WO2011017160A1

WO2011017160A1 - Mutant human interferon proteins and genes

Info

Publication number: WO2011017160A1
Application number: PCT/US2010/043536
Authority: WO
Inventors: William A. Clark
Original assignee: Pestka Biomedical Laboratories, Inc.
Priority date: 2009-07-28
Filing date: 2010-07-28
Publication date: 2011-02-10

Abstract

A new set of interferon polypeptides and genes are disclosed. The new interferon polypeptides are active and will be useful for therapeutic purposes. Administration of the interferon genes may also be useful for therapeutic purposes.

Description

Mutant Human Interferon Proteins and Genes

5 Cross-Reference to Related Application

[0001 ] This application claims priority to U.S. Provisional Application 61/229,167, filed July 28, 2009, which application is incorporated herein by reference in its entirety.

Background of the Invention

10 |0002] The invention relates to interferons, mutant interferons and biologically active fragments of interferons. The invention also relates to methods of using interferons, mutant interferons and biologically active fragments of interferons in a pharmaceutical composition and for treating a subject infected with a virus.

[0003] Interferons (IFNs) are a well known family of cytokines secreted by a large

15 variety of eukaryotic cells upon exposure to various stimuli (Zoon KC: Human

Interferons: Structure and Function, p. 1 -12. In: Interferon 8. Academic Press, London, 1987; Walter et al., Cancer Biotherm Radiopharm 1998 June; 13(3): 143-54; Pcstka, S., Biopolymers 2000; 55(4):254-287; Pestka, S., Methods in Enzymology, 78, 1981 ; Pestka, S., Methods in Enzymology, 79, 1981 ; Pestka, S., Methods in

20 Enzymology, 1 19, 1986). The interferons have been classified by their chemical and biological characteristics. The interferons are divided into two groups designated Type 1 and Type II interferons (Pestka, S.; Langcr; J.A.; Zoon, K.C.; Samuel, CE. Ann Rev Biochem 1987, 56, 727-777; Pestka, S., Biopolymers 2000; 55(4):254-287). IFN-γ, known also as immune interferon, is the only Type II interferon whereas the

25 Type I human interferons consist of several classes: IFN-α, IFN-β, IFN-ε, IFN-ω, IFN-K and IFN-τ. There is only one human IFN-β and one human IFN-ω, but a family of multiple IFN-α species exists. IFN-τ is only found in ungulates; there is no human IFN-τ. The IFNs exhibit anti- viral, immunoregulatory, and antiproliferative activity. The clinical potential of interferons has been recognized.

[0004] Interferon was discovered by Isaacs and Lindenmann (Proc. Royal Soc. London, Ser B 147, 258, 1957). Efforts to purify and characterize human leukocyte interferon have led to the preparation of homogeneous leukocyte interferons (now called IFN-αs) derived from normal or leukemic (chronic myelogenous leukemia or "CML") donor leukocytes (Pestka, S., Biopolymers 2000; 55(4):254-287; Rubinstein, M.; Levy, W.P.; Moschera, J.A.; Lai, C-Y.; Hershberg, R.D.; Bartlett, R.T.; Pestka, S. Arch Biochem Biophys 1981, 210, 307-318.). Homogeneous fibroblast interferon (now called IFN-β) was also purified to homogeneity (Friesen, H.-J.; Stein, S.;

Evinger, M.; Familletti, P. C; Moschera, J.; Meienhofer, J.; Shively, J.; Pestka, S. Arch Biochem Biophys 1981, 206, 432-450). These interferons are a family of proteins characterized by a potent ability to confer a virus-resistant state in their target cells. In addition, interferon can inhibit cell proliferation, modulate immune responses and alter expression of proteins. These properties have prompted the clinical use of leukocyte interferon as a therapeutic agent for the treatment of viral infections and malignancies.

[0005] During the past several decades, a large number of human and animal interferons have been produced, identified, purified, and cloned. Several of the interferon preparations have been prepared for clinical trial in crude form as well as in purified form. Several individual recombinant interferon-α species have been cloned and expressed. The proteins have then been purified by various procedures and formulated for clinical use in a variety of formulations. Most of the interferons in clinical use that have been approved by various regulatory agencies throughout the world are mixtures or individual species of human α interferon (Hu-IFN-α). In some countries Hu-IFN-β and γ have also been approved for clinical trial and in some cases approved for therapeutic use. The major thesis underlying clinical use of these interferons was that they were natural molecules produced by normal individuals. Indeed, the specific thesis was that all the interferons prepared for clinical use, be they natural- or recombinant-generated products, represented interferons that were produced naturally by normal people. This is true for a large number of interferons as well as specific growth factors, lymphokines, cytokines, hormones, clotting factors and other proteins that have been produced.

Summary of the Invention [0006] In one aspect, the invention comprises a biologically active interferon polypeptide or fragment thereof encoded by a mutant interferon polynucleotide, wherein the interferon polypeptide is selected from the group consisting of:

a) any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20;

b) an amino acid sequence at least 95% identical to any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20;

c) an amino acid sequence that differs from any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 by at least 1 amino acid;

d) an amino acid sequence encoded by a polynucleotide having any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19;

e) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to any one of SEQ ID NOS: 1, 3, 5, 7, 9, 1 1, 13, 15, 17, and 19; and

f) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to a sequence that is complementary to any one of SEQ ID NOS: 1 , 3, 5, 7, 9, 11, 13, 15, 17, and 19.

[0007] The the biological activity may be selected from the group consisting of antiviral activity, antiproliferative activity, induction of interferon gamma in NK-92 cells, and MHC class I antigen expression induction activity. The biologically active Interferon polypeptide or fragment thereof may also be used in a pharmaceutical composition with a suitable excipient.

[0008] In another aspect, the invention provides a method of treating a virus- infected subject or reducing a subject's risk of infection by a virus, comprising administering to the subject the biologically active interferon polypeptide or fragment thereof described above, wherein the virus is selected from the group consisting of severe acute respiratory syndrome-associated coronavirus (SARS), coronavirus, influenza, smallpox virus, cowpox virus, monkeypox virus, encephalitis-causing viruscs, hepatitis A, hepatitis B, hepatitis C, other non-A/non-B hepatitis, herpes virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex, human herpes virus type 6 (HHV-6), West Nile virus, vaccinia virus, respiratory syncytial virus, rhinovirus, arterivirus, filovirus, picornavirus, reovirus, rotavirus, rabies, papilloma virus, retroviruses including human immunodeficiency viruses (HIV), human T lymphotropic virus-type 1 and 2 (HTLV- 1/-2), papovavirus, herpesvirus, poxvirus, hepadnavirus, astrovirus, coxsackie virus, paramyxovirus, orthomyxovirus, echovirus, enterovirus, cardiovirus, togavirus, rhabdovirus, bunyavirus, arenavirus, bornavirus, adenovirus, parvovirus, and flavivirus.

[0009] In some embodiments, the interferon polypeptide comprises any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20. In other embodiments, the mutant interferon polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1 , 3, 5, 7, 9, 11, 13, 15, 17, and 19. The subject may be a human being, non-human primate, feline, canine, fish, and farm animal.

[0010] The interferon may be administered nasally, orally, parenterally, topically, rectally, by injection, by inhalation, by eye lotion, by ointment, by suppository, by controlled release patch, by infusion, or by inhalation. In some embodiments, the interferon is administered to the subject's nasopharyngeal mucosa or lung epithelium. The amount of the interferon polypeptide administered may be an amount effective to reduce the concentration of the virus particles in the subject. In some embodiments, the amount of the interferon polypeptide administered is an amount effective to prevent or reduce an increase in the concentration of virus particles in the subject.

[0011] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. Brief Description of the Drawings

[0012] Figure 1: Sequences of novel interferon genes (SEQ ID NOS: 1, 3, 5, 7, 9, 1 1, 13, 15, 17, and 19) and proteins (2, 4, 6, 8, 10, 12, 14, 16, 18, and 20).

[0013] Figure 2: Relative biological activities of novel interferon proteins in several bioassays in comparison to human IFN alpha 2a. The effectiveness of IFN α2a and novel interferons was tested in four separate bioassays. In all cases the activity was standardized to that of IFN α2, which was set as one. If the IFN is more potent than IFN α2, then the value is greater than one and if less potent, then the value is less than one.

|0014] Figure 3: Relative biological activities of each novel interferon protein in several bioassays in comparison to the congener wild-type interferon proteins. The effectiveness of IFN α2a and novel interferons was tested in four separate bioassays. In all cases the activity was standardized to that of the congener wide type which was set as one. If the IFN is more potent than congener wide type, then the value is greater than one and if less potent then it is less than one.

Detailed Description of the Invention [0015] Unless otherwise defined, 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. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. Throughout this specification and claims, the word "comprise," or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0016] The practice of the invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987);

Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, VoIs. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds.,

1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1986).

[0017] In the nucleotide and amino acid sequences described herein within the sequence listing, with respect to a nucleotide sequence, an "n" or "x" can refer to any nucleotide, whereas with respect to a protein sequence, an "n" or "x" can refer to any amino acid.

[0018] As used herein, the term "comprising" is an open-ended term that includes the specific elements and may include additional, unrecited elements. "Comprising" may be synonymous with "including" or "containing". "Comprising" may also, separately and independently of the above definition, be read as "consisting essentially of or "consisting of. As used herein, "consisting of is a closed term that includes only the specific elements recited, and "consisting essentially of includes the specific elements recited and may include additional unrecited, nonmaterial elements.

[0019] The term "sequence" includes nucleotide and amino acid sequences.

[0020] As used herein, a fragment is a portion of a polypeptide or polynucleotide sequence that is at least 2 amino acids in length or at least 6 nucleotides in length, respectively. The term oligopeptide refers to an amino acid sequence between 2 and about 20 amino acids in length. The term oligonucleotide refers to a nucleotide sequence between 2 and about 50 nucleotides in length. 10021) The term "farm animal" includes, but is not limited to, artiodactyla, lagomorpha, carnivora, reptilia, aves, bovine, equine, avian, ovine, caprine, lagomorpha, and swine.

|0022] The term "fish" includes, but is not limited to, chondrichthyes, osteichthyes and agnatha.

|0023] The term "mutant interferons" includes mutant interferon polypeptides and mutant interferon polynucleotides. As used herein, "mutant" interferon refers to an interferon polypeptide or polynucleotide that is not naturally occurring. Mutant interferons retain biological activity. In some embodiments, mutant interferons may have enhanced biological activity or activities. In some embodiments, mutant interferons may have some ^"decreased biological activity or activities. Mutant interferon polypeptides include interferons with an amino acid mutation, substitution or deletion, or a truncated polypeptide, compared to a naturally occurring interferon. Mutant interferon polynucleotides also include polynucleotide sequences with a nucleotide substitution or deletion that may or may not encode a mutant interferon polypeptide. Thus, mutant interferon polynucleotides include polynucleotide sequences encoding mutant interferon polypeptides, but also include, for example, codon-optimized polynucleotide sequences that encode a naturally occurring interferon polypeptide or a mutant polypeptide. As used herein, "mutant" and "variant" are used interchangeably. Also as used herein, "naturally occurring" and

"wild-type" and "wild type" are used interchangeably. Therefore, a mutant interferon is neither naturally occurring nor a wild-type.

|0024| As used herein, "biological activity" includes one or more of the functions or activities of a naturally occurring or wild-type polypeptide and/or polynucleotide interferon. Interferon biological activities may include, but are not limited to, antiviral activity, antiproliferative activity, induction of interferon gamma in NK 92 cells, and major histocompatibility complex (MHC) class I antigen expression induction activity.

|0025] The term "isolated" as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule. The term isolated also refers to a nucleic acid or peptide, polypeptide or protein that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an "isolated nucleic acid" is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.

