acetate, pH 4.5 and loaded on a Fractogel column at 2.0 mg total protein/ml of resin. The column was washed with equilibration buffer (50mM sodium acetate, pH 4.5) first and subsequently with 50mM acetate buffer containing lOOmM NaCl and 50mM acetate buffer containing 250 mM NaCl. The monopegylated product was then subjected to ultrafiltration diafiltration (UF/DF) and equilibrated with the formulation buffer (25mM sodium phosphate containing lOOmM NaCl, pH 7.0) and concentration adjusted to desired amounts for fill-finish activities. Production of PEG-modified IFN-α

[00213] Various preparations of PEG-modified CIFN were generated. These preparations of

CIFN monopegylated with 30 IcDa PEG are referred to below by product codes such as IM-002 or sometimes with an additional prefix, for example, IM-396-002.

[00214] Preparation 1 (IM-002): Purified consensus α- interferon at a concentration of 3.02 mg/ml [Prepared by diluting the protein in solution A (25 mM sodium phosphate buffer containing 100 mM NaCl at pH 7.0) with sufficient amounts of 100 mM sodium phosphate buffer to provide a final pH of 6.5] was treated with SPA-linear 30 kD mPEG at a molar ratio of 5: 1 at a pH 6.5 for 5.5 hours at ambient temperature. The monopegylated product mixture assayed by SEC chromatography accounted for 47.0% of the crude mixture. This experiment was conducted close to physiological conditions. The reaction conditions and the product antiviral activities with the samples from the early bench scale materials are summarized in Tables 1 and 2. The preliminary antiproliferative activity obtained with the early small scale products is summarized in Table 3.

[00215] Preparation 2 (IM-003): CIFN at a concentration of 2.73 mg/ml [prepared by diluting the protein in solution A ( 25 mM sodium phosphate buffer containing lOOmM NaCl at pH 7.0) with sufficient amounts of 50mM sodium phosphate dibasic solution to provide pH 7.4] was stirred with the peg reagent at a molar ratio of 1 :3 at ambient temperature for 3.5 hours and then at 2-8 °C for -15 hours. Product analysis showed that the monopegylated products accounted for -46% (Table 1) and the retention of antiviral activity was 14.5% (Table 2). This reaction was conducted at physiologic conditions and the product retains a significant amount of the parent IFN activity. Preparation 2 retained 60% of the antiproliferative activity of the CIFN molecule. [00216] Preparation 3 (IM-004): CIFN at a concentration of 3.0 mg/ml in 25mM sodium phosphate buffer containing lOOmM sodium chloride at pH 7.0 was treated with a five fold molar excess of the PEG reagent at ambient temperature for 1.8 hours. This reaction also conducted at the physiologic pH provided a mixture of monopegylated compounds that accounted for a 41.1% yield in the crude mixture (Table 1) and the antiviral activities (Table 2) and antiproliferative activities (Table 3) of this material show that this compound also is comparable to others discussed above.

[00217] Preparation 4 (IM-005): CIFN at a concentration of 2.57 mg/ml [ prepared by diluting the protein in 25mM sodium phosphate buffer containing lOOmM sodium chloride at pH7.0 with sufficient amounts of 15mM sodium hydroxide to provide pH 8.0] was treated with a three fold molar excess of the PEG reagent at a pH of 8.0 ±0.2 at 2-8 °C for a period of approximately 15 hours Analysis of the product mixture showed that the yield of monopegylated compounds accounted for 47.0%) and the product retained significant amounts of the antiviral (13.8% of CIFN, see Table 2) and antiproliferative activities (150% of CIFN activity in the early experiments, see Table 3) of the parent molecule. An optimization of the reaction conditions for this product showed that the same material can be obtained by using 1 :2 molar ratio of protein to reagent and carrying out the conjugation at 20 deg ("°C") for just one hour. There was a slight increase in yield of the product (47 to 52%).

[00218] Preparation 5 (IM-006; also referred to as IM-396-006): CIFN at a concentration of

2.55 mg/ml [prepared by diluting the protein in 25mM sodium phosphate buffer containing lOOmM sodium chloride at pH 7.0 with sufficient amounts of 20mM sodium hydroxide solution to provide pH 9.4] was treated with a 3 fold molar excess of the PEG reagent at a pH 9.4 at 2-8 °C for 3 hours. Monopegylated product yield was 47.2%(Table 1) and the retention of antiviral (Table 2) and antiproliferative (Table 3) activities are similar to the examples above.

