RU2392963C2 - Synthetic versions of hyperglycolised protease-resistive polypeptide, oral compositions and methods of using such versions - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and concerns a hyperglycolised version of parental interferon type 1 wherein the hyperglycolised version of parental interferon type 1 has been modified to include at least three additional glycolised sites.

EFFECT: invention provides prolonged preservation in the subject in comparison with parental interferon type 1 that allows decreasing a dosage.

93 cl, 2 ex, 32 dwg, 12 tbl

Description

FIELD OF TECHNOLOGY

[0001] The present invention relates to the field of drugs based on glycosylated, protease resistant and glycosylated protease resistant proteins.

BACKGROUND

[0002] The use of proteins as therapeutic agents has acquired clinical significance. However, various difficulties and disadvantages for their use remain, including immunogenicity; the destruction of therapeutic proteins by enzymes that are produced by the host organism; insufficient pharmacokinetic properties; etc. For example, the immunogenicity of a therapeutic protein can lead to the neutralization of protein activity by neutralizing antibodies, which, over time, are produced in a subject receiving treatment. Also, the immunogenicity of a therapeutic protein can lead to an inflammatory response. The destruction of the therapeutic protein by the enzymes of the host organism may preclude the use of certain routes of administration. For example, oral administration of therapeutic proteins may be useful in treating certain diseases; however, the therapeutic protein can be destroyed by enzymes in the gastrointestinal tract of the individual receiving the treatment. In addition, the therapeutic protein may have a short serum half-life, due, for example, to the rapid elimination of the protein by the reticuloendothelial system of the host; as a result, the pharmacokinetic profile of the therapeutic protein can be so repeated that there is a need for its repeated administration.

[0003] Many therapeutic proteins include one or more glycosylation centers, for example, amino acid sequences that are glycosylated by a eukaryotic cell. Several reports have been made of attempts to increase the degree of glycosylation of therapeutic proteins in order to ensure 1) a decrease in immunogenicity; 2) multiple administration of protein; 3) increased serum half-life; and 4) reduction of harmful side effects such as inflammation.

[0004] Disruption of the therapeutic protein by the enzymes of the host organism may preclude the use of certain routes of administration. For example, oral administration of therapeutic proteins may be useful in treating certain diseases; however, the therapeutic protein can be destroyed by enzymes in the gastrointestinal tract and / or in the serum of the individual receiving the treatment. Such proteolytic enzymes include, for example, α-chymotrypsin, carboxypeptidase, Arg-C endoproteinase, Asp-N endoproteinase, Glu-C endoproteinase, Lys-C endoproteinase, and trypsin.

[0005] There is a need for therapeutic proteins in oral dosage forms having suitable pharmacokinetic properties. The claimed invention is aimed at solving this need.

Information sources

[0006] Patent U.S. No. 6,685,933; U.S. Patent No. 4,695,623; and 4,897,471; U.S. Patent No. 6703225; U.S. Patent No. 6,569,420; U.S. Patent No. 6,299,877; U.S. Patent No. 6,586,398; U.S. Patent No. 6,531,122; U.S. Patent No. 6646110; Egrie and Brown, Br J Cancer. 2001 Apr; 84 Suppi 1: 3-10; U.S. Patent No. 6,127,332; WO 00/26354; WO 02/081507; WO 01/36001; U.S. Patent No. 5,041,376; U.S. Patent No. 5,520,911; U.S. Patent No. 6673580; U.S. Patent No. 5,853,724; Application for invention EU No. 640,619; WO 04/022747; and WO 004/0222593. Nyman et al. (1998) Eur. J. Biochem. 253: 485-493; Runkel et al. (1998) Pharmaceutical Research 15: 641; Adolfet al. (1990) J. Biol. Chem. 265: 9290-9295.

SUMMARY OF THE INVENTION

[0007] The present invention provides insights into non-native glycosylation centers, oral dosage forms of protease-resistant polypeptide variants and protease-resistant or protease-resistant, hyperglycosylated polypeptide variants, where the polypeptide variants contain at least one modified proteolytic cleavage site at the site of the native proteolytic cleavage site found parent polypeptide, which, thus, manifests an increase in resistance to proteases by cf an understanding of the parent polypeptide, wherein the polypeptide variants further include (1) a hydrocarbon group covalently linked to at least one non-native glycosylation site not found in the original therapeutic protein, or (2) a hydrocarbon group covalently linked to at least with at least one native glycosylation site found but not glycosylated in the original therapeutic protein. The present invention further provides compositions comprising oral dosage formulations containing glycosylated or protease resistant or protease resistant hyperglycosylated polypeptide variants. Further, the present invention provides nucleic acids comprising nucleotide sequences encoding a polypeptide agonist of a subject; and cells of the host organism containing the nucleic acids of the subject. The present invention further provides methods for treating viral infections, methods for treating fibrotic disorders and methods for treating proliferative disorders, methods typically including administering to the subject, if necessary, an effective amount of a polypeptide agonist in question. Further, the present invention provides containers, devices and kits comprising hyperglycosylated or protease-resistant or protease-resistant, hyperglycosylated polypeptide variants. The present invention further provides methods of treatment comprising administering to a subject an effective amount of an oral pharmaceutical composition comprising a hyperglycosylated or protease-resistant or protease-resistant, hyperglycosylated polypeptide variant, if necessary.

DISTINCTIVE SIGNS OF THE INVENTION

[0008] On the one hand, the invention provides oral pharmaceutical compositions containing a known hyperglycosylated or protease-resistant or protease-resistant, hyperglycosylated version of the parent therapeutic protein.

[0009] On the other hand, the invention provides an oral pharmaceutical composition that contains the first number of moles of a known hyperglycosylated or protease-resistant or protease-resistant, hyperglycosylated version in a first standard form, where it is confirmed that a parenteral pharmaceutical composition containing a second number of moles of the original therapeutic protein, is effective in treating a disease in a patient when administered to a patient by subcutaneous bolus injection in in which the patient received the second number of moles of the starting therapeutic protein, the selected interval between doses, where the first number of moles is greater than the second number of moles, and where, after oral administration of the first standard form to the patient, the time required to isolate the first number of moles of hyperglycosylated or protease -resistant or protease-resistant, hyperglycosylated variant, not more than the time period of the selected dosage interval.

[0010] In another aspect, the invention provides an oral composition that contains an initial dose of a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant in a first standard form, where the parenteral pharmaceutical composition containing a second dose of the starting therapeutic protein confirms that it is effective for treating a disease in a patient, when administered to the patient by subcutaneous bolus injection of a second dose from a selected interval between doses and, where the amount of the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant in moles of the drug per kilogram of the patient’s body weight in the first dose is greater than the amount of the initial therapeutic protein in moles of the drug per kilogram of the patient’s body weight in the second dose, when the first and second doses are calculated according to the average body weight of the patient in the general group of patients suffering from the disease, and where after oral administration of the first dose to the patient, the time required to isolate all of protease-resistant or protease-resistant, hyperglycosylated polypeptide variant at the first dose no greater than the time period between doses in the selected interval between doses. In some embodiments, the parenteral pharmaceutical composition is effective for treating a disease in a patient when administered to a patient in a dose calculated by body weight from a selected interval between doses, i.e. the second dose is a dose calculated by body weight, and the parenteral pharmaceutical composition corresponds to a form that provides a dosage calculated by body weight.

[0011] The present invention further provides methods of treatment comprising administering an effective amount of an oral pharmaceutical composition containing a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant to a subject, if necessary.

[0012] In another aspect, the invention provides a method of treating a disease in a patient, comprising administering to the patient an oral pharmaceutical composition comprising a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant of the parent therapeutic protein, wherein the oral pharmaceutical composition is orally administered in an amount in which the patient receives a first dose of a known protease-resistant or protease-resistant, hyperglycosylated polypeptide in the first interval between doses, where it is proved that a parenteral pharmaceutical composition containing a starting therapeutic protein is effective in treating a disease in a patient when it is administered to a patient by subcutaneous bolus injection in an amount in which the patient receives a second dose of the starting therapeutic protein, in the second interval between doses, where the first dose in moles of a protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is The patient’s body weight is greater than the second dose in moles of the initial therapeutic protein per kilogram of the patient’s body weight, when the first and second doses are calculated for the same patient’s body weight, and where after oral administration of the first dose to the patient, the time required to release protease-resistant or the protease-resistant, hyper-glycosylated polypeptide variant in the first dose is not more than the time period between doses in the second period between doses. In some examples, it is proved that the parenteral pharmaceutical composition is effective when administered to a patient in a dose calculated by body weight in the second interval between doses, i.e. the second dose corresponds to a dose calculated by body weight, and the parenteral pharmaceutical composition is presented in a form that assumes a dosage calculated by body weight. In some of the above examples, the first dose is a dose calculated by body weight, and the oral pharmaceutical composition is presented in a form that assumes a dosage calculated by body weight.

[0013] In another aspect, the invention provides a method of treating a disease in a patient, comprising administering to the patient an oral pharmaceutical composition comprising a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant of the parent therapeutic protein, wherein the oral pharmaceutical composition is orally administered in an amount in which the patient received the first dose of a known protease-resistant or protease-resistant, hyperglycosylated polypeptide option in the first interval between doses, where it is proved that a parenteral pharmaceutical composition containing a parental therapeutic protein is effective in treating a disease in a patient when administered to the patient by subcutaneous bolus injection in an amount in which the patient received a second dose of the starting therapeutic protein in the second interval between doses, where the first dose in moles of a protease-resistant or protease-resistant, hyperglycosylated polypeptide variant per kilogram of mass t ate the patient more than the second dose in moles of the initial therapeutic protein per kilogram of the patient’s body weight, while the first and second doses are calculated for the same patient’s body weight, and where the time period between doses in the first interval between doses is the same or shorter, than the time period between doses in the second interval between doses. In some examples, it is proved that a parenteral pharmaceutical composition is effective for treating a disease in a patient by administering it to a patient in a dose calculated by body weight in a second interval between doses, i.e. the second dose is a dose calculated by body weight, and the parenteral pharmaceutical composition is presented in a form that allows dosing by weight. In some of the above examples, the first dose is a dose calculated by weight, and the oral pharmaceutical composition is presented in a form that allows for dosing by weight.

[0014] In another aspect, the invention provides a method for treating a disease in a patient, comprising administering to the patient an oral pharmaceutical composition in a first standard form containing a first mole of a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant of the parent therapeutic protein, wherein the first number of moles there are more protease-resistant or protease-resistant, hyperglycosylated polypeptide variants than the second mole and the parent therapeutic protein in the parenteral pharmaceutical composition, wherein the parenteral pharmaceutical composition is an immediate release dosage form suitable for subcutaneous bolus administration, where the first standard form is administered orally to the patient in the first interval between doses, which corresponds to one or shorter than the second interval between doses, and where it is proved that the parent therapeutic protein is effective in treating a disease in a patient when administered the patient by subcutaneous bolus injection of the parenteral pharmaceutical composition in an amount in which the patient receives a second number of moles of the parent therapeutic protein in the second interval between doses.

DETAILED DESCRIPTION OF THE DRAWINGS.

[0015] Figure 1 represents the amino acid sequence of human mature IFN-α2a.

[0016] Figure 2 represents the amino acid sequence of human mature IFN-α2b.

[0017] Figure 3 represents the amino acid sequence of human IFN-β.

[0018] Figure 4 represents the amino acid sequence of mature, native human IFN-γ.

[0019] Figure 5 represents the amino acid sequence of G-CSF.

[0020] Figure 6 represents the amino acid sequence of human growth hormone.

[0021] Figure 7 represents the amino acid sequence of erythropoietin.

[0022] Figure 8 represents the amino acid sequence of GM-CSF.

[0023] Figure 9 represents the amino acid sequence of consensus IFN-α

[0024] Figure 10 represents the amino acid sequence of IFN-αc.

[0025] Figure 11 represents the amino acid sequence of IFN-α2c.

[0026] Figure 12 represents the amino acid sequence of IFN-αd.

[0027] Figure 13 represents the amino acid sequence of IFN-α5.

[0028] Figure 14 represents the amino acid sequence of IFN-α6.

[0029] Figure 15 represents the amino acid sequence of IFN-α4.

[0030] Figure 16 represents the amino acid sequence of IFN-α4b.

[0031] Figure 17 represents the amino acid sequence of IFN-α1.

[0032] Figure 18 represents the amino acid sequence of IFN-αJ.

[0033] Figure 19 represents the amino acid sequence of IFN-αH.

[0034] Figure 20 represents the amino acid sequence of IFN-αF.

[0035] Figure 21 represents the amino acid sequence of IFN-α8.

[0036] Figure 22 represents the amino acid sequence of IFN-β1

[0037] Figure 23 represents the amino acid sequence of IFN-β2a.

[0038] Figure 24 is a comparison of Infergen amino acid sequences (SEQ ID NO: **) and Interferon type 1 species (human IFN-α2b, SEQ ID NO: 2; human IFN-α14, SEQ ID NO: **; human IFN- β1, SEQ ID NO: **; human IFN-ω1, SEQ ID NO: **), which have been reported to be glycosylated by nature. Amino acid residues where glycosation takes place are labeled with bold italicized cells. Aspartic residues are binding sites for N-linked glycosylation sites and the threonine residue is a binding site for O-linked glycosylation sites. Figure 24 also represents the dominant sequence (SEQ ID NO: **) subject to comparison.

[0039] Figure 25 is a comparison of the amino acid sequences of Infergen amino acids 61-120 (SEQ ID NO: **) and typical synthetic type 1 interferon receptor polypeptide agonists. Sites 1, 2, and 3 are examples of positions where glycosylation centers are formed. N-linked glycosylation centers are formed on Sites 1 and 2. Both N-linked and O-linked glycosylation centers are formed on Site 3.