(0026| The term "high stringent hybridization conditions" is well understood in the art to encompass conditions of hybridization that allow hybridization of structurally related, but not structurally dissimilar, nucleic acids. The term "stringent" is a term of art which is understood by the skilled artisan to describe any of a number of alternative hybridization and wash conditions which allow annealing of only highly complementary nucleic acids.

[0027] As used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology".

[0028] The term "antibody" as it is used herein with respect to the invention, includes an isolated, recombinant or synthetic antibody, antibody conjugate or antibody derivative. The term "antibody" is often intended to include an antibody fragment, including an antigen-binding fragment, unless otherwise indicated or understood by context. An antigen-binding fragment competes with the intact antibody for specific binding. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989)). Antigen-binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some embodiments, antigen-binding fragments include Fab, F(ab)2, Fab', F(ab')2, F(ab')3, Fd, Fv, domain antibodies (dAb), other monovalent and divalent fragments, complementarity determining region (CDR) fragments, single- chain antibodies (e.g., scFv, scFab, and scFabΔC), chimeric antibodies, diabodies, triabodies, minibodies, nanobodies, and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide; and fusions and derivatives of the foregoing. See, e.g., Holliger and Hudson, Nature Biotechnology 23: 1126-1 136 (2005) and Hust et al., BMC Biotech 7: 14 (2007).

[0029| As used herein, "biologically acceptable medium" includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation. The use of such media for pharmaceutically active substances is known in the art. [0030| As used herein, the terms "pharmaceutical preparation", "pharmaceutical formulation" and "pharmaceutical composition" are interchangeable.

[0031) The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0032] The term "treatment" is intended to encompass also prophylaxis, therapy, cure, and prevention of spread of infection.

[0033] The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, the urinary bladder or other compartment of the body, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration. The phrase "therapeutically effective amount" as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal and thereby blocking the biological consequences of that pathway in the treated cells, at a reasonable benefit/risk ratio applicable to any medical treatment.

[0034| The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0035| The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agonists from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

[0036] As used herein, a "pharmaceutical salt" includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention.

|0037] Liposomes, as used herein, refer to lipid vesicles having an outer lipid shell, typically formed on one or more lipid bilayers, encapsulating an aqueous interior.

[0038) As used herein, "vesicle-forming lipid" refers to any amphipathic lipid having hydrophobic and polar head group moieties and which by itself can form spontaneously into bilayer vesicles in water, as exemplified by phospholipids.

[0039] As used herein, the term "microparticles" includes microspheres (uniform spheres), microcapsules (having a core and an outer layer of polymer), and particles of irregular shape.

[0040] As used herein, chemical derivatives thereof refer to substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art.

Interferon Polypeptides and Polynucleotides of the Invention

[0041] The present invention provides nucleic acids comprising a polynucleotide encoding an interferon polypeptide including an amino acid sequence shown in at least one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20, or a fragment thereof. Thus, one aspect of the invention provides a polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; (b) a nucleotide sequence comprising one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19; (c) a nucleotide sequence encoding a biologically active interferon fragment of a polypeptide of (a) or (b) above; and (d) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b) or (c) above. The fragments consist essentially of biologically active fragments of the interferon polypeptides. Further embodiments of the invention include (1) a polynucleotide comprising a nucleotide sequence with at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity to any of the nucleotide sequences in (a), (b), (c) or (d), above; (2) a polynucleotide that differs from any of the nucleotide sequences in (a), (b), (c) or (d), above, by 1 nucleotide, by 2 nucleotides, by 3 nucleotides, by 4 nucleotides, by 5 nucleotides, by 6 nucleotides, by 7 nucleotides, by 8 nucleotides, by 9 nucleotides, by 10 nucleotides, by 1 1 nucleotides, by 12 nucleotides, by 13 nucleotides, by 14 nucleotides, by 15 nucleotides, by 16 nucleotides, by 17 nucleotides, by 18 nucleotides, by 19 nucleotides, by 20 nucleotides, by 21 nucleotides, by 22 nucleotides, by 23 nucleotides, by 24 nucleotides, by 25 nucleotides, by 26 nucleotides, by 27 nucleotides, by 28 nμcleotides, by 29 nucleotides or by 30 nucleotides; or (3) a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c) or (d) above. SEQ ID NOS 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 are human interferon gene and protein sequences.

[0042] Generally, stringent hybridization conditions are selected to be about 5-10 ⁰C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 ⁰C for short probes (e.g. 10 to 50 nucleotides) and at least about 60 ⁰C for long probes (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In other embodiments, less stringent hybridization conditions may be used; for example, moderate or low stringency conditions may be used, and are well known in the art.

[0043] Exemplary high stringent hybridization conditions are equivalent to about 20-27 ⁰C below the melting temperature (T_m) of the DNA duplex formed in about 1 M salt. Many equivalent procedures exist and several popular molecular cloning manuals describe suitable conditions for highly stringent hybridization and, furthermore, provide formulas for calculating the length of hybrids expected to be stable under these conditions (see, e.g., Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1 6 or 13.3.6; or pages 9.47-9.57 of Sambrook, et al. (1989) Molecular Cloning, 2nd ed., Cold Spring Harbor Press). In a certain embodiment, "high stringency" refers to hybridization and/or washing conditions at 68°C in 0.2 x SSC, at 42 ⁰C in 50 % formamide, 4 x SSC, or under conditions that afford levels of hybridization equivalent to those observed under either of these two conditions.

|0044| Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T_m, for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher T_m) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNAiRNA, DNA: DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating T_n, have been derived (see Sambrook et al., supra, 9.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.8). A minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides.

[0045] Suitable hybridization conditions for oligonucleotides (e.g., for

oligonucleotide probes or primers) are typically somewhat different than for full- length nucleic acids (e.g., full-length cDNA), because of the oligonucleotides' lower T_m. Because the T_m of oligonucleotides will depend on the length of the

oligonucleotide sequences involved, suitable hybridization temperatures will vary depending upon the oligonucleotide molecules used. Exemplary temperatures may be 37 ⁰C (for 14-base oligonucleotides), 48 ⁰C (for 17-base oligonucleotides), 55 ⁰C (for 20-base oligonucleotides) and 60 ⁰C (for 23-base oligonucleotides). Exemplary suitable hybridization conditions for oligonucleotides include washing in 6 x SSC, 0.05 % sodium pyrophosphate, or other conditions that afford equivalent levels of hybridization.

[0046] With respect to polypeptide, peptide and protein fragments of the invention, these may be biologically active fragments and may be of any length less than the full lengths of the sequences described in the sequence identifiers provided herein. In certain embodiments, a fragment is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, or 170 amino acids in length.

|0047] With respect to nucleic acid fragments, these include those which may be useful as diagnostic probes and primers as discussed herein. Such sequences may include those which specifically identify the interferon nucleotide sequences described herein, and they may be of any length. For example, they include those of at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides, and more preferably at least about 20 nucleotides, still more preferably at least about 30 nucleotides, and even more preferably, at least about 40 nucleotides in length. Of course, larger fragments such as those from 50-300 nucleotides in length are also useful according to the present invention as are fragments corresponding to at least one of the nucleotide sequences shown in at least one of SEQ ID NOS provided herein. By a fragment at least 20 nucleotides in length, for example, is intended fragments which include 20 or more contiguous bases from at least one of the nucleotide sequences as shown in at least one of SEQ ID NOS provided herein. Fragments of 50-300 nucleotides in length include, for example, those which are about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 bases in length.

(0048] A polynucleotide that hybridizes to a portion of a polynucleotide includes a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides, and more preferably at least about 20 nucleotides, still more preferably at least about 30 nucleotides, and even more preferably about 30-70 nucleotides, including any length in between (e.g. 50 bases), of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below. [0049] The present invention also provides nucleic acids comprising a

polynucleotide encoding at least a portion of a human interferon polypeptide including an amino acid sequence shown in at least one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20, or a biologically active fragment thereof. Thus, one aspect of the invention provides a polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; (b) a nucleotide sequence comprising one of SEQ ID NOS: 1, 3, 5, 7, 9, 1 1, 13, 15, 17, and 19; (c) a nucleotide sequence encoding a biologically active interferon fragment of a polypeptide of (a) or (b) above; and (d) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b) or (c) above. The fragments consist essentially of biologically active fragments of the interferon polypeptides. Further embodiments of the invention include (1) a polynucleotide comprising a nucleotide sequence with at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity to any of the nucleotide sequences in (a), (b), (c) or (d), above; (2) a polynucleotide that differs from any of the nucleotide sequences in (a), (b), (c) or (d), above, by 1 nucleotide, by 2 nucleotides, by 3 nucleotides, by 4 nucleotides, by 5 nucleotides, by 6 nucleotides, by 7 nucleotides, by 8 nucleotides, by 9 nucleotides, by 10 nucleotides, by 11 nucleotides, by 12 nucleotides, by 13 nucleotides, by 14 nucleotides, by 15 nucleotides, by 16 nucleotides, by 17 nucleotides, by 18 nucleotides, by 19 nucleotides, by 20 nucleotides, by 21 nucleotides, by 22 nucleotides, by 23 nucleotides, by 24 nucleotides, by 25 nucleotides, by 26 nucleotides, by 27 nucleotides, by 28 nucleotides, by 29 nucleotides or by 30 nucleotides; or (3) a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c) or (d) above. According to the invention, each of these sequences and/or fragments is biologically active. [0050] In another aspect, any of the nucleic acids of the present invention which encode interferon polypeptides may include, but are not limited to, those encoding the amino acid sequence of the complete polypeptide, by itself; and the coding sequence for the complete polypeptide and additional sequences, such as those encoding an added secretory leader sequence, such as a pre-, or pro- or prepro-protein sequence.

[0051] Also provided are the nucleic acids described herein further comprising additional, non-coding sequences, including, for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example-ribosome binding and stability of mRNA; and an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.

[0052) Thus, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif. 91311), among others, many of which are commercially available. For instance, hexa- histidine as described by Gentz et al. provides for convenient purification of the fusion protein (Gentz et al. (1989) Proc. Natl. Acad. Sci. USA 86: 821-824). The "HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al. (1984) Cell 37: 767). Other examples of fusion tags include the following: (1) MBP tag is a portion of maltose binding protein (vectors available from Roche™ (Example vector designation: pIVEX MBP), New England Biological™ (Example vector designation: pMAL-p2X); (2) HA tag is a portion of the hemagglutinin of human influenza virus (vectors available from Roche™ (Example vector designation: pIVEX HA-tag), BD Bioscienccs™ (pCMV-HA)); (3) FLAG is a particular 8 amino acid epitope (vector from Sigma Chemical Co.™ (Example vector designation: gWiz/GFP); (4) CBP tag is a portion of calmodulin binding protein (vector available from Stratagene™: Example vector designation: pDual); and (5) GFP is a portion of green fluorescent protein (vector available from Stratagene™: Example vector designation: pDual). Othcr common epitope tags for creating fusion proteins: c-myc, GST, AUl, AU5, DDDDK, E epitope, E2 tag, Glu-Glu, Sl, KT-3, T7 epitope tag, V5 epitope tag, VSV-G, BFP, CFY, YFP. As discussed below, other such fusion proteins include an interferon fused to Fc at the N- or C-terminus.