[00219] Preparation 6 (IM-007): CIFN at 2.57 mg/ml [prepared by diluting the protein in

25mM sodium phosphate buffer containing lOOmM sodium chloride at pH 7.0 with sufficient amounts of 20mM sodium hydroxide solution to provide pH 8.8] was treated with a three fold molar excess of the PEG reagent at a pH 8.8 for 6 hours at 2-8 °C. Results obtained with this product appear in Tables 1, 2 and 3.

[00220] In all cases cited above, PEG reagent was added to the protein solution immediately and the mix rate was maintained at 60-70 rpm on the platform Roto Mix. Scale-up Production of PEG-Alfacon 1

[00221] In subsequent work requiring supplies for toxicological and clinical investigations

(starting with about a gram of the interferon drag), the PEG-Alfacon 1 drug substance was produced by a slight modification of the conditions used above. Thus, CIFN was concentrated by a prior ultrafiltration/diafiltration step to ~3mg/mL protein concentration into the reaction buffer (25mM sodium phosphate, lOOmM NaCl, pH 8.0). The conjugation reaction used 2:1 molar ratio of the PEG reagent to protein at pH 8.0 and incubation at ambient temperature for one hour. The reaction was quenched by the addition of excess glycine followed by purification using cation exchange chromatography, anion exchange and formulation prior to fill-finish. The mono pegylated products (a mixure of regioisomers) were obtained in > 90%) purity after these steps. Purities of the different lots of products ranged from 93-97%. Cation Exchange Chromato raphy

[00222] Separation of the reaction products was accomplished by loading the mixture on a

Fractogel column preequilibrated with acetate buffer at pH 4.5. The products were separated by washing with buffers differing in salt concentrations. The monopegylated isomers were isolated as a fraction and the product was subjected to ultrafiltration/diafiltration using 25mM sodium phosphate buffer containing lOOmM sodium chloride at pH 7.0. Final buffer exchange and dilution was carried out by an anion exchange over a Q Sepharose column preequilibrated with the formulation buffer.

[00223] The monopegylated product mixture was analyzed by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Reversed Phase High Performance Liquid Chromatography (RP-HPLC), and Size Exclusion Chromatography (SEC). SDS-PAGE

[00224] A 4-12% Bis-Tris NuPaGE acrylamide gel was used to perform the electrophoresis.

Bovine serum albumin was used as a standard along with molecular weight markers. The bands were visualized by colloidal blue staining method. The protein(s) were reduced prior to loading on the gel. Compared to CIFN standard, the product(s) ran as high molecular weight band, indicative of pegylation. RP-HPLC

[00225] A Phenomenex Synergi 4μ MAX-RP column was used for analysis. The product(s) were eluted with varying TFA-Acetonitrile combinations. The individual peaks were visualized by monitoring the DAD1 ultraviolet (uv) absorption at 214 nm and DAD2 wavelength of 280 nm. The major peak corresponded to monopegylated product and was used as the reference peak characteristic of the product. This method was used to characterize the preparations 1-6 described above. When the optimal pegylated product was identified for further scale-up manufacture, the HPLC analytical method was modified. Test material was injected on to a Zorbax 300 SB-C3 column ( 4.6 x 150 mm, Agilent Technologies, Palo Alto, CA). Two solvents (A: 0.1% TFA in water; B: 0.1% TFA in 80% acetonitrile) were used as mobile phases. The product was eluted using a gradient composed of the two mobile phases over 26 minutes. A flow rate of 1.0 mL/min was used and samples were detected by uv absorbance at 214 nm. PEG-Alfaconl eluted as a main peak with a retention time of 11.2 minutes (see Figure 4). SEC-HPLC

[00226] The identity, purity and strength analyses of the pegylated interferon(s) are carried out using a size exclusion HPLC method. A Shodex KW-803 and a Shodex KW-804 columns were used in tandem in the early characterization of preparations 1-6. The columns were run in 5mM sodium phosphate buffer at pH 7.25 at a flow rate of lml/minute. The peaks were monitored by uv absorption at 214 nm. Judged on the basis of the molecular weights and the hydrodynamic radii, the peak assigmnents were made for the mixture of isomers eluting as a single peak corresponding to monopegylated products. During scale-up manufacture of PEG- Alfacon 1, this method was modified. The product in formulation buffer was injected on a Superose 12HR 10/30 column attached to a HP Model 1100 HPLC system. Flow rate was adjusted to 0.5 mL/min and the products were detected by uv absorbance at 214 and 280 nm. Pegylated CIFN eluted at 19.5 minutes under these conditions (Figure 2).

[00227] The molecular weights of the pegylated products were further analyzed by matrix- assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS).