[0040] Figure 26 represents a synthetic mammalian Infergen nucleic acid sequence with predominant use of the human codon; and a translated open reading frame (SEQ ID NO: **). An open reading frame indicates the translated Infergen amino acid sequence (SEQ ID NO: 1). Six pairs of complementary primers A to F are shown in alternating italic and bold text. The upper sense strands of the primer pairs are identified with an odd number and the lower sense strands are identified with an even number. A short GCCACC sequence, a Kozak consensus sequence, was designed in the 3'-5 'direction of the ATG start codon to increase eukaryotic translation efficiency. Two tandem stop codons - TAA and TGA - are used to ensure completion of broadcast termination.

[0041] Figure 27 is a comparison of the mammalian Infergen nucleic acid sequence and their glycosylated mutants. Nucleotides that differ are presented in cells. The codons used taking into account the use of the preferred codon are presented in Table 8.

[0042] Figure 28 is a comparison of the amino acid sequences of amino acids 81-140 of human IFN-β1 (SEQ ID NO: **) and exemplary glycosylated variants of human IFN-β1. Sites 1 and 2 correspond to positions where glycosylated mutants are formed. Typically, only N-linked glycosylation centers are formed on Site 1. Both N-linked and O-linked glycosylation centers are formed on Site 2. Natural N-linked glycosylation centers in human IFN-β1 and mutants are present in the cells.

[0043] Figure 29 is a comparison of the amino acid sequence of amino acids 81-140 of human IFN-ω1 (SEQ ID NO: **) and exemplary glycosylated variants of human IFN-col. Sites 1 and 2 correspond to the positions where glycosylated mutants are formed. In general, only N-linked glycosylation centers are formed on Site 1. Both N-linked and O-linked glycosylation centers are formed on Site 2. Natural N-linked glycosylation centers in human IFN-ω1 mutants are present in cells.

[0044] Figure 30 is a comparative analysis of the primary amino acid sequence Infergen (SEQ ID NO: **), human IFN-α14 (SEQ ID NO: **), human IFN-β1 (SEQ ID NO: **) and exemplary fusion proteins with human IFNα14 and human IFN-β signal peptides (SEQ ID NOs: ** and **, respectively). Most sequences are shown above (SEQ ID NO: **).

[0045] Figure 31 represents the amino acid sequence of a mature, native human IFN-γ (SEQ ID NO: **).

[0046] Figure 32 is a Western blot analysis of exemplary peptides synthesized by Cos-7 cells.

DEFINITIONS

[0047] The term "polypeptide" refers to polymers of amino acids and does not refer to the specific length of the product; thus, peptides, oligopeptides and proteins are included in the definition of a polypeptide. This term also does not refer to or exclude post-translational modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, etc. Included in the term "polypeptide", for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids, non-coding amino acids, etc.), polypeptides with substituted bonds, as well as other modifications known from the prior art as natural never found in nature. The term "polypeptide" includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically labeled proteins; etc.

[0048] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein to refer to polymeric forms of nucleotides of any length. Polynucleotides may consist of deoxyribonucleotides, ribonucleotides and / or their analogues. Nucleotides can have any spatial structure and can perform any function, known or unknown. The term "polynucleotide" includes mono-, double-stranded and tri-coiled molecules. An "oligonucleotide" usually refers to polynucleotides with between about 5 and about 100 nucleotides of single or double stranded DNA. However, in the context of this summary of the invention, there is no upper limit on the length of the oligonucleotide. Oligonucleotides are also known as oligomers or oligos and can be isolated from genes, or chemically synthesized using methods known in the art. The term "polynucleotide" includes double-stranded DNA, found, in particular, in linear DNA molecules (eg, restriction fragments), viruses, plasmids, and chromosomes.

[0049] The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and examples. The nucleic acid molecule may also contain modified nucleic acid molecules, for example, such as methylated nucleic acid molecules and nucleic acid analogs. Analogues of purines and pyrimidines are known in the art. Nucleic acids may be natural, for example DNA or RNA, or may be synthetic analogues, as is known in the art. Such analogs may be preferred for use as probes because of better stability under analytical conditions. Modifications of native structures, including alterations of the backbone, polysaccharide or heterocyclic bases, show an increase in intracellular stability and binding affinity. Among the applicable chemical alterations of the skeleton are phosphorothioates; phosphorodithioates, where both non-bridging oxygen are replaced by sulfur; phosphoramidites; alkyl phosphotriethers and boranophosphates. Ahiral phosphate derivatives include 3'-O'-5'-S-phosphorothioate, 3'-S-5'-O-phosphorothioate, 3'-CH ₂ -5'-O-phosphonate and 3'-NH-5'- O-phosphoroamidate. In peptide nucleic acids, the entire ribose phosphodiester backbone is replaced by a peptide bond.

[0050] A polynucleotide or polypeptide has a certain percentage of "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, the percentage of bases or amino acids is the same when comparing two sequences. Sequence similarity can be determined in a number of different ways. To determine sequence identity, the sequences can be aligned using methods and computer programs, including BLAST, which is publicly available worldwide at ncbi.nlm.nih.gov/BLAST. See, e.g., Altschul etal. (1990), J. Mol. Biol. 215: 403-10. Another alignment algorithm is the FASTA program, available from the Genetics Computing Group (GCG), from Madison, Wisconsin, USA, a subsidiary of Oxford Molecular Group, Inc. Other alignment methods are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., Harcourt Brace & Co., San Diego, California, USA. Alignment programs that allow you to space in sequences are noteworthy. The Smith-Waterman algorithm is the same type of algorithm that allows you to do intervals in aligned sequences. See Meth. Mol. Biol. 70: 173-187 (1997). Also, a GAP program using the alignment method of Needleman and Wunsch can be used to align sequences. See J Mol. Biol. 48: 443-453 (1970).

[0051] The term "host cell" includes an individual cell or cell culture, which may be or is a recipient of any (s) recombinant (s) vector (s) or a synthetic or exogenous polynucleotide. The host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in the set of total DNA) with the original parent cell due to a natural, accidental or intentional mutation and / or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a synthetic or exogenous polynucleotide. A host cell that contains a recombinant vector is referred to as a "recombinant host cell" in the invention. In some examples, the host cell is a prokaryotic cell. In other examples, the host cell is a eukaryotic cell.

[0052] The terms "DNA regulatory sequences" and "regulatory elements", used interchangeably herein, refer to transcriptional and translational regulatory sequences, for example, promoters, enhancers, polyadenylation signals, terminators, protein breakdown signals, and the like, which provide and / or regulate the expression of the coding sequence and / or production of the encoded polypeptide in the host cell.

[0053] The term "transformation" is used herein to be equivalent to the term "genetic modification" and refers to a permanent or transient genetic change induced in a cell following the introduction of a new nucleic acid (eg, exogenous DNA into the cell). A genetic change (“modification”) can be made either by incorporating new DNA into the genome of the host cell, or by temporarily or permanently maintaining the new DNA as an episomal element. If the cell is a mammalian cell, a permanent genetic change is usually achieved by incorporating DNA into the genome of the cell.

[0054] The term “operably linked”, as used herein, refers to a relationship in which the components depicted in this manner are in a relationship that allows them to function in an established manner. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

[0055] The term "construct" as used herein refers to a recombinant nucleic acid, in particular a recombinant DNA that is formed to express a specific nucleotide sequence (s), or to be used in the construction of other recombinant nucleotide sequences.

[0056] For the present invention, the terms "treatment", "being treated" and other similar, refer to obtaining the desired pharmacological and / or physiological effect. The effect may be prophylactic in the form of a complete or partial prevention of the disease or its symptom, and / or may be therapeutic in the form of a partial or complete cure of the disease and / or collateral damage inherent in the disease. A “treatment” as used herein encompasses any treatment for a disease in a mammal, especially a human, and includes: (a) an increase in life time; (b) reducing the risk of death due to illness; (c) preventing the occurrence of a disease in a subject who may be predisposed to a disease, but who has not yet been diagnosed as having such a disease; (d) suppression of the disease, i.e. suppression of its development (e.g., a decrease in the rate of disease progression); and (e) alleviating the disease, i.e. weakening the symptoms of the disease.

[0057] The terms "individual", "host", "subject" and "patient", used here as equivalent, refer to a mammal, including primates, rodents, livestock, domestic animals, horses, etc. In some examples, the individual is person.

[0058] The term "therapeutically effective amount" means an amount of a therapeutic substance or a consumption of a therapeutic substance sufficient to provide the desired therapeutic effect. The exact desired therapeutic effect will differ depending on the condition of the patient, the composition that is used, and various other factors that are understandable to the average person skilled in the art.

[0059] As used herein, the terms "proven as effective" in the context of drug therapy for treating a disease, or any other expression of similar meanings, are meant to mean that in the drug therapy described in this manner was considered to be safe and effective, alone or in combination with one or more additional pharmaceutical agent (s), for treating a disease in a clinical trial under controlled conditions or an assigned clinical trial that The raw materials provide one or more early clinical results of the study (s) with statistical significance p <0.05. Typically, studies of drugs that prove to be effective for drugs include: (1) any study of the indication (s) for the drug indicated in the license for the drug sold by the authority; and (2) any study of the indication (s) for the drug described in the bulletin issued by a recognized body of medical experts (e.g. NIH Consensus Statement).

[0060] The term "specifically bound", in the context of antibody binding, refers to a highly avid and / or high affinity binding of an antibody to a specific region of a polypeptide, i.e. an epitope of a polypeptide, for example, a synthetic type 1 interferon receptor synthetic polypeptide agonist. For example, the binding of an antibody to a specific epitope of a synthetic type 1 interferon receptor synthetic polypeptide agonist or fragment thereof is stronger than the binding of such an antibody to any other epitope, especially one that may be present in molecules in association with, or in such a sample, as a specific polypeptide under study, for example, binds more strongly to the specific epitope of the synthetic type I interferon receptor polypeptide agonist under consideration than to any other epitopes of an interferon type 1 receptor polypeptide agonist, so that when binding conditions are selected, the antibody binds almost exclusively to the specific epitope of the synthetic type 1 interferon receptor polypeptide agonist in question, and not to any other epitope of an interferon type I receptor polypeptide agonist, or any another polypeptide that does not contain an epitope. Antibodies that bind specifically to the polypeptide may be capable of binding to other polypeptides at a weak, barely detectable level (eg, 10% or less binding to the test polypeptide is indicated). Such weak binding, or background binding, is readily noticeable with specific binding of antibodies to the polypeptide of interest, for example, using adequate controls. Typically, specific antibodies bind to this polypeptide with a binding agent of 10 ^-7 M or more, e.g. 10 ^-8 M or more (e.g. 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, etc.). Typically, an antibody with a binding affinity of 10 ^-6 M or less is not suitable, because it will not bind the antigen at a detectable level, using the generally accepted methodology currently in use.

[0061] The "fibrotic condition", "fibrotic disease", and "fibrotic disorder" are used interchangeably to refer to a condition, disease, or disorder that is susceptible to treatment by administering a composition having antifibrotic activity. Fibrotic disorders include, but are not limited to, pulmonary fibrosis, including idiopathic pulmonary fibrosis (IFL) and pulmonary fibrosis of unknown etiology, liver fibrosis, and kidney fibrosis. Other exemplary fibrotic conditions include musculoskeletal fibrosis, cardiac fibrosis, postoperative adhesions, scleroderma, glaucoma, and skin lesions, such as keloids.

[0062] The terms "proliferative disorder" and "proliferative disease" are used interchangeably to refer to any disease or condition characterized by abnormal cell enlargement or proliferation, in particular cancer.

[0063] The terms "cancer", "neoplasm" and "tumor" are used with the same meaning to refer to cells that exhibit relatively autonomous propagation, so that they represent an impaired cultural phenotype, characterized by a significant loss of control over cell proliferation. Cancer cells can be benign or malignant.

[0064] The term "viral hepatitis infection" refers to infection of one or more hepatitis A, B, C, D, or E viruses, hepatitis viral infection transmitted through blood of particular interest, especially hepatitis C viral infection.

[0065] The term "sustained virologic response" (SVR; also refers to a "sustained response" or "persistent response"), as used herein, refers to an individual's response to a treatment regimen for HCV infection, in the form of an HCV serum titer. Typically, a “stable virologic response" refers to undetected HCV RNAs (eg, less than about 500, less than about 200, or less than about 100 genomic copies per milliliter of serum) found in patient serum for at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, or at least about six months following treatment discontinuation .

[0066] The term "treatment of non-responding patients" (or "treatment of non-responding"), as used here, usually refers to HCV-infected patients who were not able to accept previous HCV treatment (refers, for example, to "non- responsive ") or who initially responded to previous treatment, but in which the susceptibility to drug therapy has not been preserved (refers, for example, to" recurrent "). The preceding treatment usually may include IFN-α treatment alone or IFN-α combination therapy, where the combination therapy may include the use of IFN-α and an antiviral agent such as ribavirin.

[0067] The term "dosing process", as used here, refers to the administration of an antiviral agent to a patient, if necessary, where the process can be carried out with one or more release of the antiviral agent from the drug, dosed agent. Thus, the term "dispensing process", as used here, includes, but is not limited to, the installation of a permanent delivery vehicle (eg, pump-action or other injection-controlled injection device); and a single subcutaneous administration following the installation of a permanent delivery vehicle.

[0068] The “systemic” or “temporary” that is used in connection with drug delivery is intended for a drug delivery system, typically a regular system, for a pre-selected period of time (eg, a different period associated, for example, with injection shock dose of the substance). “Systemic” or “temporary” drug delivery means delivering drugs at an increased, reduced, almost constant or pulsating speed or speed range (eg, the amount of drug per unit time or the volume of the drug composition per unit time), and further delivery, which is constant or almost constant, or lingering.

[0069] The term "drug delivery control device" is intended to mean any device that releases (eg, rapid, temporary release) a drug or other necessary substance contained therein, controlled or measured by the device itself and not significantly affecting the system used , or emitted at a speed that is reproducible in the system in use.