10053] The present invention also provides recombinant vectors, which comprise the nucleic acids of the present invention, and host cells containing the recombinant vectors, as well as methods of making such vectors and host cells and for using them for production of interferon polypeptides or peptides by recombinant techniques. |00541 The invention further provides an interferon polypeptide comprising an amino acid sequence selected from: (a) the amino acid sequence of an interferon polypeptide comprising an acid sequence shown in at least one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; and (b) the amino acid sequence of a biologically active interferon fragment of a polypeptide of (a). The fragments consist essentially of biologically active fragments of the interferon polypeptides. Further embodiments of the invention include (1) biologically active polypeptides having at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity to any of the polypeptide sequences in (a) or (b) above; and (2) an amino acid sequence that differs from any of the amino acid sequences in (a) or (b), above, by 1 amino acid, by 2 amino acids, by 3 amino acids, by 4 amino acids, by 5 amino acids, by 6 amino acids, by 7 amino acids, by 8 amino acids, by 9 amino acids, by 10 amino acids, by 11 amino acids, by 12 amino acids, by 13 amino acids, by 14 amino acids, by 15 amino acids, by 16 amino acids, by 17 amino acids, by 18 amino acids, by 19 amino acids, by 20 amino acids, by 21 amino acids, by 22 amino acids, by 23 amino acids, by 24 amino acids, by 25 amino acids, by 26 amino acids, by 27 amino acids, by 28 amino acids, by 29 amino acids or by 30 amino acids. According to the invention, each of these sequences and/or fragments is biologically active.

|0055] An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which comprises the amino acid sequence of an epitope-bearing portion of an interferon polypeptide having an amino acid sequence described herein. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of an interferon polypeptide of the invention include portions of such polypeptides with at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14 or at least 15, or more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above are included in the invention.

[0056| In another aspect, the invention further provides a human interferon polypeptide comprising an amino acid sequence selected from: (a) the amino acid sequence of an interferon polypeptide comprising an amino acid sequence shown in at least one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20; and (b) the amino acid sequence of a biologically active interferon fragment of a polypeptide of (a). The fragments consist essentially of biologically active fragments of the interferon polypeptides. Further embodiments of the invention include (1) biologically active polypeptides having at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity to any of the polypeptide sequences in (a) or (b) above; and (2) an amino acid sequence that differs from any of the amino acid sequences in (a) or (b), above, by 1 amino acid, by 2 amino acids, by 3 amino acids, by 4 amino acids, by 5 amino acids, by 6 amino acids, by 7 amino acids, by 8 amino acids, by 9 amino acids, by 10 amino acids, by 11 amino acids, by 12 amino acids, by 13 amino acids, by 14 amino acids, by 15 amino acids, by 16 amino acids, by 17 amino acids, by 18 amino acids, by 19 amino acids, by 20 amino acids, by 21 amino acids, by 22 amino acids, by 23 amino acids, by 24 amino acids, by 25 amino acids, by 26 amino acids, by 27 amino acids, by 28 amino acids, by 29 amino acids or by 30 amino acids. According to the invention, each of these sequences and/or fragments is biologically active. [0057| Another embodiment of the invention provides mutant interferons. In one embodiment, mutant interferon polypeptides of the invention differ from naturally occurring interferon polypeptides by 1 amino acid, by 2 amino acids, by 3 amino acids, by 4 amino acids, by 5 amino acids, by 6 amino acids, by 7 amino acids, by 8 amino acids, by 9 amino acids or by 10 amino acids. In other embodiments, mutant interferon polypeptides of the invention differ from naturally occurring interferon polypeptides by 15 or fewer amino acids, by 20 or fewer amino acids or by 25 or fewer amino acids. According to the invention, each of these sequences and/or fragments is biologically active.

[0058] In another embodiment, mutant interferon polypeptides of the invention share at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity with any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. According to the invention, each of these sequences and/or fragments is biologically active.

|0059| In one embodiment, mutant interferon polynucleotides of the invention differ from naturally occurring interferon polynucleotides by 1 nucleotide, by 2 nucleotides, by 3 nucleotides, by 4 nucleotides, by 5 nucleotides, by 6 nucleotides, by 7 nucleotides, by 8 nucleotides, by 9 nucleotides, by 10 nucleotides, by 11 nucleotides, by 12 nucleotides, by 13 nucleotides, by 14 nucleotides, by 15 nucleotides, by 16 nucleotides, by 17 nucleotides, by 18 nucleotides, by 19 nucleotides, by 20 nucleotides, by 21 nucleotides, by 22 nucleotides, by 23 nucleotides, by 24 nucleotides, by 25 nucleotides, by 26 nucleotides, by 27 nucleotides, by 28 nucleotides, by 29 nucleotides or by 30 nucleotides. In other embodiments, mutant interferon polypeptides of the invention differ from naturally occurring interferon polypeptides by 35 or fewer nucleotides, by 40 or fewer nucleotides or by 45 or fewer nucleotides. According to the invention, each of these sequences and/or fragments is biologically active. 10060] In another embodiment, mutant interferon polynucleotides of the invention share at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity with any of SEQ ID NOS: 1, 3, 5, 7, 9, 11 , 13, 15, 17, or 19. According to the invention, each of these sequences and/or fragments is biologically active.

[0061] In one embodiment, mutant interferon polynucleotides include the backtranslated versions of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19.

[0062] In one embodiment, mutant interferon polynucleotides include the reverse- backtranslated versions of SEQ ID NOS: 1, 3, 5, 7, 9, 1 1, 13, 15, 17, or 19.

[0063| It is anticipated that certain mutant interferon polypeptides of the invention may act as agonist or antagonists. While not wishing to be bound to any particular theory, it is well known that mutant forms of protein signaling factors are capable of binding to the appropriate receptor and yet not capable of activating the receptor. Such mutant proteins act as antagonists by displacing the wild-type proteins and blocking the normal receptor activation. Additionally, it is well known that one or more amino acid substitutions can be made to many proteins in order to enhance their activity in comparison to wild type forms of the protein. Such agonists may have, for example, increased half-life, binding affinity, stability, or activity in comparison to the wild type protein. There are many well known methods for obtaining mutants (or variants) with a desired activity.

[0064] Methods for generating large pools of mutant or variant proteins are well known in the art. In one embodiment, the invention contemplates using interferon polypeptides generated by combinatorial mutagenesis. Such methods, as are known in the art, are convenient for generating both point and truncation mutants, and can be especially useful for identifying potential variant sequences (e.g., homologs) that are functional in a given assay. The purpose of screening such combinatorial libraries is to generate, for example, interferon variants or homologs that can act as either agonists or antagonists. Thus, combinatorially derived variants can be generated to havc an increased potency relative to a naturally occurring form of the protein.

Likewise, interferon variants can be generated by the present combinatorial approach to act as antagonists, in that they are able to mimic, for example, binding to other extracellular matrix components (such as receptors), yet not induce any biological response, thereby inhibiting the action of interferon polypeptides or interferon agonists. Moreover, manipulation of certain domains of interferon by the present method can provide domains more suitable for use in fiαsion or chimeric proteins, for example, domains demonstrated to have specific useful properties.

[0065] To further illustrate the state of the art of combinatorial mutagenesis, it is noted that the review article of Gallop et al. (1994) J Med Chem 37: 1233 describes the general state of the art of combinatorial libraries as of the earlier 1990's. In particular, Gallop et al state at page 1239 "[screening the analog libraries aids in determining the minimum size of the active sequence and in identifying those residues critical for binding and intolerant of substitution". In addition, the Ladner et al. PCT publication WO90/02809, the Goeddel et al. U.S. Patent 5,223,408, and the Markland et al. PCT publication WO92/ 15679 illustrate specific techniques which one skilled in the art could utilize to generate libraries of variants which can be rapidly screened to identify variants/fragments which possess a particular activity. These techniques are exemplary of the art and demonstrate that large libraries of related variants/truncants can be generated and assayed to isolate particular variants without undue experimentation. Gustin et al. (1993) Virology 193:653, and Bass et al. (1990) Proteins: Structure, Function and Genetics 8:309-314, also describe other exemplary techniques from the art which can be adapted as a means for generating mutagenic variants of the interferon polypeptides of the invention.

|0066] Table 1 below illustrates the common alleles of the human interferon α family of genes/proteins and was constructed based on Pestka, S. (1983) Arch Biochem Biophys 221 : 1-37; Diaz, M.O., Pomykala, H.M., Bohlander, S.K., Maltepe, E., Malik, K., Brownstein, B., and Olopade, O.I. (1994) Genomics 22:540-52; and Pestka, S. (1986) Meth. Enzymol 1 19:3-14, and reviewed in Krause, CD., Lunn, C.A., Izotova, L.S., Mirochnitchenko, O., Kotenko, S.V., Lundell, D.J., Narula, S. K., and Pestka, S. (2000) J Biol Chem. 275:22995-3004. Tabic 1

Note: ψ indicates pseudogene

[0067] Allelic variants of human interferon-α genes and interferon-α mutants have been reported in the following applications: WO 2002/095067; WO 02/079249; WO 02/101048; WO 02/095067; WO 02/083733; WO 02/086156; WO 2002/083733; WO 03/000896; WO 02/101048; WO 02/079249; WO 03/000896; WO 2004/022593; WO 2004/022747; WO 03/023032; WO 2004/022593. See also the following publications: (1 ) Kim et al, Cancer Lett. 2003 Jan 28;189(2):183-8; (2) Hussain et al., J Interferon Cytokine Res. 2000 Sep;20(9):763-8; (3) Hussain et al., J Interferon Cytokine Res. 1998 Jul; 18(7):469-77; (4) Nyman et al., Biochem J. 1998 Jan 15;329 ( Pt 2):295-302; (5) Golovleva et al, J Interferon Cytokine Res. 1997 Oct;17(10):637- 45; (6) Hussain et al, J Interferon Cytokine Res. 1997 Sep;17(9):559-66; (7) Golovleva, I., Saha, N., Beckman, L. Hum Hered. 1997 Jul-Aug;47(4): 185-8; (8) Golovlcva et al, 1997 Apr;18(4):645-7; (9) Kita et al, J Interferon Cytokine Res. 1997 Mar; 17(3): 135-40; (10) Golovleva et al, Am J Hum Genet. 1996 Sep;59(3):570-8; (11) Hussain et al, J Interferon Cytokine Res. 1996 Jul;16(7):523-9 ; (12) Linge et al, Biochim Biophys Acta. 1995 Dec 27;1264(3):363-8 ; (13) Gewert et al, J Interferon Cytokine Res. 1995 May;15(5):403-6; (14) Lee et al, J Interferon Cytokine Res. 1995 Apr;15(4):341-9; (15) Kaluz et al, Acta Virol. 1994 Apr;38(2): 101-4; (16) Emanuel et al, J Interferon Res. 1993 Jun;13(3):227-31; (17) Kaluz et al, Acta Virol. 1993 Feb;37(l):97- 100; (18) Shekhter et al, Dokl Akad Nauk SSSR. 1990;314:998-1001 ; (19) Li et al, Sci China B. 1992 Fcb;35(2):200-6.

[0068] Indeed, it is plain from the combinatorial mutagenesis art that large scale mutagenesis of interferon proteins, even without knowing which residues were critical to the biological function, can generate wide arrays of variants having equivalent biological activity. Alternatively, such methods can be used to generate a wide array of variants having enhanced activity or antagonistic activity. Indeed, it is the ability of combinatorial techniques to screen billions of different variants by high throughout analysis that removes any requirement of a priori understanding or knowledge of critical residues.

[0069] A preferred method for determining the best overall match between a query sequence and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of

Brutlag et al. {Comp. App. Biosci., 6:237-245 (1990)). In a nucleotide or amino acid sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is presented in terms of percent identity. Preferred parameters used in a FASTDB search of a DNA sequence to calculate percent identity are:

Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining Penalty=30, Randomization Group Length=0, and Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, and Window Size=500 or query sequence length in nucleotide bases, whichever is shorter. Preferred parameters employed to calculate percent identity and similarity of an amino acid alignment are: Matrix=PAM 150, k-tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score= I , Gap Penalty=5, Gap Size Pcnalty=0.05, and Window Size=500 or query sequence length in amino acid residues, whichever is shorter.