[00228] The biological characteristics of the products were evaluated by an antiviral assay, an assay for antiproliferative activity, and pharmacokinetics in rats. 3 OK MPEG-α-amino-monopegylated CIFN

[00229] CIFN was reductively alkylated with 30 lcD linear monomethoxypolyethylene glycol aldehyde, essentially as described in Example 3 of U.S. Pat. No. 5,985,265, except that a 30 lcD linear PEG reagent (a reactive aldehyde) was substituted for the 12 kD linear PEG reagent specified in Example 3. 3 OK MPEG-α-amino-monopegylated CIFN was recovered and purified from the alkylation reaction mixture as described in Example 3 of U.S. Pat. No. 5,985,265. This preparation (IM-001) is referred to as N-terminal PEG-CIFN below. Antiviral Assay

[00230] Madin-Darby Bovine Kidney (MDBK) cells were infected with Vesicular Stomatitis

Virus (VSV) and treated with CIFN or Peg-Alfacon or N-terminal PEG-CIFN. The activity is based on the inhibition of viral replication and is normalized for the molar concentrations of the individual proteins. Percent inhibition was calculated by the following formula: Percent Inhibition = (Test-VC)/(CC-VC) where Test represents the cell staining observed in the presence of test article, VC represents the cell staining in the presence of viras but no test article and CC represents the cell staining in the absence of virus. When tested for antiviral activity using the MDBK cell line, PEG-Alfacon and N-terminal PEG-CIFN exhibited a dose dependent antiviral activity against VSV viras. Similar antiviral activity has also been measured with human lung carcinoma (A549) cells infected with Endomyocarditis (EMC) virus. Antiproliferative Assay

[00231] Human Burkitt Lymphoma or Daudi cells were treated with CIFN or PEG-Alphacon or

N-terminal PEG-CIFN at predetermined concentrations for three days and cell proliferation was measured by the incorporation of tritiated thymidine. Percent inhibition was calculated by the following formula:

[00232] Percent Inhibition = [l-(test cpm/total cpm)] x 100 where test counts per minute (cpm) represents the value measured for the test compound at the concentrations indicated and total cpm represents the value measured for proliferation of cells in the absence of test compound. When tested for antiproliferative activity using the Daudi cell line, PEG-Alfacon and N- terminal PEG-CIFN exhibited dose-dependent antiproliferative activity and the results also showed the retention of significant amount of the parent CIFN activity by the monopegylated isomer mixture. Rat Pharmacokinetics

[00233] The in vivo clearance of pegylated CIFN products was studied in 3 male Sprague-

Dawley rats. Pegylated interferons were administered by subcutaneous route (50 - 250 μg/kg) in rats. Serum concentrations were monitored over a period of one week. Cmax, AUC and the elimination t-_1/2 were determined using a pharmacokinetic analysis. A significant increase in half-life was seen with all the monopegylated CIFN mixtures. Consequently, PEG-Alfacon and N-terminal PEG-CIFN demonstrate a significantly increased plasma exposure. A similar study has also been carried out in cynomolgus monkeys and the PK parameters as well as the levels of 2 '-5' Oligoadenylate synthetase (OAS) following subcutaneous injections of PEG-Alfacon and N-terminal PEG-CIFN were monitored over 10 day time period. OAS is a surrogate marker for the potency of alpha interferons. RESULTS AND DISCUSSION

PEG-modified CIFN

Table 1. Summary of the preparation process and yield for the various pegylated CIFN molecules

] Elution with 50mM sodium acetate buffer containing 250 mM NaCl, pH 4.5 gave the

PEG-Alfacon monopegylated product(s) as a mixture with purity of 97% and greater. The purity was ascertained by a SEC-HPLC and SDS-PAGE gel electrophoresis. Data obtained are shown in Figures 2 and 3. A reversed phase HPLC analysis (Figure 4) showed that the purified product appeared as a single major peak with a retention time of 12 minutes. The average molecular mass of pegylated CIFN was measured using MALDI-TOF mass spectrometry. The observed mass of 52260 is consistent with one unit of nominal 30kd PEG (average MW of 32700 Da as measured by gel permeation chromatography (GPC) attached to one molecule of CIFN. Antiviral activity

Table 2. Antiviral activities of the pegylated alpha interferons

Antiviral Activity

[00235] In cytopathic effect inhibition assays using the MDBK/VSV system, the pegylated

CIFN products (any of preparation nos. IM-002, IM-003, IM-004, IM-005, IM-006 and IM- 007) and the N-terminal PEG-CIFN product (preparation no. IM-001) exhibited approximately 10%o of the antiviral activity of the parent molecule (CIFN). Of these, the derivative, IM-005 was chosen as the lead candidate for further development and designated as PEG-Alfacon. The antiviral activities measured depended on the actual cell lines used and infecting virus combinations. In cytopathic effect inhibition assays using the A549/EMC virus system, the PEG-Alfacon product and in the N-terminal PEG-CIFN product exhibited somewhat less than 10%) retention of CIFN antiviral activity.