[0070] By “substantially constant,” as used herein, for example, in the context of “substantially constant infusion” or “substantially constant delivery,” is meant the designation of drug delivery in a manner that is substantially continuous during a pre-selected delivery period medicines, where the amount of medicine received by the patient during any 8-hour interval in a pre-selected period never reaches zero. In addition, “substantially constant” drug delivery may also include drug delivery at a substantially constant, pre-selected speed or speed range (eg, drug amount per unit time or drug volume per unit time), which are substantially continuous during the pre-selected period of drug delivery.

[0071] For the purposes of the present invention, the term "pirphenidone" refers to 5-methyl-1-phenyl-2- (1H) -pyridone. As used here, the term "pirphenidone analogue" refers to any composition of Formula I, IIA, or IIB below. The “specific analog of pirfenidone” and all grammatical derivatives thereof refer to, and are limited to, each and every analog of pirfenidone indicated in Table 10.

[0072] The term "antifibrotic" substance, drug, or composition is intended to mean substances that inhibit or reduce fibrosis, including: type II interferon receptor agonists (eg, interferon-gamma); pirfenidone and analogues of pirfenidone; antiangiogenic substances such as VEGF antagonists, VEGF receptor antagonists, bFGF antagonists, bFGF receptor antagonists, TGF-beta antagonists, and TGF-beta receptor antagonists; and anti-inflammatory agents, including tumor necrosis factor (TNF) antagonists, such as anti-TNF antibodies (e.g., REMICADE ^™ anti-TNF monoclonal antibody) and soluble TNF receptor (e.g. ENBREL ^TM TNF Ig immunoadhesin receptor), and IL antagonists -1, such as IL-1Ra.

[0073] The terms "angiogenic substance", "angiogenic composition" and "angiogenic factor" mean substances that contribute to the formation of new vessels, such as VEGF, bFGF, and TGF-beta.

[0074] The terms “anti-angiogenic” or “angiostatic” substance, drug or composition, or “angiogenesis inhibitor” mean substances that inhibit or reduce revascularization, such as VEGF antagonists, VEGF receptor antagonists, bFGF antagonists, antagonists, antagonists bFGF receptor, TGF-beta antagonists and antagonists of the TGF-beta receptor.

[0075] As used here, the term "nucleoside" refers to a compound containing any pentose or modified pentose moiety attached to a specific position of the heterocycle or to the natural position of purines (9-position), or pyrimidines (1-position), or equivalent position in the analogue.

[0076] As used here, the term "nucleotide" refers to a phosphate ester substituted at the 5'-position with a nucleoside.

[0077] As used herein, the term “heterocycle” refers to a monovalent saturated or unsaturated isocyclic radical having at least one hetero atom, for example, N, O, S, Se or P, on a ring, each position of which may be optionally substituted independently with, for example, hydroxyl, oxo, amino, imino, lower alkyl, bromo, chloro and / or cyano. Purines and pyrimidines are included in the term “heterocycle”.

[0078] As used herein, the term "purine" refers to nitrogenous bicyclic heterocycles.

[0079] As used herein, the term "pyrimidine" refers to nitrogenous monocyclic heterocycles.

[0080] As used herein, the term "L-nucleoside" refers to a nucleoside compound that has an L-ribose sugar moiety.

[0081] The term "antitumor" substance, drug, or composition means the relation to any substance, including any chemotherapeutic substance, a biological response modifier (including without limitation (1) protein, ie peptide, molecules capable of developing or modifying biological reactions and (2) non-protein, i.e. non-peptide, molecules capable of developing or modifying biological reactions), a cytotoxic substance, or a cytostatic substance, which reduces the proliferation of neoplastic cells.

[0082] The term "anti-fibrous" substance, drug, or composition is intended to include substances that inhibit or reduce fibrosis, including: Type II interferon receptor agonists (eg, interferon-gamma); pirfenidone and analogues of pirfenidone; anti-angiogenic substances such as VEGF antagonists, VEGF receptor antagonists, bFGF antagonists, bFGF receptor antagonists, TGF-beta antagonists and TGF-beta receptor antagonists; and anti-inflammatory substances, including tumor necrosis factor (TNF) antagonists, such as anti-TNF antibodies (e.g., REMICADE ^™ anti-TNF monoclonal antibody) and soluble TNF receptor (e.g. ENBREL ^TM TNF Ig immunoadhesin receptor), and IL antagonists -I, such as IL-1Ra.

[0083] The term "chemotherapeutic agent" or "chemotherapeutic" (or "chemotherapy", in the case of treatment with a chemotherapeutic agent) is intended to mean any non-protein (ie, non-peptide) chemical compounds suitable for treating cancer. Examples of chemotherapeutic agents are alkylated substances such as thiotepa and cyclophosphamide (CYTOXAN ^™ ); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodopa, carboquion, meturedopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethyl lomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue of topotecan); bryostatin; callistatin; SS-1065 (including its synthetic analogues, adolesin, carzelesin and biselezin); cryptophycin (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pankratistatin; sarcodictin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride oxide, melphalan, novembikhine, phenesterol, prednimustine, trophosphamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fororemustine, lomustine, nimustine, ranimustine; antibiotics such as enediin antibiotics (e.g. calicheamicin, especially calicheamicin gamma-1I and calicheamicin phiI1, see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994); dynemycin, including dynemycin A ; bisphosphonates such as clodronate; esperamycin; same as neocarcinostatin chromophore and related chromoprotein enediin antibacterial chromophores), aclacinomycins, actinomycin, autramycin, azaserin, bleomycin, caraficinomycin, carcincinum, carcincinum, carcincinum, carcincinum, carcincinum, carcincinum, carcincinum, carcincinum, carcincinum, carcincinum, carcincinum, carcincinum, carcincinoma diazo-5-oxo-l-norleuci , Doksorubintsin (Adramycin ^TM) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-deoksidoksorubitsin and doxorubicin), epirubicin, esorubicin, idarubicin, martsellomitsin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin , potfiromycin, pyromycin, quelamycin, rhodorubicin, streptonigrin, streptozycin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as demopterin, methotrexate, pteropterin, trimerexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, karmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, phloxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostan, testolactone; anti-adrenal, such as aminoglutethimide, mitotan, trilostane; a folic acid replenisher such as frolinic acid; Aceglaton; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisanthrene; edatraxate; defofamine; demecolcin; diaziquon; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitogvazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pyrarubicin; losoxantrone; podophyllic acid; 2-ethyl hydrazide; procarbazine; PSK ^® ; razoxane; rhizoxin; sisofiran; spiro germanium; tenuazonic acid; triaziquon; 2,2 ', 2''-trichlorotriethylamine; trichotecenes (especially T-2 toxin, verracurin A, roridin A and angvidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacitosin; arabinoside ("Ara-C");cyclophosphamide;thiopeta; taxoids, e.g. paclitaxel (TAXOL ^® , Bristol Meyers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE ^® , Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (Gemzar ^TM); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etopside (VP-16); ifosfamide; mitroxantron; vancristine; vinorelbine (Navelbine ^TM ); novantron; teniposide; edatrxate; daunomycin; aminopterin; xeoloda; ibandronate; SRT-11; topoisomerase inhibitor RFS 2000; difluromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included in the definition of “chemotherapeutic agent” are anti-hormonal drugs that are directed to regulate or inhibit the hormonal effect on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex ^TM ), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifen, LY1 17018, onapristone and toremifene (Fareston ^TM ); inhibitors of the aromatase enzyme, which regulates the production of estrogen in the adrenal glands, such as, for example, 4 (5) -imidazoles, aminoglutethimide, megestrol acetate (Megace ^TM ), exemestane, formestane, fadrazole, Vorozol (Rivisor ^TM ), letrozole (Femara ^TM ), and anastrozole (Arimidex ^TM); and antiestrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserilin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

[0084] The term "anti-blastoma" agent, drug or compound is intended to mean any agent, including any chemotherapeutic agent, biological response modifier (including without limitation (i) protein-like, ie peptide, molecules capable of developing or modifying biological reactions and (ii) non-protein-like, i.e. non-peptide, molecules capable of developing or modifying biological reactions), a cytotoxic agent, or a cytotoxic agent that slows down the proliferation of tumors x cells.

[0085] The term “biological response modifier” refers to any protein-like (ie, peptide) molecule or any non-protein-like (ie, non-peptide) molecule capable of developing or modifying a biological response related to the treatment of cancer. Examples of biological response modifiers include antagonists of tumor associated antigens, such as antibodies to antitumor antigens, cell receptor antagonists capable of inducing cell proliferation, cell receptor agonists capable of inducing apoptosis, such as Apo-2 ligands, type I interferon receptor agonists, such as interferon-α molecules and interferon-β molecules, type II interferon receptor agonists, such as interferon-γ molecules, type II interferon receptor agonists, such as IL-28A, IL-28 B, and IL-29, antagonists of inflammatory cytokines, including tumor necrosis factor (TNF) antagonists, such as anti-TNF antibodies (e.g., REMICADE ^™ anti-TNF monoclonal antibody) and a soluble receptor Name (e.g. ENBREL ^™ TNF receptor-Ig immunoadhesin), cytokine growth factor, such as hematopoietic cytokines, including erythropoietins, such as EPOGEN epoetin alfa, granulocyte colony stimulating factors (G-CSFs), such as NEUPOGEN ^TM granulocytogram, colony stimulating factors (GM-CSFs), and thrombopoietins, lymph tsitarny growth factor cytokines, such as interleukin-2, growth factor and cytokine antagonists, including antagonists of angiogenic factors, eg. endothelial vascular growth factor (VEGF) antagonists such as AVASTIN ^™ bevacizumab (anti-VEGF monoclonal antibody).

[0086] As used herein, the term "HCV enzyme inhibitor" refers to any agent that inhibits the enzymatic activity of an enzyme encoded by HCV. The term "HCV enzyme inhibitor" includes, but is not limited to, agents that inhibit the activity of HCV NS3 protease; agents that inhibit the activity of helicase NS3 HCV; and agents that inhibit the activity of an RNA-dependent RNA polymerase of HCV NS5B.

[0087] As used herein, the terms "HCV NS3 protease inhibitor" and "NS3 protease inhibitor" refer to any agent that inhibits the protease activity of the HCV NS3 / NS4A complex. Except for other special purposes, the term "NS3 inhibitor" is used interchangeably with the terms "HCV NS3 protease inhibitor" and "NS3 protease inhibitor".

[0088] As used herein, the terms "NS5B HCV inhibitor", "NS5B inhibitor", "RNA dependent HCV NS5B RNA polymerase inhibitor", "HCV RDRP inhibitor" and "RDRP inhibitor" refer to any agent that inhibits the activity of an RNA dependent RNA polymerase NS5B HCV.

[0089] Before further describing the present invention, it should be noted that the present invention is not limited to the specific examples described, which, of course, of course, may include changes. Also, it should be noted that the terminology used here is intended only to describe particular examples and is not intended to be limiting, since the scope of protection of the present invention will be limited only by the attached claims.

[0090] Where the range of values is presented, it is obvious that each intermediate value, up to the tenth unit of measurement of the lower limit, unless the context clearly stipulates otherwise, between the upper and lower limit of such a region and any other set or intermediate value in this set interval covered by this invention. The upper and lower limits of these narrow ranges can be independently included in narrow ranges, and are also covered by this invention, allowing any specifically excluded limit in the specified range. Where the established range includes one or both of the limits, ranges excluding each or both of these including limits are also included in the invention.

[0091] In addition to redistributed other cases, all technical and scientific terms used herein have the same meaning as is commonly understood by any person skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein may also be practiced or tested on the present invention, preferred methods and materials are now described. All publications cited here are supported here by reference to disclosure of the essence and description of the methods or materials in connection with which the publications are cited.

[0092] It must be pointed out that the singular forms "a", "and" and "the" used here and in the attached claims include plural designations, unless the context implies the opposite. For example, a reference to a “protease-resistant or protease-resistant, hyperglycosylated polypeptide variant” includes a plurality of such polypeptide variants and a reference to an “oral composition” refers to one or more oral compositions and their analogs known to one skilled in the art, etc.

[0093] The publications discussed herein are provided solely for their description prior to the filing date of this application. Nothing here can be construed as confirmation that the present invention is subject to an earlier date of such publication, predominantly by an earlier invention. Further, the publication dates presented may be different from the true publication dates, which may need independent confirmation.

DETAILED DESCRIPTION OF THE INVENTION

[0094] The present invention provides oral pharmaceutical compositions comprising a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant of the parent therapeutic protein. A known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant comprises (1) a carbohydrate moiety covalently attached to at least one non-native glycosylation site that is not present in the parent therapeutic protein, or (2) a carbohydrate moiety that is covalently attached at least one native glycosylation site that is present but not glycosylated in the parent therapeutic protein. Also, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant contains at least one mutant protease cleavage site at the site of the native cleavage site found in the parent therapeutic protein, and thus exhibits increased protease resistance compared to with parental therapeutic protein.

[0095] The present invention further provides therapeutic methods for treating a disease in a patient, comprising orally administering to the patient a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant in the oral form and at a dosage interval that enables delivery of a larger amount of drug (in molar expression) per dose, and at least it has been proven that such a large number of doses per unit time are received by the patient in the method are effective for treating the disease using a subcutaneous bolus injection of the parent polypeptide in the form for parenteral administration.

[0096] The present invention further provides synthetic type 1 interferon receptor polypeptide agonists that contain one or more glycosylation sites; and compositions, including pharmaceutical compositions containing agonists. The present invention further provides nucleic acids comprising nucleotide sequences encoding the subject polypeptide agonists; and host cells comprising nucleic acids of the subject. The present invention further provides containers and kits containing the subject polypeptide agonist.