[0070] Protein and/or nucleic acid sequence homologies may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN,

BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988, Proc.

Natl. Acad. Sci. USA 85(8):2444-2448; Altschul et al., 1990, J. MoI. Biol.

215(3):403-410; Thompson et al., 1994, Nucleic Acids Res. 22(2):4673-4680;

Higgins et al., 1996, Methods Enzymol. 266:383-402; Altschul et a]., 1990, J. MoI. Biol. 215(3):403-410; Altschul et al., 1993, Nature Genetics 3:266-272).

[0071| In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST") which is well known in the art (see, e.g., Karlin and Altschul, 1990, Proc. Natl. Acad

Sci. USA 87:2267-2268; Altschul et al., 1990, J. MoI. Biol. 215:403-410; Altschul et al., 1993, Nature Genetics 3:266-272; Altschul et al., 1997, Nuc. Acids Res. 25:3389-

3402). In particular, five specific BLAST programs are used to perform the following task:

(1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database;

(2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database;

(3) BLASTX compares the six- frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database;

(4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and

(5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six- frame translations of a nucleotide sequence database.

[0072] The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., 1992, Science 256: 1443-1445; Henikoff and Henikoff, 1993, Proteins 17:49-61). Less preferably, the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).

[0073] The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably select those segments which satisfy a user- specified threshold of significance, such as a user-specified percent homology.

Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (see, e.g., Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268).

|0074| The parameters used with the above algorithms may be adapted depending on the sequence length and degree of homology studied. In some embodiments, the parameters may be the default parameters used by the algorithms in the absence of instructions from the user.

[0075] In some embodiments, the FASTDB algorithm described in Brutlag et al. Comp. App. Biosci. 6:237-245, 1990, is used. In such analyses the parameters may be selected as follows: Matrix=Unitary, k-tuple=4, Mismatch Penalty=l , Joining Penalty=30, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the sequence which hybridizes to the probe, whichever is shorter.

[0076] Using the above methods and algorithms such as FASTA with parameters depending on the sequence length and degree of homology studied, for example the default parameters used by the algorithms in the absence of instructions from the user, one can obtain nucleic acids encoding proteins having at least 90%, at least 91% sequence homology, at least 92% sequence homology, at least 93% sequence homology, at least 94% sequence homology, at least 95% sequence homology, at least 96% sequence homology, at least 97% sequence homology, at least 98% sequence homology, at least 99% sequence homology, at least 99.25% sequence homology, at least 99.50% sequence homology, at least 99.75% sequence homology, or 100% sequence homology to a protein encoded by a nucleic acid. In some embodiments, the homology levels can be determined using the "default" opening penalty and the "default" gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoffet al., in: Atlas of Protein Sequence and Structure, Vol. 5, Supp. 3 (1978)).

|0077] Alternatively, the level of polypeptide homology may be determined using the FASTDB algorithm described by Brutlag et al. Comp. App. Biosci. 6:237-245, 1990. In such analyses the parameters may be selected as follows: Matrix=PAM 0, k- tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Window Size=Sequence Length, Gap Penalty=5, Gap Size

Penalty=0.05, Window Size=500 or the length of the homologous sequence, whichever is shorter.

[0078] The invention also encompasses sequences having a lower degree of identity than that described herein but having sufficient similarity so as to perform one or more of the same functions. Similarity is determined by conserved amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, VaI, Leu, and He, interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and GIu, substitution between the amide residues Asn and GIn, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent is found in Bowie et al., Science 247:1306-1310 (1990).

[0079] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0080| The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in

Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J MoI. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web site at GCG.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available on the world wide web site at GCG.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4: 1 1-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0081) The nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J. MoI. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Antibodies to Interferons of the Invention

|0082] In another embodiment, the invention provides an antibody that binds specifically to an interferon polypeptide of the invention. The invention further provides methods for isolating antibodies that bind specifically to an interferon polypeptide of the invention. Such antibodies are useful therapeutically as described below.

[0083] The antibodies of the invention include whole antibodies and any antigen- binding fragments thereof, antibody derivatives or variants that may contain one or more modifications (e.g., an amino acid insertion, deletion, substitution, a post- translational modification or lack thereof, etc.), including antibody conjugates (i.e., antibody or antigen-binding fragment thereof conjugated to or associated with a functional moiety). The antibody derivatives, including antibody conjugates, may be based on or may comprise an antigen-binding fragment of the invention that specifically binds an epitope or polypeptide of the invention. Additionally, the aforementioned antibody embodiments may be murine, mouse, rat, hamster, goat, camel, rabbit, chimeric, humanized, or fully human antibodies, fragments, derivatives, or conjugates. It is understood that in certain aspects of the invention, the term "antibody" may exclude one or more of the antibody embodiments recited above; such conditions will be evident to the skilled artisan.

[0084] An antibody may be a monoclonal antibody, a polyclonal antibody, a murine antibody, mouse antibody, rat antibody, hamster antibody, goat antibody, camel antibody, rabbit antibody, a chimeric antibody, a primatized antibody, a humanized antibody, a (fully) human antibody, a multimeric antibody, a heterodimeric antibody, a hemidimeric antibody, a bi-, tri-, or tetravalent antibody, a bispecific antibody, a single chain antibody (e.g., scFv, scFab, and scFabΔC), Bis-scFv, a diabody, triabody or tetrabody, nanobody, minibody, single domain antibodies, and modified Fab fragments. In certain embodiments, the antibody comprises only a single variable immunoglobulin domain. Accordingly, monovalent antibodies include antibodies that comprise only one immunoglobulin variable domain (i.e., a single light or heavy variable chain) and that specifically bind to an epitope or polypeptide of the invention. In addition, antibodies of the invention may be monovalent, divalent, or multivalent for an epitope or polypeptide of the invention.

[0085] Antibody fragments include, for example, an Fab fragment, an F(ab)2 fragment, an Fab' fragment, an F(ab')2 fragment, an F(ab')3, fragment, a single chain F(v) fragment or an F(v) fragment and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9): 1 126-1136). Antibody fragments of the invention are described in more detail below.

[0086] The antibody molecules of the invention can be of any class (e.g. IgG, IgE, IgM, IgD or IgA) or subclass of immunoglobulin molecule. The constant region domains of the antibody, if present, may be selected having regard to the proposed function of the antibody molecule. For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. In particular, human IgG constant region domains may be used, especially IgGl, IgG2, IgG3, and IgG4. IgG2 and IgG4 isotypes may be used in certain embodiments where the antibody molecule is intended for therapeutic uses for which reduced or eliminated antibody effector functions are desired. Alternatively, IgGl and IgG3 isotypes may be used when the antibody molecule is intended for therapeutic purposes for which antibody effector functions are required.

[0087] In certain embodiments, one or more of the CDRs of a polypeptide of the invention may be incorporated into one or more immunoglobulin domains, universal frameworks, protein scaffolds or other biocompatible framework structures based on protein scaffolds or skeletons other than immunoglobulin domains (Nygren & Uhlen, 1997, Curr. Opin. Struct. Biol. 7:463-469; Saragovi et al, 1992, Bio/Technology 10:773-779; Skerra, 2000, J. MoI. Recognition 13: 167-187). In certain embodiments, the CDRs of an antibody are incorporated into a universal framework (i.e., a framework which can be used to create the full variability of functions, specificities, or properties which are originally sustained by a large collection of different frameworks, see U.S. 6,300,064). In other embodiments, alternative scaffolds (see, for example, Binz et al. 2005 Nat Biotech 23: 1257-1268 and Hosse et al. 2006 Protein Science 15: 14-27) may be used.

[0088] In certain embodiments, the antibody is a synthetic antibody or a recombinant antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. An antibody of the invention may also include an antibody that has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology that is available and well known in the art.

[0089] In another embodiment, the invention includes antibodies that can act as interferon antagonists. Antibodies can have extraordinary affinity and specificity for particular epitopes. The binding of an antibody to its epitope on a protein may antagonize the function of that protein by competitively or non-competitively inhibiting the interaction of that protein with other proteins necessary for proper function.

[0090] Antibodies with interferon antagonist activity can be identified in much the same way as other interferon antagonists. For example, candidate antibodies can be administered to cells expressing a reporter gene, and antibodies that cause decreased reporter gene expression are antagonists.

[0091] In one variation, antibodies of the invention can be single chain antibodies (scFv), comprising variable antigen binding domains linked by a polypeptide linker. Single chain antibodies are expressed as a single polypeptide chain and can be expressed in bacteria and as part of a phage display library. In this way, phage that express the appropriate scFv will have interferon antagonist activity. The nucleic acid encoding the single chain antibody can then be recovered from the phage and used to produce large quantities of the scFv. Construction and screening of scFv librarics is extensively described in various publications (U.S. Patents 5,258,498; 5,482,858; 5,091,513; 4,946,778; 5,969,108; 5,871,907; 5,223,409; 5,225,539).

Pharmaceutical Compositions and Methods of Treatment of the Invention

|0092] In another aspect, the invention further provides compositions comprising any of the interferon polynucleotides or interferon polypeptides, described herein, for administration to cells in vitro, to cells ex vivo, and to cells in vivo, or to a multicellular organism. In certain preferred embodiments of this aspect of the ^■ invention, the compositions comprise an interferon polynucleotide for expression of an interferon polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with loss or lack of activity of an endogenous interferon.

|0093| Thus, the invention contemplates methods of treatment using interferon polynucleotides and pharmaceutical compositions comprising interferon

polynucleotides. Interferon polynucleotides may be delivered to cells using any technique known in the art including, for example, viral packaging, lipofection or microinjection. Nucleic acids may also be carried to cells via polymeric

complexes. Any methods for controlled expression of vectors comprising interferon polynucleotides may be used. Methods for regulating expression vectors are well known in the art. For example, vectors used to deliver interferon polynucleotides may be cell type-specific, cell status-specific or inducible.

|0094| The invention also provides pharmaceutical compositions comprising interferon polypeptides of the invention and methods which may be employed, for instance, to treat or prevent immune system-related disorders such as viral infection, parasitic infection, bacterial infection, cancer, autoimmune disease, multiple sclerosis, lymphoma and allergy. Methods of treating individuals or subjects in need of interferon polypeptides are also provided. In certain preferred embodiments, the subject pharmaceutical composition is a veterinary composition for administration to a non-human animal, preferably a non-human primate.

Exemplary conditions which may be treated with an interferon include but are not limited to viral infections. Without limitation, treatment with interferon may be used to treat conditions which would benefit from inhibiting the replication of intcrfcron-sensitive viruses. Viral infections which may be treated in accordance with the invention include severe acute respiratory syndrome-associated

coronavirus (SARS), coronavirus, influenza, smallpox virus, cowpox virus, monkeypox virus, encephalitis-causing viruses, hepatitis A, hepatitis B, hepatitis C, other non-A/non-B hepatitis, herpes virus, Epstein-Barr virus (EBV),

cytomegalovirus (CMV), herpes simplex, human herpes virus type 6 (HHV-6), West Nile virus, vaccinia virus, respiratory syncytial virus, rhinovirus, arterivirus, filovirus, picornavirus, reovirus, rotavirus, rabies, papilloma virus, retroviruses including human immunodeficiency viruses (HIV), human T lymphotropic virus- type 1 and 2 (HTLV-I /-2), papovavirus, herpesvirus, poxvirus, hepadnavirus, astrovirus, coxsackie virus, paramyxovirus, orthomyxovirus, echovirus,

enterovirus, cardiovirus, togavirus, rhabdovirus, bunyavirus, arenavirus,

bornavirus, adenovirus, parvovirus, and flavivirus.