[00236] Roferon®interferon-alfa2a exhibits an antiviral activity that is 5-10 fold lower than that of Infergen® alfacon-1 when measured in the MDBK/VSV system [Ozes ON et al. J Interferon Res 12: 55-59 (1992)]. Extrapolating from the data in Table 2 above, it is evident that Pegasys®peg-interferon-alfa2a retains approximately 1.5-3.0 % of the antiviral activity of Roferon®interferon-alfa2a (compared to 0.3%> of the antiviral activity of Infergen® or Interferon Alfacon-1) when measured in the MDBK/VSV system. Thus, the pegylated consensus interferon molecules' percent retention of the parental interferon molecule's (Infergen®alfacon-l's) antiviral activity is approximately 10-fold greater than Pegasys®peg- interferon-alfa2a's percent retention of the parental interferon molecule's (Roferon®interferon- alfa2a's) antiviral activity. Similar comparisons were also made with a reference standard of PEG-Alfacon (pegylated CIFN or Infergen) and other commercial products such as PEG- Intron or PEGASYS (see Tables 4 and 5, below) in several cell lines using different infecting viruses. Results obtained support the earlier antiviral data presented above for pegylated CIFN product. Based on these CPE assay systems, PEG-Alfacon appears to have superior antiviral potency compared to PEGASYS and comparable potency to that of PEG-Intron. It is therefore anticipated to have sufficient antiviral activity for use in therapy of chronic hepatitis C. Antiproliferative activity Table 3. Antiproliferative activities of the pegylated alpha interferons

[00237] Antiproliferative activity, another measure of biological activity in interferons, was less affected by pegylation. The PEG-alfacon product exhibited approximately 40-150%) of the antiproliferative activity of the parent molecule (CIFN). The N-terminal PEG-CIFN product exhibited approximately 45% of the antiproliferative activity of the parent molecule (CIFN). Nevertheless, the pegylated consensus interferon molecules exhibited approximately 5-15 fold higher antiproliferative activity than that exhibited by PEGASYS ®PEG-interferon-alfa2a in the Daudi cell-based antiproliferation assay. Pharmacokinetics

[00238] A comparison of the various analogs synthesized as part of the invention were evaluated in the rat PK model and demonstrated significantly longer circulation times (Figure 5). In Figure 5, IM-001 denotes N-terminal PEG-CIFN and IM-003, IM-005 and IM-006 denote pegylated CIFN preparations 2, 4 and 5, respectively. The semilogarithmic plot of the serum concentration-time profile for one of these analogs designated IM-006 (or IM-396-006) is shown in Figure 6. The pharmacokinetic profile of the pegylated consensus interferon molecules exhibited characteristics very similar to pegylated interferon α-2a (Pegasys®). Thus the Cmax was reached after 24 hours and the Cmax was 633 ng/mL. The elimination half life was about 27 hours while the clearance of CIFN has been shown to occur with a tι_/ value of approximately 6 hours. It is interesting to note that all pegylated CIFN molecules i.e., N- terminal modified (IM-001) or internal lysine derivatized analogs (IM-003, IM-005 and IM- 006) exhibited very similar pharmacokinetic (PK) profiles. A similar study conducted in cynomolgus monkeys showed that the pegylated CIFN molecule (IM-005) persisted in blood for up to one week (Figure 7) and the serum at all time points examined elicited 2 '-5' OAS activity. The pharmacodynamic marker could be seen elevated in blood for at least five days. The half-life for the terminal elimination of Peg-Alfaconl was computed to be about 30 hours in cynomolgus monkeys. Thus the pegylated CIFN generated from a linear 30kd PEG reagent unexpectedly gave the desired PK characteristics for once a week administration in vivo.