[0097] The subject synthetic type 1 interferon receptor polypeptide agonist comprises a hybrid or consensus type 1 interferon receptor polypeptide agonist containing at least one glycosylation site. The glycosylation site (s) is such a site for binding the carbohydrate moiety to the synthetic polypeptide agonist in question, such that when the synthetic polypeptide agonist in question is produced in a eukaryotic cell capable of glycosylation, the synthetic polypeptide agonist in question is glycosylated. Glycosylation provides one or more advantages for the synthetic polypeptide agonist in question relative to the parent type 1 interferon receptor polypeptide agonist or compared to the naturally occurring type 1 interferon receptor polypeptide agonist. These benefits include increased serum half-life; decreased immunogenicity; increased active in vivo half-life; reduced splitting in the gastrointestinal tract; and increased rate of absorption by intestinal epithelial cells. An increased rate of absorption by intestinal epithelial cells and a reduced digestion in the gastrointestinal tract are important for parenteral (e.g. oral) dosage forms of the synthetic type 1 interferon receptor synthetic polypeptide agonist.

[0098] The subject synthetic type 1 interferon receptor polypeptide agonists are useful in the treatment of various diseases, including viral infections, fibrotic disorders, and proliferative disorders. In this connection, the present invention further provides methods for treating viral infections, methods for treating fibrotic disorders and methods for treating proliferative disorders, methods typically including administering to the individual, if necessary, an effective amount of a synthetic polypeptide agonist in question. In some examples, the subject treatment method further includes administering at least one additional therapeutic agent for treating a viral infection, fibrotic disorder, or proliferative disorder. In some examples, a contemplated treatment method further includes administering at least one side-effect control agent to reduce side effects caused by one or more therapeutic substances.

[0099] In another aspect, synthetic type 1 interferon receptor polypeptide agonists of the invention find use as reagents for detecting and isolating type 1 interferon receptor receptors, such as detecting expression of type 1 interferon receptor in various types of cells and tissues, including determining the density of interferon receptors Type 1 and distribution in the cell population, and sorting cells based on the expression of type 1 interferon receptors. In yet another aspect, the subject synthetic type 1 interferon receptor agonists are useful for developing substances capable of binding to the type 1 interferon receptor receptor or activating types similar to the subject synthetic type 1 interferon receptor agonists. Synthetic interferon type 1 receptor agonists of the invention can be used in signal transduction methods of type 1 interferon receptors to study small molecule agonists or antagonists of type 1 interferon receptor signal transduction.

POLYPEPTIDE OPTIONS

[00100] The present invention relates to protease-resistant or protease-resistant, hyperglycosylated polypeptide variants. Protease-resistant or protease-resistant, hyperglycosylated polypeptide variants contain at least one mutant protease cleavage site at the site of the native protease cleavage site present on the parent therapeutic protein and, therefore, exhibits increased protease resistance compared to with parental therapeutic protein.

[00101] A protease cleavage site that is present in a parent therapeutic protein and that is mutated in a protease-resistant or protease-resistant, hyperglycosylated polypeptide variant, such as the site is not cleaved, or exhibits increased cleavage resistance (ie is a worse substrate than the native site for proteolytic processing) by a protease that cleaves the site for cleavage of the protease in the parent protein, refers to the so-called "mutated site d I protease cleavage "or" mutant cleavage site. " The protease cleavage site, which is found in the parent therapeutic protein, refers to the so-called "native protease cleavage site".

[00102] Also, a protease-resistant or protease-resistant, hyperglycosylated polypeptide variant includes (1) a carbohydrate moiety covalently attached to at least one non-native glycosylation site not found in the parent therapeutic protein, or (2) a carbohydrate moiety, covalently attached to at least one native glycosylation site found, but not glycosylated, in the parent therapeutic protein. A glycosylation site that is not found in the parent therapeutic protein refers to the so-called "non-native glycosylation site". A glycosylation site that is found but not glycosylated in the parent therapeutic protein refers to the so-called "native glycosylation site". Therefore, a protease-resistant or protease-resistant, hyperglycosylated polypeptide variant includes (1) a carbohydrate moiety covalently attached to at least one non-native glycosylation site, and / or (2) a carbohydrate moiety covalently attached, at least at least one native glycosylation site. A protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide, which includes (1) a carbohydrate moiety covalently attached to a non-native glycosylation site or (2) a carbohydrate moiety covalently attached to a native glycosylation site, and which contains at least one mutant protease cleavage site at the site of the native protease cleavage site found in the parent therapeutic protein refers to the so-called “protease-resistant or protease-resistant” ezistentnomu, hyperglycosylated polypeptide variant ".

[00103] By a "known" protease-resistant or protease-resistant, hyperglycosylated variant polypeptide is meant any protease-resistant or protease-resistant, hyperglycosylated polypeptide variant existing, currently created or created in the future, which (1) retains the necessary pharmacological activity of the parent therapeutic protein and (2) exhibits a longer half-life from serum or a larger area under the curve of the concentration of the drug in serum as a function of time (PPC) compared with those of the parent therapeutic protein when administered to the patient in the same form and in the same dose, frequency of administration and route of administration. The present invention provides compositions, including oral pharmaceutical compositions containing known protease-resistant or protease-resistant, hyperglycosylated polypeptide variants.

[00104] A known hyperglycosylated, protease-resistant polypeptide variant is provided in a composition suitable for oral administration. The parent therapeutic protein is typically administered as an immediate-release formulation suitable for subcutaneous bolus administration. Typically, a dosage form of a known protease-resistant or protease-resistant, hyperglycosylated version of an oral polypeptide contains the first number of moles; and the parent therapeutic protein is present in a parenteral dosage form that contains a second number of moles. In general, the first number of moles is greater than the second number of moles. However, a protease-resistant or protease-resistant, hyperglycosylated oral polypeptide variant is released after a period of time that lasts no longer than the dosing interval when the parental therapeutic protein is administered in a regimen that is found to be effective for treating a patient’s disease .

[00105] The parent therapeutic protein is typically provided in a parenteral dosage form, administered by subcutaneous bolus injection, which provides a “sustained” effect by slowly releasing the therapeutic protein into the bloodstream by diffusing the drug from the tissues surrounding the injection site.

[00106] The method of the invention under consideration replaces the "prolonged" action of a subcutaneous bolus injection with a similar pharmacokinetic profile, achieved by oral delivery of a long-acting substance (a known hyperglycosylated, protease-resistant variant of a polypeptide with a longer half-life from serum and / or PPC than its parent protein), which is not part of a delayed or prolonged action. This means that the time required to release the first mole of a known hyperglycosylated, protease-resistant variant of the polypeptide when administered orally is not more than the time period between doses of the parent therapeutic protein when administered by subcutaneous bolus injection in a method that is proven to be effective for treatment diseases. Thus, in some examples, the known hyperglycosylated, protease-resistant polypeptide variant is introduced, at least as often, or, in many cases, more often and at higher doses (in molar value) than the parent therapeutic protein. Structural features

[00107] The protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide has an amino acid sequence containing one or more mutant protease cleavage sites at the site (s) of the native protease cleavage found in the corresponding parent therapeutic protein; and has an amino acid sequence that contains (1) one or more non-native glycosylation sites and / or (2) one or more native glycosylation sites. So, for example, the desired variant of the polypeptide has an amino acid sequence that contains one or more mutant sites for protease cleavage at the site of the native site (s) for cleavage of the protease found in the parent therapeutic protein; and has an amino acid sequence that contains one or more glycosylation sites not found in the parent therapeutic protein or found but not glycosylated in the parent therapeutic protein. The parent therapeutic protein is, in some examples, a natural polypeptide. In other examples, the parent therapeutic protein is a non-natural polypeptide (e.g., a synthetic polypeptide, a hybrid polypeptide, a consensus polypeptide, a fusion polypeptide, a recombinant polypeptide, or a variant of a natural polypeptide). As used herein, the terms “polypeptide variant” and “polypeptide variant” both refer to any polypeptide that contains one or more mutant protease cleavage sites at the site of the native protease cleavage site (s) found in the parent therapeutic protein; and which contains (1) one or more glycosylation sites not found in the parent therapeutic protein, or (2) one or more glycosylation sites found but not glycosylated in the parent therapeutic protein.

[00108] Non-native and native glycosylation sites include N-linked glycosylation sites and O-linked glycosylation sites. TV-linked glycosylation sites include, for example, Asn-X-Ser / Thr, where the aspartic moiety represents the site for N-linked glycosylation, and where X corresponds to any amino acid. O-linked glycosylation sites include at least one serine or threonine residue. The number of O-linked glycosylation sites is known in the art and described in the literature. See, e.g., Ten Hagen et al. (1999) J Biol. Chem. 274 (39): 27867-74; Hanisch et al. (2001) Glycobiology 11: 731-740; and Ten Hagen et al. (2003) Glycobiology 13: 1R-16R.

[00109] In all examples, the polypeptide variant is hyperglycosylated, eg, the polypeptide variant contains (1) a carbohydrate moiety covalently linked to a non-native glycosylation site, and / or (2) a carbohydrate moiety covalently linked to a native glycosylation site. In many examples, a known protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide contains a carbohydrate moiety covalently linked to a native glycosylation site; and a carbohydrate moiety covalently linked to a non-native glycosylation site. In some examples, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant forms O-linked glycosylation. In other examples, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant forms N-linked glycosylation. In other examples, a known protease-resistant or protease-resistant, hyperglycosylated variant of the polypeptide form both O-linked and N-linked glycosylation.

[00110] In some examples, a known protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide contains one, two, three, four or five carbohydrate moieties, each of which is linked to different glycosylation sites. In other examples, a known protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide is glycosylated at a non-native glycosylation site. In some of these examples, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is hyperglycosylated at one non-native glycosylation site. In other examples, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is glycosylated at more than one non-native glycosylation site, e.g., a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is glycosylated in two, three or four glycosylation sites.

[00111] In other examples, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is glycosylated at a non-native glycosylation site. In some of these examples, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is glycosylated at a single native glycosylation site. In other examples, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is hyperglycosylated at more than one native glycosylation site, e.g., a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is glycosylated at two, three or three glycosylation.

[00112] In other examples, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is glycosylated at both native glycosylation site (s) and non-native glycosylation site (s).

[00113] A known protease-resistant or protease-resistant, hyperglycosylated variant of the polypeptide may contain at least one additional carbohydrate moiety not found in the parent therapeutic protein, each being synthesized in a eukaryotic cell capable of N- and / or O -related protein glycosylation. So, for example, in comparison with the parent therapeutic protein, the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant may contain at least one, at least two, at least three, or at least four or more additional carbohydrate parts. For example, where the parent therapeutic protein has one covalently linked carbohydrate moiety, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant may have two, three, four or more covalently linked carbohydrate moieties. In some examples, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant does not have a carbohydrate moiety covalently linked to a non-native glycosylation site; and has instead, at least one, at least two, at least three, or at least four or more additional carbohydrate moieties attached to native glycosylation sites. In other examples, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant does not have a carbohydrate moiety covalently linked to a native glycosylation site; and instead contains at least two, at least three, or at least four or more carbohydrate moieties attached to non-native glycosylation sites.

Glycosylated Type 1 Interferons.

[00114] The claimed synthetic type 1 interferon receptor polypeptide agonist may include a consensus or hybrid amino acid sequence of a type 1 interferon receptor polypeptide agonist that contains one or more non-native glycosylation sites. So, for example, the claimed synthetic interferon 1 receptor polypeptide agonist can have an amino acid sequence that contains one or more glycosylation sites not found in a naturally occurring type 1 interferon receptor polypeptide agonist, e.g. not found in any known naturally occurring natural IFN-α, IFN-β, or IFN-ω). As used herein, the term "non-native glycosylation site" is defined as a glycosylation site located at the amino acid sequence of a synthetic type 1 interferon receptor polypeptide agonist, such that the glycosylation site / position does not contain a homologous glycosylation / position site that is present in the amino acid sequence of a natural polypeptide agonist type 1 interferon receptor.

[00115] Alternatively, the claimed synthetic interferon 1 receptor polypeptide agonist may have a consensus or hybrid amino acid sequence of an interferon 1 receptor polypeptide agonist that contains one or more natural or native glycosylation sites. As used herein, the term "native glycosylation site" is defined as a glycosylation site located at a position in the amino acid sequence of a synthetic type 1 interferon receptor polypeptide agonist, such that the glycosylation site / position contains a homologous glycosylation site / position that is present at least in one amino acid sequence of a natural type 1 interferon receptor polypeptide agonist.

[00116] As used herein, the term “synthetic type 1 interferon receptor polypeptide agonist” is defined as any consensus or hybrid type 1 interferon receptor polypeptide agonist that contains one or more glycosylation sites. Thus, a “synthetic type 1 interferon receptor polypeptide agonist" refers to any consensus or hybrid type 1 interferon receptor polypeptide agonist that contains one or more glycosylation sites, including any type 1 interferon receptor consensus or hybrid polypeptide agonist that contains one or more native sites glycosylation and / or one or more non-native glycosylation sites.

[00117] A “type 1 interferon receptor polypeptide parent agonist” is a type 1 interferon receptor polypeptide agonist that is used as a reference for comparison. In some examples, the claimed synthetic type 1 interferon receptor polypeptide agonist contains at least one additional glycosylation site not found in the parent type 1 interferon receptor polypeptide agonist. For example, in some instances, the parent polypeptide receptor agonist type 1 interferon consensus is ^{IFN-α Infergen ® (InterMune,} Inc., Brisbane, Calif.). As shown in Figure 25, a synthetic polypeptide claimed interferon receptor agonist, Type 1 comprises one or more glycosylation sites not found in the parent consensus IFN-α Infergen ^®.

[00118] The claimed synthetic type 1 interferon receptor polypeptide agonist has a length of from about 150 amino acids to about 200 amino acids, eg, from about 150 amino acids to about 155 amino acids, from about 155 amino acids to about 160 amino acids, from about 160 amino acids to about 165 amino acids, from about 165 amino acids to about 170 amino acids, from about 170 amino acids to about 175 amino acids, from about 175 amino acids to about 180 amino acids, from about 180 amino acids to about 185 amino acids, from about 185 amino acids to about 190 amino acids, from about 190 aminok slot to about 195 amino acids, or about 195 amino acids to about 200 amino acids.