10095] The method of the invention may also be used to modify various immune responses. The interferon polypeptides described herein may be used for treating viral infections caused by viruses such as those described herein. These interferon polypeptides may also used in a prophylactic manner, such as by preventing the infection or preventing the subject from exhibiting symptoms associated with the infection. Subjects who may benefit from such preventive treatment include those with an elevated risk of being infected, such as subjects who have become exposed to the virus or to individuals who have been infected by or exposed to the virus.

[0096] In one embodiment, an interferon may be used as an anti- viral agent.

Interferons have been used clinically for the treatment of acquired immune disorders, viral hepatitis including chronic hepatitis B, hepatitis C, hepatitis D, papilloma viruses, herpes, viral encephalitis, and in the prophylaxis of rhinitis and respiratory infections.

[0097] In another embodiment, an interferon of the invention may be used as an anti-parasitic agent. The interferons may be used, for example, for treating

Cryptosporidium parvum infection. In still another embodiment, an interferon of the- invention may be used as an anti-bacterial agent, interferons have been used clinically for anti-bacterial therapy. For example, interferons may be used in the treatment of multidrug-resistant pulmonary tuberculosis. [0098 J In yet another embodiment, an interferon of the invention may be used as part of an immunotherapy protocol. The interferons of the present invention may be used clinically for immunotherapy or more particularly, for example, to prevent graft vs. host rejection, or to curtail the progression of autoimmune diseases, such as arthritis, multiple sclerosis, or diabetes.

|0099] In another embodiment, an interferon of the invention may be used as part of a program for treating allergies. In still another embodiment, interferons can be used as vaccine adjuvants, interferons may be used as an adjuvant or coadjuvant to enhance or stimulate the immune response in cases of prophylactic or therapeutic vaccination.

[0100] In addition to the treatment of animals in general, the interferons of the invention may be used for the treatment of primates as part of veterinary protocols. |01011 In addition to the treatment of animals in general, the interferons of the invention may be used for the treatment of fish.

[0102] An interferon of the invention may be used for treating cats. In still another embodiment, an interferon of the invention may be used to treat dogs or other household pets, (de Mari K, Maynard L, Eun HM, Lebreux B. Vet Rec. (2003) 152: 105-8). In still another embodiment, an interferon of the invention may be used to treat farm animals. In yet another embodiment, an interferon of the invention may be used to treat fish. In yet another embodiment, an interferon of the invention may be used to treat humans.

[0103] This invention further provides a method of treating a subject infected with a virus selected from the group consisting of severe acute respiratory syndrome- associated coronavirus (SARS), coronavirus, influenza, smallpox virus, cowpox virus, monkeypox virus, encephalitis-causing viruses, hepatitis A, hepatitis B, hepatitis C, other non-A/non-B hepatitis, herpes virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex, human herpes virus type 6 (HHV-6), West Nile virus, vaccinia virus, respiratory syncytial virus, rhinovirus, arterivirus, filovirus, picomavirus, reovirus, rotavirus, rabies, papilloma virus, retroviruses including human immunodeficiency viruses (HIV), human T lymphotropic virus-type 1 and 2

(HTLV-I /-2), papovavirus, herpesvirus, poxvirus, hepadnavirus, astrovirus, coxsackie virus, paramyxovirus, orthomyxovirus, echovirus, enterovirus, cardio virus, togavirus, rhabdovirus, bunyavirus, arenavirus, bornavirus, adenovirus, parvovirus, and flavivirus, comprising administering to the subject an amount of an interferon polypeptide that is effective to reduce the concentration of virus particles in the subject, thereby treating the subject.

|0104] This invention contemplates any of the treatment methods described herein also as methods for preventing the subject from becoming afflicted or infected, or as methods of reducing the subject's risk of a affliction or infection, or as protecting the subject against disorders/conditions related to a particular virus, or as preventing the subject from exhibiting symptoms associated with a viral infection. For instance, the above methods for treating subject infected with a virus may also be used to prevent the subject from becoming infected with the virus, or to reduce the subject risk of viral infection.

|0105] This invention provides a method of reducing a subject's risk of viral infection comprising administering to the subject an interferon polypeptide. In one embodiment, this method comprises preventing the subject from being infected with the virus. In one embodiment, this method comprises preventing the subject from exhibiting symptoms associated with a viral infection. In one embodiment, this method comprises protecting the subject against disorders/conditions related to a particular virus. This protection may be conferred by preventing or lessening the severity of a disorder/condition resulting from the infection. In another

embodiment, the protection may also be conferred by reducing the spread of infection to others by lessening the severity of a disorder/condition resulting from the infection in the patient. In another embodiment, the prevention or reduction of risk is effected by causing the subject's cells to become less susceptible to infection. The methods and embodiments described herein are not necessarily mutually exclusive. The viral infections include but are not limited to those caused by severe acute respiratory syndrome-associated coronavirus (SARS), coronavirus, influenza, smallpox virus, cowpox virus, monkeypox virus, encephalitis-causing viruses, hepatitis A, hepatitis B, hepatitis C, other non-A/non-B hepatitis, herpes virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex, human herpes virus type 6 (HHV-6), West Nile virus, vaccinia virus, respiratory syncytial virus, rhinovirus, arterivirus, filovirus, picornavirus, reovirus, rotavirus, rabies, papilloma virus, retroviruses including human immunodeficiency viruses (HIV), human T lymphotropic virus-type 1 and 2 (HTLV-I /-2), papovavirus, herpesvirus, poxvirus, hepadnavirus, astrovirus, coxsackie virus, paramyxovirus,

orthomyxovirus, echovirus, enterovirus, cardiovirus, togavirus, rhabdovirus, bunyavirus, arenavirus, bornavirus, adenovirus, parvovirus, and flavivirus.

[0106] This invention provides a method of treating a subject afflicted with influenza (orthomyxovirus), comprising administering to the subject an amount of an interferon polypeptide that is effective to reduce the concentration of influenza virus particles in the subject, wherein the interferon polypeptide comprises an amino acid sequence that has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity to at least one of the SEQ ID NOS described herein, thereby treating the subject. According to the invention, each of these sequences and/or fragments is biologically active.

[01071 This invention provides a method of treating a subject afflicted with Hepatitis C, comprising administering to the subject an amount of an interferon polypeptide that is effective to reduce the concentration of Hepatitis C virus particles in the subject, wherein the interferon polypeptide comprises an amino acid sequence that has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity to at least one of the SEQ ID NOS described herein, thereby treating the subject. According to the invention, each of these sequences and/or fragments is biologically active.

[0108] This invention provides a method of treating a subject afflicted with rhinovirus, coronavirus, or arenavirus, comprising administering to the subject an amount of an interferon polypeptide that is effective to reduce the concentration of rhinovinis, coronavirus, or arenavirus virus particles in the subject, wherein the interferon polypeptide comprises an amino acid sequence that has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity to at least one of the SEQ ID NOS described herein, thereby treating the subject. According to the invention, each of these sequences and/or fragments is biologically active.

[01091 The interferon polypeptides referred to herein, such as in the context of treatment and/or prevention, include but are not limited to IFN-α polypeptides, IFN-β polypeptides, IFN-γ polypeptides, and IFN- ω polypeptides (Zoon KC: Human Interferons: Structure and Function, p. 1-12. In: Interferon 8. Academic Press, London, 1987; Walter et al, Cancer Biotherm Radiopharm 1998 June; 13(3): 143-54; Pestka, S., Biopolymers 2000; 55(4):254-287; Biopolymers 2000; 55(4):254-287; Pestka, S., Methods in Enzymology, 78, 1981; Pestka, S., Methods in Enzymology, 79, 1981; Pestka, S., Methods in Enzymology, 119, 1986; Pestka, S., Langer; J. A., Zoon, K.C., Samuel, CE. Ann Rev Biochem 1987, 56, 727-777).

[0110] This invention also provides a method of preventing a subject from becoming afflicted with a syndrome caused by a virus selected from the group consisting of severe acute respiratory syndrome-associated coronavirus (SARS), coronavirus, influenza, smallpox virus, cowpox virus, monkeypox virus, encephalitis-causing viruses, hepatitis A, hepatitis B, hepatitis C, other non-A/non- B hepatitis, herpes virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex, human herpes virus type 6 (HHV-6), West Nile virus, vaccinia virus, respiratory syncytial virus, rhinovirus, arterivirus, filovirus, picornavirus, reovirus, rotavirus, rabies, papilloma virus, retroviruses including human

immunodeficiency viruses (HIV), human T lymphotropic virus-type 1 and 2

(HTLV- 1/-2), papovavirus, herpesvirus, poxvirus, hepadnavirus, astrovirus, coxsackie virus, paramyxovirus, orthomyxovirus, echovirus, enterovirus, cardiovirus, togavirus, rhabdovirus, bunyavirus, arenavirus, bornavirus, adenovirus, parvovirus, and flavivirus, comprising administering to the subject an amount of an interferon polypeptide.

[0111] This invention provides a method of preventing a subject from becoming afflicted with influenza, comprising administering to the subject an amount of an interferon polypeptide described herein. This invention provides a method of reducing the risk of a subject from becoming infected with influenza, comprising administering to the subject an amount of an interferon polypeptide described herein. In one embodiment of the methods described herein, the subject is a human being. The subjects include but are not limited to dogs, cats, fish, monkeys, and farm animals.

[0112] This invention provides a method of preventing a subject from becoming afflicted with Hepatitis C, comprising administering to the subject an amount of an interferon polypeptide described herein. This invention provides a method of reducing the risk of a subject from becoming infected with Hepatitis C, comprising administering to the subject an amount of an interferon polypeptide described herein. In one embodiment of the methods described herein, the subject is a human being. The subjects include but are not limited to dogs, cats, fish, monkeys, and farm animals.

[0113] This invention provides a method of preventing a subject from becoming afflicted with rhino virus, coronavirus, or arenavirus comprising administering to the subject an amount of an interferon polypeptide described herein. This invention provides a method of reducing the risk of a subject from becoming infected with rhinovirus, coronavirus, or arenavirus comprising administering to the subject an amount of an interferon polypeptide described herein. In one embodiment of the methods described herein, the subject is a human being. The subjects include but are not limited to dogs, cats, fish, monkeys, and farm animals.

[0114] This invention provides the use of an interferon polypeptide of the invention for the preparation of a medicament for any of the methods of treatment, prevention, prophylaxis, and reduction of risk described herein.

[0115] This invention provides a pharmaceutical package comprising an interferon composition with instructions for administering the composition to a subject. The instructions may include written and/or pictorial instructions. The instructions may bc for any of the methods or treatment, prevention, prophylaxis, and reduction of risk described herein. •

[0116] The subject invention also contemplates functional antagonists, e.g., wherein one or more amino acid residues are different from the naturally occurring interferon, which inhibit one or more biological activities of the naturally occurring interferon. Such antagonists can be used to treat disorders resulting from aberrant overexpression or other activation of an endogenous interferon. The functional antagonists may be formulated in a pharmaceutical preparation.

[0117] The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a biological activity of an interferon polypeptide, which involves contacting a receptor which is enhanced by an interferon polypeptide with the candidate compound in the presence of an interferon polypeptide, assaying, for example, anti- viral activity in the presence of the candidate compound and an interferon polypeptide, and comparing the activity to a standard level of activity, the standard being assayed when contact is made between the receptor and interferon in the absence of the candidate compound. In this assay, an increase in activity over the standard indicates that the candidate compound is an agonist of interferon activity and a decrease in activity compared to the standard indicates that the compound is an antagonist of interferon activity.

[0118] An additional aspect of the invention is related to a method for treating an animal in need of an increased level of interferon activity in the body comprising administering to such an animal a composition comprising a therapeutically effective amount of an isolated interferon polypeptide of the invention or an agonist thereof.

[0119] A still further aspect of the invention is a method for treating an animal in need of a decreased level of interferon activity in the body comprising, administering to such an animal a composition comprising a therapeutically effective amount of an interferon antagonist. Preferred antagonists for use in the present invention are interfcron-specific antibodies.