Example 2: Antiviral activity characterization of PEG-modified CIFN and other PEGylated alpha-interferons

[00239] In order to fully characterize the pegylated CIFN of this invention in other human cell lines for antiviral activity and also compare to other similar products, experiments were conducted to screen such molecules in A549, HeLa and ME180 cells using the same infecting viruses such as VSV and EMCV. MATERIALS AND METHODS

[00240] HeLa or ME 180 cells were grown in DMEM or RPMI-1640 medium supplemented with 10%) heat-inactivated serum (calf or fetal as required) (Hyclone Laboratories, Inc., Logan, UT), L-glutamine (2 mM), streptomycin (100 μg/ml), and penicillin (100 u/ml). Cells were grown at 37°C in a 5% CO₂ humidified incubator. HeLa or ME 180 cells (2 x 104 cells per well in 96-well microtiter plates) were treated with IFN for 24 hours prior to the addition of the virus. Vesicular Stomatitis Virus (VSV) or endomyocarditis virus (EMCV) at a MOI of 0.1 was added and the plates incubated for an additional 48 hours. At this time, the cell monolayer was stained with 0.5% crystal violet in 20% (vol/vol) methanol. All cytopathic effect (CPE) inhibition assays were done in duplicate. One unit of IFN was defined as that amount of IFN that inhibited cytopathic effect by 50%>. The activity of all IFNs was measured against a NIH standard human IFN-α (Namalwa/Sendai) (Ga23-901-532).

[00241] A549/EMCV CPE assays were performed according to the standard protocol described in the literature. RESULTS

[00242] In the initial screen, CIFN or Infergen was compared to PEG-Alfacon 1 (also known as

PEG-CIFN or pegylated Infergen) in different cell lines with VSV in the cytopathic effect (CPE) inhibition assay. Data obtained from this study are shown in Table 4. Specific activity was calculated by comparing the activity of the test interferon to NIH standard, GA23-902- 530. Table 4: Comparison of Antiviral Activity of IFN- Alfacon-1 and PEG-Alfacon- 1

[00243] Comparisons of the specific activities for the cell lines ME180 and HeLa showed that

Infergen or Interferon Alfacon-1 had significantly higher antiviral activity (P < 0.001) for all results in Table 4. In contrast, the specific activity of Interferon alfacon-1 and PEG-Alfacon-1 were quite similar when A549 cells were used.

[00244] Antiviral activities of the product of this invention were compared with the parent molecule and other marketed products in different cell lines. The experimental data from such studies are analyzed using one way Anova statistical analysis program. The graphic presentation of the results shown in Figures 8-10 indicate the statistical significance of the data. The specific activity data obtained from this comparative study in A549, HeLa and ME 180 cell lines using the EMC viras are also summarized in Table 5. Table 5. Mean Antiviral Specific Activities

[00245] It is clear from all the data presented in this example that PEG-Alfacon 1 retains a significant amount of the antiviral activity of the parent, Infergen molecule. In the head-to- head comparison, it is clear that PEG-Alfacon 1 retains comparable level of activity to PEG- Intron and significantly higher level of activity than Pegasys. However, in terms of the pharmacokinetic properties (Figure 5), PEG-Alfacon 1 does much better compared to PEG- Intron and the half-life for terminal elimination of the drag is comparable to Pegasys. Thus PEG-Alfacon is expected to have a superior in vivo activity based on a combination of antiviral activity and pharmacokinetic properties. [00246] The antiviral activity assay data obtained for Infergen® (Interferon Alfacon- 1 , PEG- alfacon- 1, Pegasys® (peg-interferon-alfa2a), and PEG-Intron® (peg-interferon-alfa2b) in the A549, HeLa and Me 180 cell systems (Tables 4 and 5, above) above are consistent with the antiviral activity assay data obtained for several pegylated CIFN molecules, infergen® (Interferon Alfacon-1) and Pegasys® (peg-interferon-alfa2a) in the MDBK cell system (Table 2 above). Both sets of data indicate that PEG-alfacon- 1 (and other pegylated CIFN molecules) exhibits an antiviral activity that is at least 10-fold greater than that of Pegasys®peg-interferon- alfa2a. In addition, the MDBK assay data in Table 2 and the A549 and ME 180 data in Tables 4 and 5 indicate that PEG-Alfacon-1 retains approximately 10%) of the antiviral activity of the parental interferon molecule (CIFN) whereas Pegasys®peg-interferon-alfa2a retains approximately 1.0% of the parental interferon molecule's activity (i.e. Roferon®interferon- alfa2a's activity) in the same assay systems based on the data presented here and published in the literature which has repeatedly shown a 5-10 fold greater activity of Infergen vs Roferon. Thus, PEG-Alfacon- l's percent retention of the parental interferon molecule's antiviral activity (i.e. the antiviral activity of Infergen®alfacon-l) is approximately 10-fold greater than Pegasys®peg-interferon-alfa2a's percent retention of the parental interferon molecule's antiviral activity (i.e. the antiviral activity of Roferon®interferon-alfa2a).