[00119] In some examples, the amino acid sequence of a naturally occurring type 1 interferon receptor polypeptide agonist is altered by the inclusion of at least one native glycosylation site. As one non-limiting example follows, where a naturally occurring type 1 interferon receptor polypeptide agonist contains the amino acid sequence KDSS, the KDSS sequence is replaced by KNSS. In another non-limiting example, where a naturally occurring type 1 interferon receptor polypeptide agonist contains the WDET amino acid sequence, the WDET sequence is replaced by WNET. In the following non-limiting example, where a naturally occurring type 1 interferon receptor polypeptide agonist contains the amino acid sequence VEET, the VEET sequence is replaced by VTET. In another non-limiting example, where a naturally occurring type 1 interferon receptor polypeptide agonist contains the amino acid sequence VEET, the VEET sequence is replaced by VNET.

[00120] In many examples, the claimed synthetic type I interferon receptor polypeptide agonist is glycosylated. In some examples, the claimed synthetic type I interferon receptor polypeptide agonist forms O-linked glycosylation. In other examples, the claimed synthetic type I interferon receptor polypeptide agonist forms N-linked glycosylation. In other examples, the claimed synthetic type 1 interferon receptor polypeptide agonist forms both O-linked and N-linked glycosylation.

[00121] In some examples, the claimed synthetic type I interferon receptor polypeptide agonist is glycosylated at a non-native glycosylation site. In some of these examples, the claimed synthetic type I interferon receptor polypeptide agonist is glycosylated at a single non-native glycosylation site. In other examples, the claimed synthetic type 1 interferon receptor polypeptide agonist is glycosylated at more than one non-native glycosylation site, e.g., the claimed synthetic type 1 interferon receptor polypeptide agonist is glycosylated at two, three, or four non-native glycosylation sites.

[00122] In other examples, the claimed synthetic type I interferon receptor polypeptide agonist is glycosylated at a native glycosylation site. In some of these examples, the claimed synthetic type I interferon receptor polypeptide agonist is glycosylated at a single native glycosylation site. In other examples, the claimed synthetic type 1 interferon receptor polypeptide agonist is glycosylated at more than one native glycosylation site, e.g., the claimed synthetic type 1 interferon receptor polypeptide agonist is glycosylated at two, three, or four native glycosylation sites.

[00123] The claimed synthetic type 1 interferon receptor polypeptide agonist is glycosylated at both the native glycosylation site (s) and the non-native glycosylation site (s).

[00124] Whether the subject synthetic type I interferon receptor polypeptide agonist forms N-linked and / or O-linked glycosylation is readily determined using standard methods. See, e.g., "Techniques in Glycobiology" R. Townsend and A. Hotchkiss, eds. (1997) Marcel Dekker; and "Glycoanalysis Protocols (Methods in Molecular Biology, Vol. 76)" E. Hounsell, ed. (1998) Humana Press. The change in the electrophoretic mobility of a protein before and after treatment with chemical or enzymatic deglycosylation (e.g., using endoglycosidases and / or exoglycosidases) is used by standard methods to determine the protein glycosylation status. Enzymatic deglycosylation can be carried out using various enzymes, including, but not limited to, peptide-N4- (M-acetyl-β-D-glucosaminyl) asparagine amidase (PNGase F); endoglycosidase F1, endoglycosidase F2, endoglycosidase F3, α (2 → 3, 6, 8, 9) neuraminidase, etc. For example, a protein is analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), both pretreated with PNGase F and untreated with PNGaseF. A significant reduction in band width and a change in transition position after processing with PNGaseF is considered an assessment of N-linked glycosylation. The carbohydrate content of a glycosylated protein can also be determined by the lectin method of protein stains (e.g., proteins separated by SDS-PAGE and transferred to a substrate such as a nylon membrane). Lectins, carbohydrate-related proteins from various plant tissues, have both high affinity and narrow specificity for a wide range of specific carbohydrate epitopes found in glycoprotein glycans. Cummings (1994) Methods in Enzymol. 230: 66-86. Lectins can be specifically labeled (each directly or indirectly), which allows the determination of the binding of lectins to carbohydrates of glycosylated proteins. For example, when conjugated to biotin or digoxigenin, lectin bound to a glycosylated protein can be easily identified on membrane blots through the utilization reaction of avidin or anti-digoxigenin antibodies conjugated to an enzyme such as alkaline phosphatase, β-galactosidase, luciferase, or peroxidase horseradish, to highlight the detected product. Testing using a lectin panel with a well-defined specificity provides a significant amount of information about glycoprotein carbohydrate complement.

Consensus polypeptide agonists of type 1 interferon receptors with non-native glycosylation sites.

[00125) In some examples, the subject synthetic type 1 interferon receptor polypeptide agonist comprises a consensus amino acid sequence and at least one non-native glycosylation site. In other examples, the subject synthetic type 1 interferon receptor polypeptide agonist comprises a consensus amino acid sequence and at least one native glycosylation site.

[00126] A consensus sequence is obtained by joining three or more amino acid sequences and establishing amino acids that are common to at least two sequences. In some examples, the synthetic Type 1 interferon receptor polypeptide agonist contains a consensus sequence derived from the determining consensus sequence of natural human IFN-α 2b, natural human IFN-α14, and natural human IFN-β1. In other examples, the synthetic type 1 interferon receptor polypeptide agonist contains a consensus sequence derived from the determining consensus sequence of natural human IFN-α2b, natural human IFN-α l4, and natural human IFN-ω1. In other examples, a synthetic type 1 interferon receptor polypeptide agonist comprises a consensus sequence derived from the determining consensus sequence of natural human IFN-α2b, natural human IFN-β1, and natural human IFN-ω1. In other examples, a synthetic type 1 interferon receptor polypeptide agonist comprises a consensus sequence derived from the determining consensus sequence of natural human IFN-α14, natural human IFN-β1, and natural human IFN-ω1. In other examples, the synthetic type 1 interferon receptor polypeptide agonist contains a consensus sequence derived from the determining consensus sequence of natural human IFN-α2b, natural human IFN-α14, natural human IFN-β1, and natural human IFN-ω1. Other examples provide further comparative analysis, including a comparison of the amino acid sequence of consensus IFN-α. Infergen ^® .

[00127] In some of these examples, the subject synthetic type 1 interferon receptor polypeptide agonist comprises a consensus sequence containing one or more glycosylation sites derived from one or more amino acid sequences of a parent type 1 interferon receptor polypeptide agonist used to obtain a consensus sequence. In further examples, the consensus sequence is further modified to include at least one non-native glycosylation site.

[00128] In one example, the subject synthetic type 1 interferon receptor polypeptide agonist comprises the amino acid sequence representing the majority sequence depicted in FIG. 24 (SEQ ID NO: **), further modified to include at least one non-native glycosylation site.

[00129] In another example, the subject synthetic type 1 interferon receptor polypeptide agonist comprises the amino acid sequence representing the majority sequence depicted in FIG. 24 (SEQ ID NO: **), further modified to include at least one the glycosylation site of the VTET IFN-α2b glycosylation site, the KNSS IFN-α14 glycosylation site, the WNET IFN-β1 glycosylation site, and the WNMT IFN-ω1 glycosylation site. In other examples, the majority sequence is further modified to include one or more non-native glycosylation sites.

[00130] In other examples, the subject synthetic type 1 interferon receptor polypeptide agonist is derived from a consensus sequence that does not contain a glycosylation site derived from the parent type 1 interferon receptor polypeptide agonist. In these examples, the consensus sequence is further modified to include at least one non-native glycosylation site to produce the synthetic type 1 interferon receptor synthetic polypeptide of interest. For example, in some examples in which the consensus sequence includes KDSS, the KDSS sequence is modified to KNSS or KNST. According to another non-limiting example, where the consensus sequence includes WDET, the WDET sequence is modified to WNET or WNES. According to another non-limiting example, where the consensus sequence includes VEET, the VEET sequence is modified to VTET, VNES, or VNET.

[00131] In specific examples, the subject synthetic type 1 interferon receptor polypeptide agonist comprises an amino acid sequence termed "majoritarian" in Figure 24, and further comprises one or more of the following modifications: KDSS modified to KNST; WDET modified to WNES; VEET is modified to VNES or VNET.

[00132] In some specific examples, the subject synthetic type 1 interferon receptor polypeptide agonist contains an amino acid sequence as indicated in any of SEQ ID NOs: * - *, as shown in Figure 25.

[00133] In one example, the subject synthetic type 1 interferon receptor polypeptide agonist comprises an amino acid sequence corresponding to the majority sequence depicted in FIG. 28 (SEQ ID NO: **), which is further modified to include at least one non-native glycosylation site. In some examples, the subject interferon type 1 receptor polypeptide agonist contains an amino acid sequence as indicated in any of SEQ ID NOs: * - *, as shown in Figure 28.

[00134] In one example, the subject synthetic type 1 interferon receptor polypeptide agonist comprises an amino acid sequence corresponding to the majority sequence shown in FIG. 29 (SEQ ID NO: *), further modified to include at least one non-native glycosylation site . In some examples, a subject type 1 interferon receptor polypeptide agonist comprises an amino acid sequence as indicated in any of SEQ ID NOs: * - *, as shown in Figure 29.

Hybrid interferon type 1 receptor polypeptide agonists with non-native glycosylation sites.

[00135] In some examples, the subject synthetic type 1 interferon receptor polypeptide agonist comprises a hybrid type 1 interferon receptor polypeptide agonist with one or more glycosylation sites. In other examples, the subject synthetic type 1 interferon receptor polypeptide agonist comprises a hybrid type 1 interferon receptor polypeptide agonist with one or more glycosylation sites not found in any naturally occurring type 1 interferon receptor polypeptide agonist. As used herein, a “type 1 interferon receptor hybrid polypeptide agonist” is a polypeptide comprising an amino acid sequence containing discrete sub sequences corresponding in identity and number of amino acids to sub sequences of other naturally occurring type 1 interferon receptor polypeptide agonists, where the amino acid sequence of the synthetic polypeptide under consideration agonist differs from that of any natural polypeptide agonist of the interferon receptor 1 t ipa. In some examples, discrete sub-sequences are selected from IFN-α2b, IFN-α14, IFN-β1 and IFN-ω, and the amino acid sequence of a polypeptide agonist is different from the amino acid sequence of a naturally occurring IFN-α2b, IFN-α14, IFN-α2b, IFN-α2b receptor polypeptide agonist, IFN-β1, and IFN-ω.

[00136] In other examples, discrete sub-sequences may be selected from IFN-α2b, IFN-α14, IFN-β1, consensus IFN-α Infergen ^® and IFN-ω, and the amino acid sequence of a polypeptide agonist differs from every amino acid sequences of polypeptide agonist interferon receptor type 1 IFN-α2b, IFN-α14, IFN-β1, consensus IFN-α Infergen ^® and IFN-ω, respectively.

[00137] In some of these examples, the synthetic type 1 interferon receptor polypeptide agonist in question is a hybrid type 1 interferon receptor polypeptide agonist whose amino acid sequence contains one or more glycosylation sites derived from one or more amino acid sequences of the parent type 1 interferon receptor polypeptide agonist, used to obtain a hybrid sequence. In further examples, the hybrid sequence is further modified to include at least one additional non-native glycosylation site (in addition to any non-native glycosylation site derived from the amino acid sequence of the parent type 1 interferon receptor polypeptide agonist).

[00138] It should be understood that synthetic type 1 interferon receptor polypeptide agonists of the claimed invention include type 1 interferon hybrid polypeptide agonists formed by substituting one or more amino acid residues in the amino acid sequence of the parent IFN-α with the amino acid residue or residues that form the native glycosylation site in the homologous position of the amino acid sequence in another parent IFN-α.

[00139] In one non-limiting example, the subject synthetic type 1 interferon receptor polypeptide agonist is a hybrid type 1 interferon receptor polypeptide agonist having a hybrid sequence formed by substituting KNSS with native KDSS residues in the interferon alpha-2a sequence or in the interferon alpha-2b sequence . These synthetic type 1 receptor polypeptide agonists are referred to herein as IFN-α2a (D99N) and IFN-α2b (D99N), respectively, where the numbering of the amino acid sequence is as shown in Figure 24.

[00140] In another non-limiting example, the subject synthetic type 1 interferon receptor polypeptide agonist is a hybrid type 1 interferon receptor polypeptide agonist comprising a hybrid sequence formed by substituting WNET for native WDET residues in the interferon alpha-2a sequence or in the interferon alpha-2b sequence . These synthetic type 1 receptor polypeptide agonists are here referred to as IFN-α2a (D105N) and IFN-α2b (D105N), respectively, where the numbering of the amino acid sequence is as shown in Figure 24.

[00141] In another non-limiting example, the subject synthetic type 1 interferon receptor polypeptide agonist is a hybrid type 1 interferon receptor polypeptide agonist comprising a hybrid sequence formed by substituting KNSS and WNET for native KDSS and WDET residues, respectively, in the interferon alpha-2a sequence or in the sequence of interferon alpha-2b. These synthetic type 1 receptor polypeptide agonists are referred to herein as IFN-α2a (D99N, D105N) and IFN-α2b (D99N, D105N), respectively, where the amino acid sequence numbering is as shown in Figure 24.

[00142] In other examples, the subject synthetic type 1 interferon receptor polypeptide agonist is derived from a hybrid sequence that does not contain any glycosylation sites derived from the amino acid sequence of the parent type 1 interferon receptor polypeptide agonist. In these examples, the hybrid sequence is then further modified by the inclusion of at least one non-native glycosylation site to produce the synthetic type 1 interferon receptor synthetic polypeptide of interest. For example, in some examples, where the hybrid sequence includes KDSS, the KDSS sequence is modified to KNSS. In another non-limiting example, where the hybrid sequence includes WDET, the WDET sequence is modified to WNET. In another non-limiting example, where the hybrid sequence includes VEET, the VEET sequence is modified to VTET or VNET.