[0120] In one embodiment, administration of the described dosages may be every other day, but is preferably once or twice a week. Doses may be administered over at least a 24 week period by injection. [0121) Administration of the dose can be intravenous, subcutaneous, intramuscular, or any other acceptable systemic method. Based on the judgment of the attending clinician, the amount of drug administered and the treatment regimen used will, of course, be dependent on the age, sex and medical history of the patient being treated, the neutrophil count (e.g. the severity of the neutropenia), the severity of the specific disease condition and the tolerance of the patient to the treatment as evidenced by local toxicity and by systemic side-effects. Dosage amount and frequency may be determined during initial screenings of neutrophil count.

[0122] Conventional pharmaceutical formulations can be also prepared using the subject interferon compositions of the present invention. The formulations comprise a therapeutically effective amount of an interferon polypeptide together with pharmaceutically acceptable carriers. For example, adjuvants, diluents, preservatives and/or solubilizers, if needed, may be used in the practice of the invention.

Pharmaceutical compositions of interferon including those of the present invention may include diluents of various buffers (e.g., Tris-HCl, acetate, phosphate) having a range of pH and ionic strength, carriers (e.g., human serum albumin), solubilizers (e.g., Polyoxyethylene Sorbitan or TWEEN™ polysorbate), and preservatives (e.g., thimerosal, benzyl alcohol). See, for example, U.S. Pat. No. 4,496,537.

[0123] The therapeutic amount of the interferon composition administered to treat the conditions described above is based on the interferon activity of the composition. It is an amount that is effective to significantly affect a positive clinical response. In the case of treatment for a viral infection, a positive clinical response may be indicated by a reduction in the concentration of virus particles in the subject, or more generally as a reduction in the symptoms of the infection. In the case of prophylactic treatment, a positive clinical response may be indicated, for example, by an absence of virus particles in the subject, by a reduction in the concentration of virus particles in the subject, or by maintaining the concentration of virus particles in the subject below the threshold concentration above which the subject exhibits the symptoms of the viral infection. As used herein, a prophylactic treatment includes preventing a subject from becoming afflicted with a disorder caused by a virus.

[0124] Although the clinical dose may cause some level of side effects in some patients, the maximal dose for mammals including humans is the highest dose that does not cause unmanageable clinically- important side effects. For purposes of the present invention, such clinically important side effects are those which would require cessation of therapy due to severe flu-like symptoms, central nervous system depression, severe gastrointestinal disorders, alopecia, severe pruritus or rash.

Substantial white and/or red blood cell and/or liver enzyme abnormalities or anemia- like conditions are also dose limiting.

J0125] Naturally, the dosages of interferon may vary somewhat depending upon the formulation selected. In general, however, the interferon composition is administered in amounts ranging from about 100,000 to about several million IU/m² per day, based on the mammal's condition. The range set forth above is illustrative and those skilled in the art will determine the optimal dosing of interferon selected based on clinical experience and the treatment indication.

[0126] The pharmaceutical compositions may be in the form of a solution, suspension, tablet, capsule, lyophilized powder or the like, prepared according to methods well known in the art. It is also contemplated that administration of such compositions will be chiefly by the parenteral route although oral or inhalation routes may also be used depending upon the needs of the artisan.

[0127] In another aspect, the present invention provides pharmaceutical preparations comprising interferons, interferon agonists or interferon antagonists (collectively referred to herein as the compounds of the invention). The interferons, interferon agonists and/or interferon antagonists for use in the subject method may be conveniently formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof. The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists. Except insofar as any conventional media or agent is incompatible with the activity of the compositions of the present invention, its use in the pharmaceutical preparation of the invention is contemplated. Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington's Pharmaceutical

Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable "deposit formulations". [0128] Pharmaceutical formulations of the present invention can also include veterinary compositions, e.g., pharmaceutical preparations of the compositions of the present invention suitable for veterinary uses, e.g., for the treatment of livestock, fish, non-human primate, or domestic animals, e.g., dogs and cats.

|0129] Rechargeable or biodegradable devices may also provide methods of introduction. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for sustained release at a particular target site.

[0130) The preparations of the present invention may be administered to humans and other animals by any suitable route of administration, including orally, nasally (as by, for example, a spray), parenterally, topically (as by powders, ointments or drops, including buccally and sublingually), intrathecally/intracerebroventricularly (ICV), intracranially, directly into the central nervous system (intracavitary), intravaginally, intracisternally, or rectally. The preparations are administered in forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, controlled release patch, etc., administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. In some embodiment, oral and topical administrations may be preferred. In one embodiment, the interferon is delivered directly to nasopharyngeal mucosa. In one embodiment, the interferon is delivered directly to the lung epithelium. Unlike using these tissues as a vehicle for systemic delivery, direct delivery to these cells may make them resistant to viruses. Moreover, it reduces the amount of systemic interferon so side effects will be minimal or eliminated.

[0131] Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms such as described below or by other conventional methods known to those of skill in the art. [0132] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

|0133] The selected dosage level will depend upon a variety of factors including: the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof; the route of administration; the time of administration; the rate of excretion of the particular compound being employed; the duration of the treatment; other drugs, compounds and/or materials used in combination with the particular composition employed; the age, sex, weight, condition, general health and prior medical history of the patient being treated; and like factors well known in the medical arts.

[0134] A physician or veterinarian having ordinary skill in the art can readily determine and-prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0135] In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0136] If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

[0137] The patient receiving this treatment may be any animal in need thereof or any animal that is the source of virus (animal vector), including: mammals such as equines, cattle, swine, rodents and sheep; poultry; and pets in general. In a preferred embodiment, the patient is a primate (in particular, a human) or another mammal.

|0138| The compound of the invention can be administered as such or in admixtures with pharmaceutically acceptable and/or sterile carriers and can also be administered in conjunction with other agents. Non-limiting examples of such agents include antimicrobial agents such as penicillins, cephalosporins, aminoglycosides, and glycopeptides. Conjunctive therapy thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutic effects of the first administered one is not entirely disappeared when the subsequent is administered.

[0139| While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). The compositions of the present invention may be formulated for administration in any convenient way for use in human or veterinary medicine. In certain embodiments, the compound included in the pharmaceutical preparation may be active itself, or may be a prodrug, e.g., capable of being converted to an active compound in a physiological setting.

[0140] Thus, another aspect of the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more of the compounds of the invention, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by

subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or (4) intravaginally or intrarectally, for example, as a pessary, cream or foam. However, in certain embodiments the subject compounds may be simply dissolved or suspended in sterile water. In certain embodiments, the pharmaceutical preparation is non-pyrogenic, i.e., does not elevate the body temperature of a patient.

[0141] Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safϊlower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (1 1) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen- free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in

pharmaceutical formulations.

[0142] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, for example, liposomes, polymers, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. The subject interferons can be provided in formulations also including penetration enhancers, carrier compounds and/or transfection agents.

|0143] The compositions of the invention also encompass any pharmaceutically acceptable salts, esters or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the compositions also include to pharmaceutically acceptable salts and other bioequivalents.

|0144] In certain embodiments, the formulations are as part of a "supramolecular complex." To further illustrate, the interferon can be contacted with at least one polymer to form a composite and then the polymer of the composite treated under conditions sufficient to form a supramolecular complex containing the interferon and a multi-dimensional polymer network. The polymer molecule may be linear or branched. Accordingly, a group of two or more polymer molecules may be linear, branched, or a mixture of linear and branched polymers. The composite may be further modified with at least one ligand, e.g., to direct cellular uptake of the expression construct or otherwise effect tissue or cellular distribution in vivo of the exprcssion construct. The composite may take any suitable form and, preferably, is in the form of particles.

[0145] In certain embodiments, the supramolecular complexes are aggregated into particles, for example, formulations of particles having an average diameter of between 20 and 5000 nanometer (nm). In another embodiment, the particles have an average diameter of between 20 and 200 nm. In another embodiment, the particles have an average diameter of between 2 and 10 microns. Use of a particle size of between 2 and 10 microns may be used for example, for delivery to the lung.

[0146] In certain embodiments, the interferons are provided in cationic, non-lipid vehicles and formulated to be used in aerosol delivery via the respiratory tract.

Formulations using poly(ethylenimine) (PEI) and macromolecules such as dsRNA and dsRNA-encoding plasmids can result in a high level of pulmonary transfection and increased stability during nebulization. PEI-nucleic acid formulations can also exhibit a high degree of specificity for the lungs.

[0147| In addition to formulating interferon with PEI, the invention also contemplates the use of cyclodextrin-modified polymers, such as cyclodextrin- modified poly(ethylenimine).

|0148| In certain embodiments, the invention provides a composition including interferons that are encapsulated or otherwise associated with liposomes. In a preferred embodiment, the liposomes are cationic liposomes composed of between about 20-80 mole percent of a cationic vesicle-forming lipid, with the remainder neutral vesicle-forming lipids and/or other components. In certain embodiments, the lipid is a vesicle-forming lipid. A preferred vesicle-forming lipid is a diacyl-chain lipid, such as a phospholipid, whose acyl chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation.

[0149] Neutral vesicle-forming lipids are those vesicle forming lipids which have no net charge or which may include a small percentage of lipids having a negative charge in the polar head group. Included in this class of lipids are the phospholipids, such as phosphatidylcholine (PC), phosphatidyl ethanolamine (PE),

phosphatidylinositol (PI), and sphingomyelin (SM), and cholesterol, cholesterol derivatives, and other uncharged sterols. [0150| The above-described lipids can be obtained commercially, or prepared according to published methods. Other lipids that can be included in the invention ^■ are glycolipids, such as cerebrosides and gangliosides.

|01511 In one embodiment of the invention, the interferon- liposome complex includes liposomes having a surface coating of hydrophilic polymer chains, effective to extend the blood circulation time of the plasmid/liposome complexes. Suitable hydrophilic polymers include cyclodextrin (CD), polyethylene glycol (PEG), polylactic acid, polyglycolic acid, polyvinyl-pyrrolid-one, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses, such as hydroxymethylcellulose or hydroxyethyl-cellulose. A preferred hydrophilic polymer chain is

polyethyleneglycol (PEG), preferably as a PEG chain having a molecular weight between 500-10,000 Daltons, more preferably between 1 ,000-5,000 Daltons. The hydrophilic polymer may have solubility in water and in a non-aqueous solvent, such as chloroform.

[0152] It will be appreciated that the hydrophilic polymer can be stably coupled to the lipid, or coupled through an unstable linkage which allows the polymer-coated plasmid- liposome complexes to shed or "release" the hydrophilic polymer coating during circulation in the bloodstream or after localization at a target site. Attachment of hydrophilic polymers, in particular polyethyleneglycol (PEG), to vesicle- forming lipids through a bond effective to release the polymer chains in response to a stimulus have been described, for example in WO 98/16202, WO 98/16201, which are hereby incorporated by reference, and by Kirpotin, D. et al. (FEBS Letters, 388:1 15-1 18 (1996).

[0153] In certain embodiments, the hydrophobic segment in the polymer-lipid conjugate is a hydrophobic polypeptide sequence. Preferably, the polypeptide sequence consists of about 5-80, more preferably 10-50, most preferably 20-30, non- polar and/or aliphatic/aromatic amino acid residues. These sequences are active in triggering fusion of certain enveloped viruses with host cells and include

parainfluenza viruses, such as Sendai, Simian Virus-5 (SV5), measles virus,

Newcastle Disease Virus (NDV) and Respiratory Syncytial Virus (RSV). Other examples include human retroviruses, such as Human Immunodeficiency Virus- 1 (HIV-I), the causative agent of AIDS, which infects cells by fusion of the virus envelope with the plasma membrane of the host cell. Fusion occurs at physiological (i.e., neutral) pH and is followed by injection of the viral genetic material

(nucleocapsid) into the cytoplasmic compartment of the host cell.