Example 3: Pharmacokinetic and pharmacodynamics analysis of PEG-alfaeon [00247] The following is a summary of the results of pharmacokinetic and pharmacodynamic data analysis that uses results from a single dose study of PEG-alfacon. Individual and mean PK profiles were examined and mean PD profiles of serum 2'5'-oligoadenylate synthetase (OAS) activity were examined. Additionally, PK profiles were used to simulate serum drug profiles predicted for multiple PEG-Alfacon doses given at 10 day intervals. [00248] The pharmacokinetic data were taken from the single dose, range finding clinical study of PEG-alfacon. Groups of 6 healthy male and female volunteers were given a single subcutaneous injection of PEG-alfacon in doses ranging from 15 to 210 μg in an escalating dose design. Samples from volunteers who received doses from 60 μg and higher were selected for this analysis. The final treatment group received a 210 μg dose that was identified in the escalation phase as the maximum tolerated dose. Baseline serum PEG-alfacon values from samples obtained 5 min before dosing were measurable in two cases (subjects 531 and 532, 120 μg dose group). Baseline values were subtracted from subsequent samples to obtain baseline corrected values for these two cases and data are shown with and without baseline correction.

[00249] Individual data were subjected to noncompartmental and compartmental pharmacokinectic analysis. Mean data were analyzed in three steps with the aid of commercially available Kinetica™ software: noncompartmental pharmacokinetic analysis, compartmental pharmacokinetic analysis and compartmental pharmacokinetic/pharmacodynamic analysis.

[00250] Results from noncompartmental pharmacokinetic analysis of serum drag concentrations are summarized in Table 6. Peak concentrations were achieved within 36 to 72 h after sc dosing and increased with dose from a mean of 665 pg/mL after a 60 μg dose to approximately 3600 pg/mL after 210 μg. Mean profiles are shown in Figure 11. Table 6 shows mean PK parameters calculated from individual profiles and PK parameters calculated from mean serum profiles. In general there was reasonable agreement between the methods although there was considerable variation between subjects within each group that could not be readily explained by sex, body mass index (BMI; kg/m²) or thigh vs abdominal injection site variables. As an example, subject 424 had one quantifiable value following a 90 μg dose and other samples were below assay detection, while samples from subject 425 were all above 6000 pg/mL for PEG-alfacon following the same dose.

[00251] Table 6 compares the mean pharmacokinetic parameters for each dose group calculated using noncompartmental methods with corresponding parameters calculated using compartmental analysis of mean serum concentrations for each dose group. Data were insufficient to calculate parameters with noncompartmental methods for subjects 318, 423, 425, 529 and 747. Both methods resulted in calculated parameters that were in general agreement with values for AUC parameters (reflecting total drug exposure over time), and elimination half-life (tι_/ ) showing similar values with each method. Peak serum concentrations tended to be slightly lower for mean data compared to means of individual maximum concentrations. There was considerable variability within groups with %CV ranging from approximately 30 to 70%> across all parameters.

[00252] Mean serum profiles for each dose group were fitted to a 1-compartment body model using commercially available software. Individual serum profiles were also fitted to a 1- compartment body model. In 6 of the 36 individual datasets drug profiles, data were insufficient to permit calculation of a 1-compartment body model (Subjects 315, 318, 321, 425, 529, 747). Table 6

Mean noncompartmental pharmacokinetic parameters*

60 μg 90 μg 120 nε^a 150 ue 180 μg 210 us n/dose 5 4 6 6 6 5

Cmax [pg/mL] 665±274 1454±467 2979±1829 2608±1492 2298±1065 3610±1179

[580] [1090] [2203] [2441] [2150] [2938]

'max InJ 72±0 70±43 49±36 37±23 54±28 62±27

[72] [72] [48] [48] [48] [72]

AUC o-last 57.9±34.2 156.2±52.9 2420.5±156.0 296.8±203.2 250.3±117.2 537.6±253.7

[ng/mL*h] (59.1) [136.1] [214.6] [320.0] [277.5] [474.3] tι/2 [h] 119±133 44±9 23±8 40±17 51±29 75±28

[39.8] [38.5] [30] [62] [65] [87]

Mean Compartmental pharmacokinetic parameters

Cmax [pg/mL] 877±257 1364± 654 2752±1425 2656±1529 2306±971 3819±1333

[619] [1288] [2572] [2557] [2116] [3027] [h] 48±19 72±59 34±16 35±22 47±118 50±18

[65] [44] [44] [44] [43] [44]