[00143] In some examples, the subject synthetic type 1 interferon receptor polypeptide agonist comprises, in order from the N-terminus to the C-terminus, from about 2 to about 90, eg, from about 2 to about 5, from about 5 to about 7, from about 7 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 30, from about 30 to about 35, from about 35 to about 40 , from about 40 to about 45, from about 45 to about 50, from about 50 to about 55, from about 55 to about 60, from about 60 to about 65, from about 65 to about 70, from about 75 to about 80, from about 80 to about 85, or from okol about 85 to about 90 neighboring amino acids of the first type 1 interferon receptor polypeptide agonist selected from natural human IFN-α2b (SEQ ID NO: *), natural human IFN-α14 (SEQ ID NO: *), natural human IFN-β1 (SEQ ID NO: *), and natural human IFN-ω1 (SEQ ID NO: *); and from about 2 to about 90, for example, from about 2 to about 5, from about 5 to about 7, from about 7 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 30, from about 30 to about 35, from about 35 to about 40, from about 40 to about 45, from about 45 to about 50, from about 50 to about 55, from about 55 to about 60, from about 60 to about 65, from about 65 to about 70, from about 75 to about 80, from about 80 to about 85, or from about 85 to about 90 neighboring amino acids of a second type 1 interferon receptor polypeptide agonist selected from natural human IFN-α2b, people ovine IFN-αl4, human IFN-β1 and human IFN-β1), where the first and second polypeptide agonists of type 1 interferon receptor are different.

[00144] In some examples, the subject hybrid synthetic type 1 interferon receptor polypeptide agonist further comprises from about 2 to about 90, eg, from about 2 to about 5, from about 5 to about 7, from about 7 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 30, from about 30 to about 35, from about 35 to about 40, from about 40 to about 45, from about 45 to about 50, from about 50 to about 55, from about 55 to about 60, from about 60 to about 65, from about 65 to about 70, from about 75 to about 80, from about 80 to about 85, or from about 85 to about 90 adjacent amino acids of a third type 1 interferon receptor polypeptide agonist selected from natural human IFN-α2b, human IFN-α14, human IFN-β1 and human IFN-ω1, where the third type 1 interferon receptor polypeptide agonist is different from the first and second interferon receptor polypeptide agonists 1 type.

[00145] In still other examples, the subject type 1 interferon receptor hybrid polypeptide agonist further comprises from about 2 to about 90, eg, from about 2 to about 5, from about 5 to about 7, from about 7 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 30, from about 30 to about 35, from about 35 to about 40, from about 40 to about 45, from about 45 to about 50, from about 50 to about 55, from about 55 to about 60, from about 60 to about 65, from about 65 to about 70, from about 75 to about 80, from about 80 to about 85, or from about 85 to about 90 neighboring amines acids of the fourth type 1 interferon receptor polypeptide agonist selected from natural human IFN-α2b, human IFN-α14, human IFN-β1, and human IFN-ω1, where the fourth type 1 interferon receptor polypeptide agonist is different from the first, second and third polypeptide agonists type 1 interferon receptor.

[00146] In separate examples, any of the synthetic examples of a type 1 interferon receptor synthetic hybrid polypeptide agonist, contains from about 4 to about 90, eg, from about 4 to about 7, from about 7 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 30, from about 30 to about 35, from about 35 to about 40, from about 40 to about 45, from about 45 to about 50, from about 50 to about 55, from about 55 to about 60, from about 60 to about 65, from about 65 to about 70, from about 75 to about 80, from about 80 to about 85, or from about 85 to eye about 90 contiguous amino acids of the polypeptide segment of human IFN-α14, which comprises at least the amino acid sequence KNSS native human IFN-α14.

[00147] In separate examples, any of the synthetic examples of a type 1 interferon receptor synthetic hybrid polypeptide agonist, contains from about 4 to about 90, eg, from about 4 to about 7, from about 7 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 30, from about 30 to about 35, from about 35 to about 40, from about 40 to about 45, from about 45 to about 50, from about 50 to about 55, from about 55 to about 60, from about 60 to about 65, from about 65 to about 70, from about 75 to about 80, from about 80 to about 85, or from about 85 to eye about 90 contiguous amino acids of the polypeptide segment of human IFN-β1, which comprises at least the amino acid sequence of native human WNET IFN IFN-β1.

[00148] In certain examples, any of the synthetic examples of a type 1 interferon receptor synthetic hybrid polypeptide agonist, contains from about 4 to about 90, eg, from about 4 to about 7, from about 7 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 30, from about 30 to about 35, from about 35 to about 40, from about 40 to about 45, from about 45 to about 50, from about 50 to about 55, from about 55 to about 60, from about 60 to about 65, from about 65 to about 70, from about 75 to about 80, from about 80 to about 85, or from about 85 to about90 neighboring amino acids of the human IFN-ω1 polypeptide segment, which includes at least the WNMT amino acid sequence of natural human IFN-β1.

[00149] In separate examples, any of the synthetic examples of the type 1 interferon receptor synthetic hybrid polypeptide agonist of the above contains from about 4 to about 90, eg, from about 4 to about 7, from about 7 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 30, from about 30 to about 35, from about 35 to about 40, from about 40 to about 45, from about 45 to about 50 , from about 50 to about 55, from about 55 to about 60, from about 60 to about 65, from about 65 to about 70, from about 75 to about 80, from about 80 to about 85, or from about 85 to about 90 from the contiguous amino acids of the human IFN-α2b polypeptide segment, which includes at least the amino acid sequence VTET of natural human IFN-α2b.

Functional signs

[00150] The synthetic polypeptide in question is a type 1 interferon receptor polypeptide agonist, for example, the synthetic type 1 interferon receptor synthetic polypeptide agonist binds to and causes signal transduction through the type 1 interferon receptor. Whether the subject synthetic type I interferon receptor polypeptide agonist functions as an type I interferon receptor agonist can be quickly determined using a known method. Such methods include methods based on the use of in vitro cells to detect the activation of genes that respond to interferon (eg, the use of a reporter gene functionally linked to a promoter containing one or more eterferon active elements); and the like. Such methods also include KJRA methods for determining the activation function of an interferon type 1 receptor, as described below in the Diagnostic Use section.

[00151] In some examples, the claimed synthetic type 1 interferon receptor polypeptide agonist exhibits one or more of the following: an antiproliferative effect, an antiviral effect, and an antifibrotic effect. Whether the claimed synthetic type I interferon receptor polypeptide agonist exhibits an antiviral effect can be readily determined using any known method, including, for example, an in vitro method based on the use of cells to determine inhibition of viral replication. See, e.g., Patick et al. (1999) Antimicrobial Agents and Chemotherapy 43: 2444-2450. Whether the claimed synthetic type I interferon receptor polypeptide agonist exhibits an antiproliferative effect can be readily determined using any known method, including, for example, an in vitro method based on the use of cells to determine inhibition of proliferation.

[00152] The subject synthetic type 1 interferon receptor polypeptide agonist exhibits one or more of the following properties: increased serum half-life; reduced in vivo immunogenicity; increased functional half-life in vivo; increased stability; reduced digestion by the conditions of the gastrointestinal tract; and improved solubility in water.

[00153] In some examples, the synthetic type 1 interferon receptor polypeptide agonist in question exhibits increased serum half-life compared to the naturally occurring type 1 interferon receptor polypeptide agonist or compared to the parent type 1 interferon receptor polypeptide agonist. The term "serum half-life" is used here interchangeably with the terms "plasma half-life" and "circulation half-life". In some examples, the subject synthetic type 1 interferon receptor polypeptide agonist has a serum half-life that is at least about 10%, at least about 15%, at least about 20%, at least , about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, p at least about 80%, at least about 90%, at least about 100% (or twice), at least about 2.5 times, at least about three times, at least about 3.5-fold, at least about four-fold, at least about 4.5-fold, or at least about five-fold longer serum half-life of a natural polypeptide receptor agonist interferon type 1 or the parent polypeptide agonist of an interferon type 1 receptor that does not contain a non-native glycosylation site. In some examples, the subject synthetic type 1 interferon receptor polypeptide agonist has a serum half-life that is at least about 10%, at least about 15%, at least about 20%, at least , about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, p at least about 80%, at least about 90%, at least about 100% (or twice), at least about 2.5 times, at least about three times, at least about 3.5-fold, at least about four-fold, at least about 4.5-fold, or at least about five-fold longer serum half-life of a natural interferon receptor polypeptide agonist Type 1 or type 1 interferon receptor polypeptide agonist that has the same amino acid sequence as the natural receptor agonist ora type 1 interferon.

[00154] The half-life of serum of the synthetic type 1 interferon receptor polypeptide agonist in question is easily determined using well-known methods. For example, the subject synthetic type 1 interferon receptor polypeptide agonist is definitely labeled and administered to an individual (e.g., an experimental animal not belonging to the human genus or to a human), and, at different times of the next administration of the agonist, a blood sample is taken and a blood sample is determined the amount of a specifically labeled synthetic type 1 interferon receptor polypeptide agonist.

[00155] In some examples, the subject synthetic type 1 interferon receptor polypeptide agonist exhibits increased resistance to digestion by the conditions of the gastrointestinal tract compared to the naturally occurring type 1 interferon receptor polypeptide agonist or compared to the parent type interferon receptor polypeptide agonist. In some examples, the subject synthetic type 1 interferon receptor polypeptide agonist exhibits at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or for less at least 90% or more, a decrease in digestion in the gastrointestinal tract compared with the digestion level of a natural type 1 interferon receptor polypeptide agonist or a parent type 1 interferon receptor polypeptide agonist that does not contain non-native glycosylation sites.

[00156] Whether the subject synthetic type I interferon receptor polypeptide agonist exhibits increased resistance to digestion by the conditions of the gastrointestinal tract can be readily determined using known methods. For example, the synthetic type 1 interferon receptor polypeptide agonist in question is contacted in vitro with digestive enzymes located in the gastrointestinal tract, and the effect of the enzymes on the structural and functional integrity of the synthetic type 1 interferon receptor polypeptide agonist is determined. A method can be used to determine resistance to digestion by gastrointestinal conditions in vivo.

[00157] A known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant suitable for use here is a protease-resistant or protease-resistant, hyperglycosylated variant of a parent therapeutic protein, where the parent therapeutic protein is any therapeutic protein that is effective for treating a disease or conditions in a patient when administered to a patient. A list of typical therapeutic proteins is provided below. A known protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide is effective for treating the same disease or condition in a patient as the corresponding parent therapeutic protein.

Variants of a Protease Resistant or Hyper-Glycosylated Resistant Polypeptide. Resistant to Protease.

[00158] A known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is a protease-resistant or protease-resistant, hyperglycosylated variant of a therapeutic protein, and, in many examples, is presented in the first dosage form. The first dosage form may contain the first number of moles of a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant in an oral pharmaceutical composition. The parent therapeutic protein in many examples may be formulated as an immediate release suitable for subcutaneous bolus administration, i.e. the second standard form, where the first number of moles in the first standard form is greater than the second number of moles of the therapeutic protein in the second standard form. For example, the first number of moles may be at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%, or at least about 75%, or at least about 100%, or at least about three times, or at least about four times, or at least about five times, or at least about six times h, or at least about seven times, or at least about eight times, or at least about nine times, or at least about ten times, or more than second number of moles.

[00159] In many examples, after orally administering the first unit dose to a patient, the time required to release the first mole of a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is no more than the period of time that elapses between doses of the parent therapeutic protein when administration in a second standard form by subcutaneous bolus injection at a selected dosage frequency in a therapeutic regimen that is proven to be effective m for treating a disease or condition of the patient. So, for example, the time required to release the first mole of a known protease-resistant or protease-resistant, hyperglycosylated variant of the polypeptide after oral administration of the first standard form can be at least about 5%, at least about 10 %, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least , about 40%, at least about 45%, or at least about 50%, or more, me earlier than the time interval between the doses of the parental therapeutic in the second standard form when administered by subcutaneous bolus injection at a selected dosing frequency. In some examples, the first unit form is an immediate release formulation suitable for oral delivery.

[00160] A known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant can be administered orally more frequently than the corresponding parent polypeptide administered by subcutaneous bolus injection. For example, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant can be administered orally, at least two times more often, at least 2 1/3 times more often, at least 2.5 times more often, at least at least three times more often, at least 3.5 times more often, or at least four times more often, or at least five times more often, or at least six times more often, or more often than the corresponding parent a polypeptide administered by subcutaneous bolus injection. So, for example, if the parent therapeutic polypeptide is administered once a week, then the corresponding variant of the protease-resistant or protease-resistant, hyperglycosylated polypeptide can be administered twice a week, three times a week, once a day, twice a day, three times a day , or more than three times daily.

[00161] In one non-limiting example, the parent therapeutic protein is IFN-γ1b, and IFN-γ1b is administered in unit dosage form suitable for subcutaneous administration at a dose of 1 × 10 ⁶ International Units (IU) / m ² (or 50 μg / m ² or 3.0 × 10 ^-9 mol / m ² ) subcutaneously three times a week, with a total weekly dose of 150 μg / m ² (or 3 × 10 ⁶ ME / m ² or 9.0 × 10 ^-9 mol / m ² ). The desired hyperglycosylated, protease-resistant variant of IFN-γ1b is in unit dosage form suitable for oral delivery; the well-known hyperglycosylated, protease-resistant variant of IFN-γ1b is administered orally, and more often than 3 times a week (e.g. 4 times a week, 5 times a week, 6 times a week, once a day, twice a day, or three times a day); and the total weekly dose of the hyperglycosylated, protease-resistant IFN-γ1b variant that is administered is greater than or equivalent to 9.0 × 10 ^-9 mol / m, e.g., the total weekly dose is from about 9.0 × 10 ^-9 mol / m to about 1.0 × 10 ^-8 mol / m, from about 1.0 × 10 ^-8 mol / m ² to about 2.5 × 10 ^-8 mol / m ² , from about 2.5 × 10 ^-8 mol / m ² to about 5.0 × 10 ^-8 mol / m ² , or from about 5.0 × 10 ^-8 mol / m ² to about 7.5 × 10 ^-8 mol / m, or from about 7.5 × 10 ⁻⁸ mol / m to about 1.0 × 10 ⁻⁷ mol / m, or from about 1.0 × 10 ⁻⁷ mol / m ² to about 1.0 × 10 ⁻⁶ mol / m ² .