[0154] In certain embodiments, the polymeric complexes, such as the

supramolecular complexes, and liposomes of the subject invention can be associated with one or more ligands effective to bind to specific cell surface proteins or matrix on the target cell, thereby facilitating sequestration of the complex to target cells, and in some instances, enhancing uptake of the interferon by the cell. Merely to illustrate, examples of ligands suitable for use in targeting the supramolecular complexes and liposomes of the present invention to specific cell types are listed in Table 2 below.

Table 2

[0155| The present invention also contemplates the derivatization of the subject polymeric and liposomal complexes with ligands that promote transcytosis of the complexes. To further illustrate, a polymeric complex, such as a supramolecular complex, can be covalently linked to an internalizing peptide which drives the translocation of the complex across a cell membrane in order to facilitate intracellular localization of the interferon. In this regard, the internalizing peptide, by itself, is capable of crossing a cellular membrane by, e.g., transcytosis, at a relatively high rate. The internalizing peptide is conjugated, e.g., as covalent pendant group, to the polymer.

[0156] Pore-forming proteins or peptides may serve as internalizing peptides herein. Pore-forming proteins or peptides may be obtained or derived from, for example, C9 complement protein, cytolytic T-cell molecules or NK-cell molecules. These moieties are capable of forming ring-like structures in membranes, thereby allowing transport of attached complexes through the membrane and into the cell interior.

[0157] Mere membrane intercalation of an internalizing peptide may be sufficient for translocation of the complexes across cell membranes. However, translocation may be improved by attaching to the internalizing peptide a substrate for intracellular enzymes (i.e., an "accessory peptide"). It is preferred that an accessory peptide be attached to a portion(s) of the internalizing peptide that protrudes through the cell membrane to the cytoplasmic face. The accessory peptide may be advantageously attached to one terminus of a translocating/internalizing moiety or anchoring peptide. An accessory moiety of the present invention may contain one or more amino acid residues. In one embodiment, an accessory moiety may provide a substrate for cellular phosphorylation (for instance, the accessory peptide may contain a tyrosine residue).

[0158| The internalizing and accessory peptides can each, independently, be added to an interferon complex or liposome by chemical cross-linking or through non- covalent interaction (e.g., use of streptavidin-biotin conjugates, His6-Ni interactions, etc). In certain instances, unstructured polypeptide linkers can be included between the peptide moieties and the polymeric complex or liposome.

[0159] It is also contemplates that such internalizing and accessory peptides can be associated directly with an interferon, such as through a covalent linkage to a hydro xyl group on the backbone of the protein. In certain embodiments, the linkage is susceptible to cleavage under physiological conditions, such as by exposure to esterases, or simple hydrolysis reactions. Such compositions can be used alone or formulated in polymeric complexes or liposomes.

[0160] Another aspect of the invention provides aerosols for the delivery of interferon to the respiratory tract. The respiratory tract includes the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli. The upper and lower airways are called the conductive airways. The terminal bronchioli then divide into respiratory bronchioli which then lead to the ultimate respiratory zone, the alveoli, or deep lung.

|0161] Administration by inhalation may be oral and/or nasal, intratracheal (trans- stomal or via tracheostomy tube), or via a breathing assistance device such as a respirator. Examples of pharmaceutical devices for aerosol delivery include metered dose inhalers (MDIs), dry powder inhalers (DPIs), and air-jet nebulizers. Exemplary delivery systems by inhalation which can be readily adapted for delivery of the subject interferons are described in, for example, U.S. patents 5,756,353; 5,858,784; and PCT applications WO98/31346; WO98/10796; WOOO/27359; WOO 1/54664; WO02/060412. Other aerosol formulations that may be used for delivering the interferons are described in U.S. Patents 6,294,153; 6,344,194; 6,071,497, and PCT applications WO02/066078; WO02/053190; WOO 1/60420; WO00/66206. [0162| The human lungs can remove or rapidly degrade hydrolytically cleavable deposited aerosols over periods ranging from minutes to hours. In the upper airways, ciliated epithelia contribute to the "mucociliary excalator" by which particles are swept from the airways toward the mouth. Pavia, D., "LungMucociliary Clearance," in Aerosols and the Lung: Clinical and Experimental Aspects. Clarke, S. W. and Pavia, D., Eds., Butterworths, London, 1984. In the deep lungs, alveolar macrophages are capable of phagocytosing particles soon after their deposition. Warheit et al. Microscopy Res. Tech., 26: 412-422 (1993); and Brain, J. D., "Physiology and Pathophysiology of Pulmonary Macrophages," in The

Reticuloendothelial System. S. M. Reichard and J. Filkins, Eds., Plenum, New York, pp. 315-327, 1985. The deep lung, or alveoli, are the primary target of inhaled therapeutic aerosols for systemic delivery of interferons.

[0163] In preferred embodiments, particularly where systemic dosing with the ■ interferon is desired, the aerosolized interferons are formulated as microparticles. Microparticles having a diameter of between 0.5 and ten microns can penetrate the lungs, passing through most of the natural barriers. A diameter of less than ten microns is required to bypass the throat; a diameter of 0.5 microns or greater is required to avoid being exhaled.

[0164] In certain preferred embodiments, the subject interferons are formulated in a supramolecular complex, as described above, which have a diameter of between 0.5 and ten microns, which can be aggregated into particles having a diameter of between 0.5 and ten microns.

|O1651 In other embodiments, the subject interferons are provided in liposomes or supramolecular complexes (such as described above) appropriately formulated for pulmonary delivery.

[0166] In addition to the supramolecular complexes described above, a number of other polymers can be used to form the microparticles.

[0167] Polymers are preferably biodegradable within the time period over which release of the interferon is desired or relatively soon thereafter, generally in the range of one year, more typically a few months, even more typically a few days to a few weeks. Biodegradation can refer to either a breakup of the microparticle, that is, dissociation of the polymers forming the microparticles and/or of the polymers themselves. This can occur as a result of change in pH from the carrier in which the particles are administered to the pH at the site of release, as in the case of the diketopiperazines, hydrolysis, as in the case of poly(hydroxy acids), by diffusion of an ion such as calcium out of the microparticle, as in the case of microparticles formed by ionic bonding of a polymer such as alginate, and by enzymatic action, as in the case of many of the polysaccharides and proteins. In some cases linear release may be most useful, although in others a pulse release or "bulk release" may provide more effective results.

[0168] The microparticles can be suspended in any appropriate pharmaceutical carrier, such as saline, for administration to a patient. In the most preferred embodiment, the microparticles will be stored in dry or lyophilized form until immediately before administration. They can then be suspended in sufficient solution, for example an aqueous solution for administration as an aerosol, or administered as a dry powder.

|0169] The microparticles can be delivered to specific cells, especially phagocytic cells and organs. Phagocytic cells within the Peyer's patches appear to selectively take up microparticles administered orally. Phagocytic cells of the reticuloendothelial system also take up microparticles when administered intravenously. Endocytosis of the microparticles by macrophages in the lungs can be used to target the

microparticles to the spleen, bone marrow, liver and lymph nodes.

[0170) The microparticles can also be targeted by attachment of ligands, such as those described above, which specifically or non-specifically bind to particular targets. Examples of such ligands also include antibodies and fragments including the variable regions, lectins, and hormones or other organic molecules having receptors on the surfaces of the target cells.

[0171] In the preferred embodiment, the microparticles are stored lyophilized. The dosage is determined by the amount of encapsulated interferon, the rate of release within the pulmonary system, and the pharmacokinetics of the compound.

|0172| The microparticles can be delivered using a variety of methods, ranging from administration directly into the nasal passages so that some of the particles reach the pulmonary system, to the use of a powder instillation device, to the use of a catheter or tube reaching into the pulmonary tract. Dry powder inhalers are commercially available, although those using hydrocarbon propellants are no longer used and those relying on the intake of a breath by a patient can result in a variable dose.

|0173| Another aspect of the invention relates to coated medical devices. For instance, in certain embodiments, the subject invention provides a medical device having a coating adhered to at least one surface, wherein the coating comprises a polymer matrix and an interferon. Such coatings can be applied to surgical implements such as screws, plates, washers, sutures, prosthesis anchors, tacks, staples, electrical leads, valves, membranes. The devices can be catheters, implantable vascular access ports, blood storage bags, blood tubing, central venous catheters, arterial catheters, vascular grafts, intra-aortic balloon pumps, heart valves, cardiovascular sutures, artificial hearts, a pacemaker, ventricular assist pumps, extracorporeal devices, blood filters, hemodialysis units, hemoperfusion units, plasmapheresis units, and filters adapted for deployment in a blood vessel.

[0174J In some embodiments according to the present invention, monomers for forming a polymer are combined with an interferon and are mixed to make a homogeneous dispersion of the interferon in the monomer solution. The dispersion is then applied to a stent or other device according to a conventional coating process, after which the crosslinking process is initiated by a conventional initiator, such as UV light. In other embodiments according to the present invention, a polymer composition is combined with an interferon to form a dispersion. The dispersion is then applied to a surface of a medical device and the polymer is cross-linked to form a solid coating. In other embodiments according to the present invention, a polymer and an interferon are combined with a suitable solvent to form a dispersion, which is then applied to a stent in a conventional fashion. The solvent is then removed by a conventional process, such as heat evaporation, with the result that the polymer and interferon (together forming a sustained-release drug delivery system) remain on the stent as a coating. An analogous process may be used where the interferon is dissolved in the polymer composition.

[0175| In some embodiments according to the present invention, the device comprises an interferon and polymer suspension or dispersion, wherein the polymer is rigid, and forms a constituent part of a device to be inserted or implanted into a body. For instance, in particular embodiments according to the present invention, the device is a surgical screw, stent, pacemaker, etc. coated with the interferon suspended or dispersed in the polymer.

[0176| As discussed above, the coating according to the present invention comprises a polymer that is bioerodible or non bioerodible. The choice of bioerodible versus non-bioerodible polymer is made based upon the intended end use of the system or device. In some embodiments according to the present invention, the polymer is advantageously bioerodible. For instance, where the system is a coating on a surgically implantable device, such as a screw, stent, pacemaker, etc., the polymer is advantageously bioerodible. Other embodiments according to the present invention in which the polymer is advantageously bioerodible include devices that are implantable, inhalable, or injectable suspensions or dispersions .of interferon in a polymer, wherein the further elements (such as screws or anchors) are not utilized. |0177] In other embodiments according to the present invention, the rate of bioerosion of the polymer is advantageously on the same order as the rate of interferon release. For instance, where the system comprises an interferon suspended or dispersed in a polymer that is coated onto a surgical implement, such as an orthopedic screw, a stent, a pacemaker, or a non-bioerodible suture, the polymer advantageously bioerodes at such a rate that the surface area of the interferon that is directly exposed to the surrounding body tissue remains substantially constant over time.

[0178) In other embodiments according to the present invention, the polymer vehicle is permeable to water in the surrounding tissue, e.g. in blood plasma. In such cases, water solution may permeate the polymer, thereby contacting the interferon. The rate of dissolution may be governed by a complex set of variables, such as the polymer's permeability, the solubility of the interferon, the pH, ionic strength, and protein composition, etc., of the physiologic fluid.

[0179] In some embodiments according to the present invention, the polymer is non-bioerodible. Non bioerodible polymers are especially useful where the system includes a polymer intended to be coated onto, or form a constituent part, of a surgical implement that is adapted to be permanently, or semi permanently, inserted or implanted into a body. Exemplary devices in which the polymer advantageously forms a permanent coating on a surgical implement include an orthopedic screw, a stent, a prosthetic joint, an artificial valve, a permanent suture, a pacemaker, etc.