AUC o-_∞ 95.2±29.8 149.6±102.4 278.8±189.3 329.6±222.2 290.0±133.4 620.7±269.6

[ng/mL*h] [53.2] [153.9] [341.3] [333.0] [293.5] [526.2] tι/2 [h]] 38±17 38±29 30±22 38±20 39±14 68±40

[27] [29] [51] [47] [57] [85]

* calculated from individual serum profiles±sd [calculated from mean serum profiles]

Calculated using baseline corrected values for subjects 531 and 532 [00253] Dose corrected Cmax and AUC values are expected to be constant for drugs that follow linear kinetics. As shown in Table 7, there was considerable variation in both dose-corrected parameters. There was no consistent trend among doses, suggesting that variation within groups may explain deviations from dose proportional increase. [00254] Table 7. Dose Corrected Cmax and AUC parameters calculated from individual means and from mean profiles (mean) for values determined using noncompartmental methods.

Table 7

Dose Cmax AUC o-last

[μg] [pg/mL] [pg/mL*h]

60 11 964

90 16 1735

120* 25 2017

150 17 1979

180 13 1390

210 17 2560 meanόO 10 986 mean90 12 1512 meanl20* 18 1789 raeanl50 16 2133 meanlSO 12 1542 mean210 14 2258

* calculated using baseline corrected values for subjects 531 and 32 [00255] Dose corrected AUCo-ia_st and C_raax values were plotted versus body mass index (BMI) to examine potential effects of body weight on drug absorption from the subcutaneous injection. Results as shown in Figures 12A and 12B, indicate no consistent trends in drug absorption with change in BMI.

[00256] Fits of mean serum concentration data with the 1-compartment model resulted in reasonable curves for single subcutaneous doses ranging from 60 tlirough 210 μg as shown in Figure 13 for mean profiles.

[00257] Simulations used pharmacokinetic parameters calculated from the fitted curves to model serum drag profiles following multiple dosing regimens. Figure 14A simulates the serum profiles expected with a dosing regimen of 60 μg administered every 10 days using mean parameters calculated from each dose group. Baseline corrected values were used for subjects 531 and 532 to calculate mean parameters in the 120 μg dose group for all simulations. In general, there is good agreement among each simulation. Steady state is reached during the first dose interval and trough levels fall below the 300 pg/mL level that is the limit of assay quantification. Simulations based on mean data from the 60 μg dose group had peak concentrations that were approximately 50% of peak concentrations in simulations calculated from mean data from subjects given a 150 μg dose of PEG-alfacon. The difference is can be attributed to variability among treatment groups, as there is no apparent treatment group (dose) related change in simulation profiles. Additional simulations are shown in Figures 14B-G for 100, 150 and 200 μg doses administered every 10 and every 7 days. Profiles show the expected pattern of change with increasing dose. There is little effect from shortening the dosing interval to 7 days.

[00258] Mean serum OAS response profiles are shown in Figure 15 where PD response is shown as %> change from baseline. Standard deviations were comparable to mean values in many cases that reflects considerable variability within each treatment group.

[00259] Figure 16 shows the results of PK-PD modeling. Calculated EC₅₀ values, which reflect the concentration of drag resulting in 50% of maximum effect, varied greatly among doses. Similarly, the calculated Emax value had large differences with dose as shown in Table 8. Table 8

Dose (meg) Emax (% OAS Change) EC50 (pg/mL)

60 313 127

90 7932 27701

120 791 1046

150 470 202

180 717 591

210 441 212 While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

What is claimed is:

1. A monopegylated consensus interferon (CIFN) molecule comprised of a single CIFN polypeptide and a single polyethylene glycol (PEG) moiety, wherein the PEG moiety is linear and about 30 kD in molecular weight and is directly or indirectly attached through a covalent linkage to a lysine residue in the CIFN polypeptide.

2. The monopegylated CIFN molecule of claim 1 , wherein the PEG moiety is linked to a surface-exposed lysine residue in the CIFN polypeptide.

3. The monopegylated CIFN molecule of claim 1, wherein the lysine residue is selected from the group consisting of lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide.

4. The monopegylated CIFN molecule of claim 3, wherein the lysine residue is ι lys of the CIFN polypeptide.

5. The monopegylated CIFN molecule of claim 3, wherein the lysine residue is lys⁵⁰ of the CIFN polypeptide.

6. The monopegylated CIFN molecule of claim 3, wherein the lysine residue is

71 lys of the CIFN polypeptide.