[00162] In another aspect, the total weekly dose of the hyperglycosylated, protease-resistant IFN-γ1b variant that is administered is greater than or equivalent to 500 μg, eg, from about 500 μg to about 750 μg, from about 750 μg to about 1000 mcg, from about 1000 mcg to about 1500 mcg, or from about 1500 mcg to about 2000 mcg.

[00163] A known protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide exhibits increased protease resistance compared to the corresponding parent polypeptide. In some examples, a known protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide is resistant to serum proteases, which is at least about 10%, at least about 15%, at least about 20 %, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least by about 50%, at least about 55%, at least about 60%, at least about 65%, at least an eye 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100% (or twice), at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least , about 5 times (at least five times), at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times, at least about 3 0 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least at least about 90 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, or at least about 1000 times, or more higher than creep resistance exact proteases corresponding parent therapeutic protein, in human blood, human serum, or in in vitro mixture containing one or more proteases.

[00164] In some examples, a known protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide exhibits at least about 10%, at least about 15%, at least about 20%, at least by about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least e, about 80%, at least about 90%, at least about 100% (or twice), at least about 2.5 times, at least about 3 times at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times (at least five times), at least at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times at least about 30 times, at least about 40 times, at least at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 600 times, at least , about 700 times, at least about 800 times, at least about 900 times, or at least about 1000 times, or more, increased resistance to the action of one or more α-chymotrypsin, Arg endoproteinase -C, Asp-N endoproteinases, Glu-C endoproteinases, Lys-C endoproteinases, and trypsin, compared to the corresponding parent therapeutic protein.

[00165] In some examples, the magnitude of the increase in protaz resistance of the polypeptide variant is determined by comparing the half-life of the polypeptide variant to the half-life of the corresponding parent therapeutic protein in human blood or human serum in vitro, or in an in vitro composition containing one or more serum proteases. For example, resistance to protease cleavage can be determined by detecting the level of biological activity of the protease-resistant polypeptide variant following the separate interaction of the polypeptide variant and the corresponding parent therapeutic protein with a mixture of proteases, with human serum, or with human blood; and comparing the activity of the selected polypeptide with that of the corresponding parent therapeutic protein. If the biological activity of a polypeptide variant is higher than that of the corresponding parent therapeutic protein, following incubation with human blood, human serum, or one or more protases, then the polypeptide variant has increased resistance to the action of the protease compared to the parent therapeutic protein.

[00166] The following is a non-limiting example of a method for determining in vitro protease resistance. In separate containers, the polypeptide variant and the corresponding parent therapeutic protein are added to the protease mixture containing 1.5 pg of each α-chymotrypsin, carboxypeptidase, Arg-C endoproteinase, Asp-N endoproteinase, Glu-C endoproteinase, Lys- endoproteinase C, and trypsin, forming a reaction mixture; and keep the reaction mixture at 25 ° C for 30 minutes. At the end of the 30-minute reaction period, a substance is added that inhibits protease activity; and determine the biological activity of the variant polypeptide and the corresponding parent therapeutic protein. The following is another non-limiting example of a method for determining in vitro protease resistance. In separate containers, a polypeptide variant and the corresponding parent therapeutic protein are added to each lysate of human blood, or human serum, to form a reaction mixture; and maintaining the reaction mixture at 37 ° C. for a suitable period of time (eg, 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 60 minutes, etc.). Then add a substance that inhibits the activity of proteases; and determine the biological activity of the variant polypeptide and the corresponding parent therapeutic protein.

[00167] An appropriate parent therapeutic protein can be any parent therapeutic protein that is proven to be effective in treating a disease or condition in a patient when administered to the patient in an immediate release formulation by subcutaneous bolus administration of a second unit dose at an acceptable dosage frequency. In these examples, a known protease-resistant or protease-resistant, hyperglycosylated variant of the polypeptide is effective for treating the same disease or condition in a patient when administered orally to a patient in a first standard dose at an acceptable dosage frequency that is no less frequent than that for parent therapeutic protein.

[00168] In many examples, a known hyperglycosylated, protease-resistant polypeptide variant exhibits the desired pharmacological activity in a mammalian host, eg, a hyperglycosylated, protease-resistant polypeptide variant can exhibit at least about 50%, at least about 60 %, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the desired pharmacological activity of the corresponding parent therapeutic protein. By non-limiting examples, a hyperglycosylated, protease-resistant polypeptide variant may exhibit one or more of the following activities: antiproliferative activity, antiviral activity, antifibrotic activity; hematopoietic activity; angiogenic activity; enzymatic activity; growth factor activity; chemokine activity; receptor agonist activity; receptor antagonist activity; and anti-angiogenic activity; where the activity is the same as the desired corresponding parent therapeutic protein.

[00169] A known hyperglycosylated, protease-resistant variant of the polypeptide exhibits an increased serum half-life or increased AUC compared with the parent therapeutic protein used under similar conditions.

[00170] In some examples, the known hyperglycosylated, protease-resistant variant of the polypeptide has an increased serum half-life compared to the corresponding parent polypeptide. The term “serum half-life” is used here interchangeably with the terms “plasma half-life” and “circulation half-life”. In some examples, the hyperglycosylated, protease-resistant polypeptide variant has a serum half-life that is at least about 10%, at least about 15%, at least about 20%, at least by about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least at least about 80%, at least about 90%, at least about 100% (or twice), at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times (at least five times), at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 600 times, at least at least about 700 times, at least about 800 times, at least about 900 times, or at least about 1000 times, or more, longer than the serum half-life of the corresponding parent therapeutically protein. In some examples, the degree of time increase in the release of the known hyperglycosylated, protease-resistant variant of the polypeptide is redistributed by comparing the time of the removal of the known hyperglycosylated, protease-resistant variant of the polypeptide with the time of the removal of the corresponding parent therapeutic protein in human blood or human serum in vivo.

[00171] In some examples, the half-life increase in the half-life of a known hyperglycosylated, protease-resistant variant of the polypeptide is determined by comparing the half-life of the known hyperglycosylated, protease-resistant variant of the polypeptide and the half-life of the corresponding parent therapeutic protein from human blood or human serum in vitro, or in an in vitro composition containing one or more serum proteases. For example, resistance to protease cleavage can be determined by detecting the level of biological activity of a known hyperglycosylated, protease-resistant variant of the polypeptide, following separate contact of the variant polypeptide and the corresponding parent therapeutic protein with a mixture of proteases, human serum, or human blood; and comparing the activity of a variant polypeptide with that of the corresponding parent therapeutic protein. If the biological activity of a polypeptide variant is higher than that of the corresponding parent therapeutic protein following incubation with human blood, human serum, or one or more protases, the polypeptide variant has an increased half-life compared to the parent therapeutic protein.

[00172] In some examples, a known hyperglycosylated, protease-resistant variant of the polypeptide has an AUC that is at least 10%, at least 15%, at least 20%, at least 25 %, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least at least 100% (or double), at least 2.5 times, at least re, 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, or at least 5 times more than the PPC of the corresponding parent therapeutic protein when used under similar conditions.

[00173] The half-life of serum or AUC of a known hyperglycosylated, protease-resistant variant of the polypeptide can be easily determined using well-known methods. For example, a known hyperglycosylated, protease-resistant variant of a polypeptide is detectably labeled and administered to an individual (e.g., non-human experimental animals or humans), and, at different times following the administration of a variant of a hyperglycosylated, protease-resistant polypeptide, selected a blood sample and determine the amount of detectably labeled hyperglycosylated, protease-resistant variant polypeptide in the blood sample.

SV Scan Methods

[00174] A glycosylated or protease-resistant or protease-resistant, hyperglycosylated polypeptide variant of the parent therapeutic protein can be determined using the RF scanning method (structural homology). Structural homology refers to the homology between the topology and the three-dimensional structure of two proteins. Quantitative methods are well known in the art for identifying structurally linked amino acid positions with a three-dimensional structure of homologous proteins. Typical methods include, but are not limited to: CATN (Class, Structure, Topology, and Superfamily Homology), which is a hierarchical classification of protein domain structures based on four different levels (Orengo et al., Structure, 5 (8): 1093-1108, 1997 ); CE (Combinatorial Optimal Path Elongation), which is a method for calculating paired structural alignments (Shindyalov et al., Protein Engineering, 11 (9): 739-747, 1998); FSSP (Classification of Layers Based on Alignment of Proteins in Spatial Structure), which is a database based on a complete comparison of all three-dimensional protein structures that are currently taken in the Protein Data Bank (PDB) (Holm et al., Science, 273: 595-602, 1996); SCOP (Structural Classification of Proteins), which presents a descriptive database based on the structural and evolutionary relationships between all proteins whose structure is known (Murzin et al., J. Mol. Biol, 247: 536-540, 1995); and VAST (Vector Homology Search Program), which compares the first established coordinates of a three-dimensional protein structure with that found in the MMDB / PDB database (Gibrat et al., Current Opinion in Structural Biology, 6: 377-385, 1995).

[00175] In one non-limiting example, IFN-α2b mutants with enhanced proteolysis resistance are generated by two-dimensional rational scanning; and the corresponding residues of members of the cytokine family that have structural homology to IFN-α2b are identified and the identified residues of other cytokines are also modified to produce cytokines with increased proteolysis resistance. See e.g. WO 04/022593. Protein Therapy

[00176] A known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is a variant of a polypeptide that exhibits a therapeutic effect in a mammalian host ("parent therapeutic protein") in treating a disease or condition in a mammalian host. A known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant treats the same disease or condition in the host as the parent therapeutic protein.

[00177] It should be noted that in the context of amino acid substitutions for the formation of protease-resistant variants of the parent therapeutic protein, the amino acid numbering used to describe the amino acid substitutions that change the protease cleavage site corresponds to the numbering of amino acids, as indicated in Figures 1-23, amino acid substitutions for the formation of protease-resistant variants of the parent therapeutic protein, the amino acid numbering used to describe the amino acid substitutions that form with The glycosylation point corresponds to the numbering of amino acids, as indicated in Figures 24-30. The corresponding amino acid positions of IFN-α depicted in, for example, Figure 1 and Figure 24, are readily detectable. For example, it will be apparent to those skilled in the art that the D99 IFN-α2b shown in Figure 24 corresponds to the D71 IFN-α2b shown in Figure 2 and corresponds to the D71 IFN-α2a shown in Figure 1. So, for example, the amino acid sequence of D99 and D105 IFN-α2b depicted in Figure 24 corresponds to the amino acid sequence, respectively, D71 and D77 IFN-α2a depicted in Figure 1 of the amino acid sequence IFN-α2b depicted in Figure 2;

the amino acid sequence R50 of IFN-α2b shown in Figure 24 corresponds to the amino acid sequence R23 of IFN-α2b shown in Figure 2; the amino acid sequence D99, D 105, and E1 34 Infergen depicted in Figure 24 corresponds to the amino acid sequence D71, D77, and D106, respectively, of the consensus IFN-α indicated in Figure 9; the amino acid positions of S99, E134, and F136 of the amino acid sequence of IFN-β1 shown in Figure 24 correspond to S74, E109, and Fill, respectively, in the amino acid sequence of IFN-β shown in Figure 3; and the positions of the amino acids E38, S40, and S99 of the amino acid sequence of IFN-γ indicated in Figure 31 correspond to E41, S43, and S102, respectively, of the amino acid sequence of IFN-γ indicated in Figure 4.