[0180] There is a multiplicity of different stents that may be utilized following percutaneous transluminal coronary angioplasty. Although any number of stents may be utilized in accordance with the present invention, for simplicity, a limited number of stents will be described in exemplary embodiments of the present invention. The skilled artisan will recognize that any number of stents may be utilized in connection with the present invention. In addition, as stated above, other medical devices may be utilized.

[0181] A stent is commonly used as a tubular structure left inside the lumen of a duct to relieve an obstruction. Commonly, stents are inserted into the lumen in a non- expanded form and are then expanded autonomously, or with the aid of a second device in situ. A typical method of expansion occurs through the use of a catheter- mounted angioplasty balloon which is inflated within the stenosed vessel or body passageway in order to shear and disrupt the obstructions associated with the wall components of the vessel and to obtain an enlarged lumen.

[0182] It should be appreciated that a stent in accordance with the present invention may be embodied in a shape-memory material, including, for example, an appropriate alloy of nickel and titanium or stainless steel.

[0183] Regardless of the design of the stent, it is preferable to have the interferon applied with enough specificity and a sufficient concentration to provide an effective dosage in the lesion area. In this regard, the "reservoir size" in the coating is preferably sized to adequately apply the interferon at the desired location and in the desired amount.

[0184] In an alternate exemplary embodiment, the entire inner and outer surface of the stent may be coated with the interferon in therapeutic dosage amounts. It is, however, important to note that the coating techniques may vary depending on the interferon. Also, the coating techniques may vary depending on the material comprising the stent or other intraluminal medical device.

[0185] The intraluminal medical device comprises the sustained release drug delivery coating. The interferon coating may be applied to the stent via a conventional coating process, such as impregnating coating, spray coating and dip coating.

[0186] The interferon may be incorporated onto or affixed to the stent in a number of ways. In the exemplary embodiment, the interferon is directly incorporated into a polymeric matrix and sprayed onto the outer surface of the stent. The interferon elutes from the polymeric matrix over time and enters the surrounding tissue. The interferon preferably remains on the stent for at least three days up to approximately six months, and more preferably between seven and thirty days.

Exemplification |0187] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example 1: Isolation of Human IFNα Clones [0188] Human interferon alpha sequences were isolated by PCR through standard methods.

[0189] The Human interferon alpha gene sequences identified using this approach are: G37 (SEQ ID NO: 1), G56 (SEQ ID NO: 3), G59 (SEQ ID NO: 5), G73 (SEQ ID NO: 7), G2O8 (SEQ ID NO: 9), G215 (SEQ ID NO: 1 1), G223 (SEQ ID NO: 13), G56M (SEQ ID NO: 15), G59M (SEQ ID NO: 17), and G215M (SEQ ID NO: 19). Amino acid sequences corresponding to each of these are also provided: PT37 (SEQ ID NO: 2), PT56 (SEQ ID NO: 4), PT59 (SEQ ID NO: 6), PT73 (SEQ ID NO: 8), PT208 (SEQ ID NO: 10), PT215 (SEQ ID NO: 12), PT223 (SEQ ID NO: 14), PT56M (SEQ ID NO: 16), PT59M (SEQ ID NO: 18), and PT215M (SEQ ID NO: 20).

101901 Example 2: Antiviral Activity of the novel IFN Proteins and Mutants

[0191] The antiviral activity of the human IFN proteins was determined using the cytopathic effect (CPE) assay, as outlined in here. Human A549 epithelial carcinoma cells (ATCC Manassas, VA) were plated in a 96 well plate at 20,000 cells/well and grown in 0.1 ml DMEM with 10% Fetal Bovine Serum. Two to four hours later 0.1 ml of serial dilutions of IFN samples were added and the plate was incubated overnight. The cells were then infected with 0.05 ml of

encephalomyocarditis virus and returned to the incubator for 40-48 hours. Once killing was complete in wells which contained no interferon the media was removed and the remaining cells were stained with crystal violet and washed gently with tapwater to remove excess dye. The dye was then released by addition of 0.1 ml of 70% methanol or ethanol and read at 570 or 562 nm on a Molecular Devices plate reader. The percent cell survival was determined by subtracting the virus control (no IFN) from each sample and taking 100% protection as the cell control (no IFN, no virus). The EC50 was determined using GraphPad Prism software using a Sigmoidal, variable slope curve fit. Units/ml was determined by comparison to a laboratory standard of human IFN alpha-2a of known unitage. The A549 cell serves as a model for prevention of viral infection in human cells. |0192| Example 3: Effectiveness of the novel IFN Proteins and Mutants in Inducing IFN gamma in NK-92 cells

[0193] The natural killer phenotype Human non-Hodgkin's lymphoma cell line NK-92 (ATCC Manassas, VA) was maintained in the ATCC-recommended media containing 100-200 U/ml 1L-2. Prior to assay with IFN protein, the cells were washed 2X with media without IL-2 and then 50,000 cells in 0.1 were added to a microtiter plate. Serial dilution of IFN samples were prepared and 0.15 ml was added to the cells. After incubation for 24 hours the plate was centrifuged to pellet the cells and the supernatant removed and frozen for later analysis. IFN-gamma production was determined by a commercial ELISA (BL InterferonSource# 41500) and the data analyzed in the GraphPad Prism software package. The curve fitting was set to place the bottom of the curve at the background of IFN-gamma produced by cells which had not been stimulated by IFN, and the top of the curve set as the maximal IFN-gamma produced in the experiment. All assays were run concurrently with a defined human IFN alpha-2a standard. The NK-92 cell serves as a model for stimulation of immune cell activity. 101941 Example 4: Antiproliferative Activity of the IFN Proteins and Mutants on Human Fetal Lung Fibroblast Cell Line HFLl

[0195] The human fetal lung fibroblast cell line HFLl (ATCC Manassas, VA) was maintained in F12K media with 10% FBS and was plated in a microtiter plate at 2500 cells in 0.1 ml. The plate was then incubated at 37 degrees C to allow the cells to adhere. The media was then removed and 0.2 ml of serially diluted IFN sample was added to the cells. The plate was then returned to the incubator and the cells were allowed to grow for 6 to 7 days. At this point 0.1 ml of the media was removed and 0.06 ml of MTS reagent (Promega) was added to each well and allowed to incubate for 4 hours. The plate was then read at 490 nm in a Molecular Devices plate reader. The data was analyzed in the GraphPad Prism software package taking 100% as the value obtained for cells grown without IFN and 0% as the background of MTS added to media with no cells. All assays were run concurrently with a defined IFN alpha-2a standard. The HFLl cell serves as a model for antiproliferative activity against putatively normal human cells.

|0196j Example 5: Antiproliferative Activity of the IFN Proteins and Mutants on Ovarian Carcinoma Cell Line OVCAR3

[0197] The human ovarian carcinoma cell line NIH:OCVAR-3 (ATCC Manassas, VA) was maintained in the ATCC recommended media. At the start of the assay 1000 cells in 0.1 ml were plated in a 96 well plate which was incubated at 37 degrees C for 4 hours to allow the cells to adhere. After 4 hours the media was removed and 0.2 ml of serial dilutions of IFN samples were added to the cells. These were returned to the incubator and allowed to grow for 6 to 7 days. At this point 0.1 ml of media was removed and 0.06ml of MTS reagent (Promega) was added to the media and allowed to incubate for 4 hours. The plate was then read at 490 nm in a Molecular Devices plate reader. The data was analyzed in the GraphPad Prism software package taking 100% as the value obtained for cells grown without IFN and 0% as the background of MTS added to media with no cells. All assays were run concurrently with a defined IFN alpha-2a standard. The OVCAR3 cell line serves as a model for antiproliferative activity against human cancer cells. [0198] Results: Sec Figures 2 and 3. The effectiveness of novel interferons was tested in vitro in several assays. Most of the novel interferons showed substantial increases as quantified by EC50 in at least one bioactivity vs. the IFN alpha 2a standard and vs. the wild type congener. Several of the novel interferon molecules showed substantial decreases in at least one bioactivity vs. the IFN alpha 2a standard or vs. the wild type congener.

[0199] The differences in relative efficacy of these IFNs in different assays suggests that selected IFNs or combinations of selected IFNs may be preferred for treatment of specific diseases.

[0200| The enhancement of certain bioactivities such as antiviral activity or NK-92- stimulatory activity combined with lower AP activity on HFLl cells suggests that several of these interferons may provide clinical benefits in vivo. Clinical benefits may include, but are not limited to, an enhanced therapeutic window, i.e., the difference between the dose required to have a beneficial clinical effect and the dose at which dose-limiting side effects become evident, apparent with these molecules as assessed in vitro. Such an enhancement in the therapeutic window may be more evident in certain patient subpopulations and may relate to patients' common or unique pharmacogenomic profiles or prior exposure either to pharmacological agents or to environmental factors.

[0201] Alternatively, it is contemplated that these IFNs may be useful as IFN antagonists is certain conditions where reduction or modulation of endogenous IFN activity is warranted.

Claims

What' is claimed is:

1. A biologically active interferon polypeptide or fragment thereof encoded by a mutant interferon polynucleotide, wherein the interferon polypeptide is selected from the group consisting of:

a) any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and

20;

e) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19; and

0 an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to a sequence that is complementary to any one of SEQ ID NOS: 1, 3, 5, 7, 9, 1 1, 13, 15, 17, and 19.

2. A biologically active interferon polypeptide or fragment thereof of claim 1 , wherein the biological activity is selected from the group consisting of antiviral activity, antiproliferative activity, induction if interferon gamma in NK-92 cells, and MHC class I antigen expression induction activity.

3. A pharmaceutical composition comprising a biologically active interferon polypeptide or fragment thereof of claim 1 and a suitable excipient.

4. A method of treating a virus- infected subject or reducing a subject's risk of infection by a virus, comprising administering to the subject a biologically active interferon polypeptide or fragment thereof of claim 1, wherein the virus is selected from the group consisting of severe acute respiratory syndrome- associated coronavirus (SARS), coronavirus, influenza, smallpox virus, cowpox virus, monkeypox virus, encephalitis-causing viruses, hepatitis A, hepatitis B, hepatitis C, other non-A/non-B hepatitis, herpes virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex, human herpes virus type 6 (HHV-6), West Nile virus, vaccinia virus, respiratory syncytial virus, rhinovirus, arterivirus, filovirus, picornavirus, reovirus, rotavirus, rabies, papilloma virus, retroviruses including human immunodeficiency viruses (HIV), human T lymphotropic virus-type 1 and 2 (HTLV-I/- 2), papovavirus, herpesvirus, poxvirus, hepadnavirus, astrovirus, coxsackie virus, paramyxovirus, orthomyxovirus, echovirus, enterovirus, cardiovirus, togavirus, rhabdovirus, bunyavirus, arenavirus, bornavirus, adenovirus, parvovirus, and flavivirus.

5. The method of claim 4, wherein the interferon polypeptide comprises any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20.

6. The method of claim 4, wherein the mutant interferon polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19.

7. The method of claim 4, wherein the subject is selected from the group consisting of a human being, non-human primate, feline, canine, fish, and farm animal.

8. The method of claim 4, wherein the interferon is administered nasally, orally, parenterally, topically, rectally, by injection, by inhalation, by eye lotion, by ointment, by suppository, by controlled release patch, by infusion, or by inhalation.

9. The method of claim 4, wherein the interferon is administered to the subject's nasopharyngeal mucosa or lung epithelium.

10. The method of claim 4, wherein the amount of the interferon polypeptide administered is an amount effective to reduce the concentration of the virus particles in the subject.

1 1. The method of claim 4, wherein the amount of the interferon polypeptide administered is an amount effective to prevent or reduce an increase in the concentration of virus particles in the subject.