7. The monopegylated CIFN molecule of claim 3, wherein the lysine residue is lys⁸⁴ of the CIFN polypeptide.

8. The monopegylated CIFN molecule of claim 3, wherein the lysine residue is lys¹²¹ of the CIFN polypeptide.

9. The monopegylated CIFN molecule of claim 3, wherein the lysine residue is lys¹²² of the CIFN polypeptide.

10. The monopegylated CIFN molecule of claim 3, wherein the lysine residue is lys¹³⁴ of the CIFN polypeptide.

11. The monopegylated CIFN molecule of claim 3, wherein the lysine residue is lys¹³⁵ of the CIFN polypeptide.

12. The monopegylated CIFN molecule of claim 3, wherein the lysine residue is lys¹⁶⁵ of the CIFN polypeptide.

13. The monopegylated CIFN molecule of any of claims 1-12, wherein the covalent linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the lysine residue in the CIFN polypeptide.

14. The monopegylated CIFN molecule of any of claims 1-13, wherein the covalent linkage comprises an amide bond formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the lysine residue in the CIFN polypeptide.

15. A composition comprising a population of monopegylated consensus interferon (CIFN) molecules, wherein the population consists of two or more species of molecules, wherein each such species is comprised of a single CIFN polypeptide and a single PEG moiety and the PEG moiety is linear and about 30 kD in molecular weight and is directly or indirectly attached tlirough a covalent linkage to either a lysine residue in the CIFN polypeptide or the N-terminal residue in the CIFN polypeptide, provided that in at least a first such species the PEG moiety is in a covalent linlcage with a lysine residue in the CIFN polypeptide, and provided that in a second such species the PEG moiety is in a covalent linlcage with the N-terminal residue in the CIFN polypeptide.

16. The composition of claim 15, wherein in the first such species the PEG moiety is in a covalent linlcage with a surface-exposed lysine residue in the CIFN polypeptide.

17. The composition of claim 15, wherein the population comprises two or more species of monopegylated CIFN in which the PEG moiety is covalently linked to a lysine residue in the CIFN polypeptide, provided that the location of the linkage site in any such species is not the same as the location of the linkage site in any other such species.

18. The composition of claim 15, wherein the lysine residue is selected from the group consisting of lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide.

19. The composition of claim 17, wherein the lysine residue of the linkage site in any such species is selected from the group consisting of lys³ , lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide.

20. The composition of claim 17, wherein the lysine residue of the linkage site in any such species is a surface-exposed lysine residue in the CIFN polypeptide.

21. The composition of any of claims 15-20, wherein the covalent linkage in the first such species comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of a lysine residue in the CIFN polypeptide, and the covalent linlcage in the second such species comprises an amide bond between a propionyl group of the PEG moiety and the alpha-amino group of the N-terminal amino acid residue in the CIFN polypeptide.

22. The composition of any of claims 15-20, wherein the covalent linkage in the first such species comprises an amide bond formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of a lysine residue in the CIFN polypeptide, and the covalent linlcage in the second such species comprises an amide bond formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the alpha-amino group of the N-terminal residue of the CIFN polypeptide.

23. A composition comprising a population of monopegylated consensus interferon (CIFN) molecules, wherein the population consists of two or more species of molecules, wherein each such species is comprised of a single CIFN polypeptide and a single PEG moiety and the PEG moiety is linear and about 30 kD in molecular weight and is directly or indirectly attached tlirough a covalent linkage to a lysine residue in the CIFN polypeptide, provided that the location of the linkage site in any such species is not the same as the location of the linkage site in any other such species.

24. The composition of claim 23, wherein the lysine residue at the linlcage site in any such species is selected from the group consisting of lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide.

25. The composition of claim 23, wherein the lysine residue at the linlcage site in any such species is a surface-exposed lysine residue of the CIFN polypeptide.

26. The composition of any of claims 15-25, wherein the CIFN polypeptide is interferon alpha-co .

27. The monopegylated CIFN molecule of any of claims 1-14, wherein the CIFN polypeptide is interferon alpha-con^

28. A product that is produced by the process of:

(a) reacting consensus interferon (CIFN) and a succinimidyl ester of alpha-methoxy, omega-propionylpoly(ethylene glycol) (mPEGspa) that is linear and has a molecular weight of about 30 kD, at a CIFN:mPEGspa molar ratio of about 1 : 1 to about 1 :5, and at a pH of about 7 to about 9; and

(b) recovering the monopegylated product of the reaction of step (a).

29. The product of claim 28, wherein in step (a) the CIFN:mPEGspa ratio is about 1 :3 and the pH is about 8.

30. The product of claim 28, wherein in step (a) the CIFN:mPEGspa ratio is about 1 :2 and the pH is about 8.0.