[00178] Suitable protease-resistant or protease-resistant, hyperglycosylated polypeptide variants include protease-resistant or protease-resistant, hyperglycosylated forms of any parental therapeutic protein that the mammalian host needs, including, but not limited to: interferon (eg, IFN- γ, IFN-α, IFN-β, IFN-ω; IFN-γ; as described in more detail below); insulin (e.g. Novolin, Humulin, Humalog, Lantus, Ultralente, etc.); erythropoietin (e.g. Procrit®, Eprex®, or Epogen® (epoetin-α); Aranesp® (darbepoetin-α); NeoRecormon®, Epogin® (epoetin-β); etc.); antibody (e.g. monoclonal antibody) (e.g. Rituxan® (rituk-simab); Remacade® (infliximab); Herceptin® (trastuzumab); Humira ^TM (adalimumab); Xolair® (omalizumab); Wehhag® (tositumomab); Rapaptiva ^TM (efalizumab); Erbitux ^TM (cetuximab); etc.), including the antigen-binding fragment of a monoclonal antibody; blood factor (e.g. tissue activator of plasminogen Activaz® (alteplase); NovoSeven® (recombinant human factor Vila); Vila factor; Factor VIII (e.g., Cogenate®); Factor IX; (3-globin; hemoglobin; etc. p.); colony-stimulating factor (e.g. Neupogen® (filgrastim; G-CSF); Neulastim (pegfilgrstim); granulocyte colony-stimulating factor (G-CSF), granulocyte monocyte colony-stimulating factor, macrophage colony-stimulating factor, mega-colony stimulating and .); growth hormone (e.g. somatotropin, e.g. Genot opin®, Nutropin®, Norditropin®, Sizen®, Serostim®, Humatrop®, etc .; human growth hormone; etc.); interleukin (e.g. IL-1; IL-2, including, for example, Proleukin®; IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9; etc.); growth factor (e.g. Regranex® (beclapermin; PDGF); Fiblast ® (trafermin; bFGF); Stemgen® (ancestim; stem cell factor); keratinocytic growth factor; acidic fibroblast growth factor, stem cell factor, main fibroblast growth factor, hepatocyte growth factor; etc.); soluble receptor (e.g., TNF-α-binding soluble receptor such as Enbrel® (etanercept); soluble VEGF receptor; soluble interleukin receptor; soluble y / S T cell receptor; etc.); enzyme (e.g. a-glucosidase; Cerazyme® (imiglucarase; β-glucocerebrosidase, Ceredase® (alglucerase); enzyme activator (e.g. tissue plasminogen activator); chemokine (e.g. IP-10; Mig; GroaAL-S, RANTES; MIP-1a; MIP-1β; MCP-1; PF-4; etc.);

angiogenic substance (e.g., vascular endothelial growth factor (VEGF); anti-angiogenic substance (e.g., soluble VEGF receptor); protein vaccine; neuroactive peptide such as bradykinin, cholecystokinin, gastin, sectretin, oxytocin, gonadoliberin, beta-beta , enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone releasing factor, bombesin, warfarin, dynorphin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, gorogne, angi, thyrotropin-producing, vasoactive intestinal peptide, sleep peptide, etc .; other proteins, such as thrombolytic substance, atrial natriuretic peptide, bone morphogenetic protein, thrombopoietin, relaxin, glial fibrous acid protein, follicle-stimulating hormone, human alpha-1, anti-alpha leukemia inhibiting, transforming growth factor, tissue factor, insulin-like growth factor, luteinizing hormone, follicle-stimulating hormone, macrophage activation factor, tumor necrosis factor, actor causing neutrophil chemotaxis, nerve growth factor, tissue inhibitor of metalloproteinases; vasoactive intestinal peptide, angiogenin, angiotropin, fibrin; hirudin; leukemia inhibiting factor; IL-1 receptor antagonist (e.g., Kineret® (anakinra)); etc. Also suitable for use are fusion proteins containing all or part of any of the above proteins.

[00179] As indicated above, a known protease-resistant or protease-resistant, hyperglycosylated protein variant exhibits at least one of the required pharmacological activities of the respective parent protein. Examples of suitable methods for specific therapeutic proteins include, but are not limited to, GMCSF (Eaves, A. C. and Eaves C. J., Erythropoiesis in culture. In: McCullock EA (edt) Cell culture techniques-Clinics in hematology. W In Saunders , Eastbourne, p. 371-91 (1984); Metcalf, D., International Journal of Cell Cloning 10: 116-25 (1992); Testa, NG, et al., Assays for hematopoietic growth factors. In: Balkwill FR ( edt) Cytokines A practical Approach, pp 229-44; IRL Press Oxford 1991) EPO (bioassay: Kitamura et al., J. Cell. Physiol. 140, p. 323 (1989)); Hirudin (analysis of platelet aggregation properties: Blood Coagul Fibrinolysis 7 (2): 259-61 (1996)); IFNa (antiviral test: Rubinstein et al., J. Virol. 37 (2): 755-8 (1981); antiproliferative test: Gao Y, et al. Mol Cell Biol. 19 (11): 7305-13 (1999) ; and biotest: Czamiecki et al., J. Virol. 49 p490 (1984)); GCSF (bioassay: Shirafuji et al., Exp. Hematol. 17 pi 16 (1989); proliferation of mouse cells of the NFS-60 line (Weinstein et al, Proc Nati Acad Sci 83: 5010-4 (1986)); insulin (test ³ H-glucose uptake: Steppan et al., Nature 409 (6818): 307-12 (2001)); hGH (Ba / P3-hGHR proliferative test: J din Endocrinol Metab 85 (11): 4274-9 (2000); International standard for growth hormone: Horm Res, 51 Suppi 1: 7-12 (1999)); factor X (factor X activity test: Van Wijk et al. Thromb Res 22: 681-686 (1981)); factor VII ( analysis of coagulating activity using prothrombin clotting time: Belaaouaj et al., J. Biol. Chem. 275: 27123-8 (2000); Diaz-Collier et al., Thromb Haemost 71: 339-46 (1994)).

Interferons

[00180] In some examples, the parent therapeutic protein is interferon, and the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant contains (1) a carbohydrate moiety covalently attached to at least one non-native glycosylation site not found in the parent interferon or (2) a carbohydrate moiety covalently attached to at least one native glycosylation site found but not glycosylated in parental interferon; and contains one or more mutated protease cleavage sites found in the parent therapeutic protein.

[00181] In some embodiments, the parent polypeptide is a type 1 interferon receptor polypeptide agonist. Type 1 interferon receptor polypeptide agonists include IFN-α, IFN-β, IFN-γt, and ifn-ωcd. So, for example, a protease-resistant or protease-resistant, hyperglycosylated polypeptide variant can be a protease-resistant or protease-resistant, hyperglycosylated variant of a type 1 interferon receptor polypeptide agonist, including hyperglycosylated variants of IFN-α, IFN-β, IFN-γ, and ifn-ω, in which at least one protease cleavage site found in the parent protein is missing.

[00182] In other examples, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is any protease-resistant or protease-resistant, glycosylated synthetic type 1 interferon receptor polypeptide agonist described in the Synthetic Interferon Receptor Agonist patent application Type 1 "(USSN 60/600202), published August 9, 2004, a full summary of the essence of this application is incorporated herein by reference.

[00183] In other examples, the parent polypeptide is a type II interferon receptor polypeptide agonist. Type II interferon receptor polypeptide agonists include interferon-gamma (IFN-γ). So, for example, a protease-resistant or protease-resistant, hyperglycosylated polypeptide variant can be a proteazoresistant or protease-resistant, hyperglycosylated variant of a type II interferon receptor polypeptide agonist, including hyperglycosylated IFN-γ, which does not contain at least one site protease cleavage found in the parent protein.

IFN-α

[00184] The amino acid sequence of any known IFN-α can be changed to form the synthetic type 1 interferon receptor synthetic polypeptide of interest. The term "interferon alpha", as used here, refers to a family of corresponding polypeptides that inhibit viral replication and cell proliferation and modulate the immune response.

[00185] Suitable alpha interferons include, but are not limited to, natural IFN-α (including, but not limited to, natural IFN-α 2a, IFN-α 2b, and IFN-αl4); IFN-α, as described in U.S. Patent No. 6704225; recombinant interferon alpha-2b, such as interferon Intron-A, obtained from Sobering Corporation, Kenilworth, N.J .; recombinant interferon alfa-2a, such as interferon Roferon, obtained from Hoffmann-La Roche, Nutley, N. J .; recombinant interferon alpha-2C, such as Berofor alpha 2 interferon, obtained from Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, Conn .; interferon alpha-n1, a purified mixture of natural alpha interferons, such as Sumiferon, obtained from Sumitomo, Japan or as Wellferon interferon alpha-n1 (INS), obtained from Glaxo-Wellcome Ltd., London, Great Britain; and interferon alpha-p3 mixture of natural alpha interferons manufactured by Interferon Sciences and obtained from Purdue Frederick Co., Norwalk, Conn., under the trade name Alferon; and IFN-α14.

[00186] Suitable known protease-resistant or protease-resistant, hyperglycosylated polypeptide variants include protease-resistant or protease-resistant, hyperglycosylated forms of any parent alpha interferon polypeptide. In one aspect, a known protease-resistant or protease-resistant, hyperglycosylated variant of the parent alpha interferon polypeptide comprises an amino acid sequence that is so different from the amino acid sequence of the parent polypeptide that the variant contains one or more glycosylation sites not found in the parent polypeptide; and further comprises at least one mutant protease cleavage site at the site of the native protease cleavage site found in the parent protein.

(00187] In another aspect, the parent polypeptide is IFN-α2a and the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is a [D99N] IFN-α2a glycopeptide, where [D99N] IFN-α2a glycopeptide is an IFN-α2a variant containing (a) an aspartic residue at the site of a native aspartic acid residue at amino acid position 99 in the amino acid sequence of IFN-α2a, and (b) a carbohydrate moiety covalently attached to the R group of said aspartic residue. the sequence of IFN-α2a is the same as the amino acid sequence of IFN-α2b shown in Figure 1, providing that the sequence of IFN-α2a contains a lysine residue at the arginine residue at amino acid position 50 in the sequence of IFN-α2b shown in Figure 1.

[00188] In another aspect, the parent polypeptide is IFN-α2a and the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is [D99N, D105N] IFN-α2a glycopeptide, where [D99N, D105N] IFN-α2a glycopeptide -α2a containing (a) an aspartic residue at the site of the native aspartic acid residue at each amino acid position 99 and 105 in the amino acid sequence of IFN-α2a; and (b) a carbohydrate moiety covalently attached to the R group of each said aspartic residue. It should be understood that the amino acid sequence of IFN-α2a is the same as the amino acid sequence of IFN-α2b shown in Figure 1, providing that the sequence of IFN-α2a contains a lysine residue at the arginine residue at amino acid position 50 in the sequence of IFN-α2b, depicted in Figure 1.

[00189] In another aspect, the parent polypeptide is IFN-α2b and the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is a [D99N] IFN-α2b glycopeptide, where [D99N] IFN-α2b glycopeptide is an IFN-α2b variant containing (a) an aspartic residue at the site of a native aspartic acid residue at amino acid position 99 in the amino acid sequence of IFN-α2b shown in Figure 1, and (b) a carbohydrate moiety covalently attached to the R group of said aspartic residue.

[00190] In another aspect, the parent polypeptide is IFN-α2b and the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is [D99N, D105N] IFN-α2b glycopeptide, where [D99N, D105N] IFN-α2b glycopeptide -α2b, containing (a) an aspartic residue at the site of the native aspartic acid residue at each amino acid position 99 and 105 in the amino acid sequence IFN-α2b shown in Figure 1; and (b) a carbohydrate moiety covalently attached to the R group of each indicated aspartic acid residue.

[00191] Suitable alpha interferons further include consensus IFN-α. Consensus IFN-α (also referred to as "KIFN" and "IFN-con" and "consensus interferon") includes, but is not limited to, the amino acid sequences designated IFN-con ₁ , IFN-con ₂ and IFN-con ₃ , which are described in US Patents No. 4695623 and No. 4897471; and consensus interferon as defined by the determination of the consensus sequence of natural alpha interferons (eg, Infergen®, InterMune, Inc., Brisbane, Calif). IFN-con ₁ is a consensus interferon substance in the Infergen® alfacon-1 product. Infergen® consensus interferon product refers to its trade name (Infergen®) or its generic name (interferon alfacon-1).

[00192] Suitable known protease-resistant or protease-resistant, hyperglycosylated polypeptide variants include hyperglycosylated forms of any parent consensus IFN-α polypeptide; wherein the variant does not contain at least the protease cleavage site found in the parent protein. In another aspect, a known protease-resistant or protease-resistant, hyperglycosylated variant of the parent consensus IFN-α polypeptide has an amino acid sequence that is different from the amino acid sequence of the parent polypeptide so that the variant contains one or more glycosylation sites not found in the parent polypeptide; and wherein the variant comprises at least one mutant protease cleavage site at the site of the native protease cleavage site found in the parent protein.

[00193] In another aspect, the parent polypeptide is an interferon alfacon-1 polypeptide and a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is a [D99N] interferon alfacon-1 glycopeptide, where [D99N] interferon alfacon-1 glycopeptide is an option an alfacon-1 polypeptide having (a) an aspartic residue substituted with a native aspartic acid residue at amino acid position 99 in the Infergen amino acid sequence (interferon alfacon-1) shown in Figure 1, and (b) carbohydrate discharge portion, covalently attached to the R-group of said asparagine residue.

[00194] In another aspect, wherein the parent polypeptide is an interferon alfacon-1 polypeptide and a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is [D99N, D105N] interferon alfacon-1 glycopeptide, where [D99N, 0105M] interferon alfacon 1 glycopeptide is a variant of the interferon alfacon-1 polypeptide containing (a) an aspartic residue substituted by a native aspartic acid residue at amino acid position 99 and 105 in the Infergen amino acid sequence shown in Figure 1, and (b) a carbohydrate the part covalently attached to the R-group of each specified aspartic residue.

[00195] In another aspect, the parent polypeptide is an interferon alfacon-1 polypeptide and the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is [D99N, D105N, E134N] interferon alfacon-1 glycopeptide, where [D99N, D4NN, E105N, interferon alfacon-1 glycopeptide is a variant of the interferon alfacon-1 polypeptide containing (a) an aspartic moiety substituted for each residual of native aspartic acid, aspartic acid, and glutamic acid at amino acid positions 99, 105 and 134, respectively, in amino islotnoy sequence Infergen, shown in Figure 1, and (b) a carbohydrate moiety covalently attached to the R-group of each of said asparagine residue.

[00196] In another aspect, the parent polypeptide is an interferon alfacon-1 polypeptide and the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is [D99N, E134N] interferon alfacon-1 glycopeptide, where [D99N, E 134N] interferon alfacon 1 glycopeptide is a variant of the interferon alfacon-1 polypeptide containing (a) an aspartic residue substituted for each residual of native aspartic acid and glutamic acid at amino acid positions 99 and 134, respectively, in the amino acid sequence Infergen depicted in Figure 1, and (b) a carbohydrate moiety covalently attached to the R group of each indicated aspartic moiety.

[00197] In another aspect, the parent polypeptide is an interferon alfacon-1 polypeptide and a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is [D105N, E134N] interferon alfacon-1 glycopeptide, where [D105N, E134N] interferon a glycopeptide is a variant of the interferon alfacon-1 polypeptide containing (a) an aspartic moiety substituted for each native aspartic acid and glutamic acid residues at amino acid positions 105 and 134, respectively, in the amino acid sequence Infergen depicted in Figure 1, and (b) a carbohydrate moiety covalently attached to the R group of each indicated aspartic moiety.

[00198] In another aspect, the parent polypeptide is an interferon-alfacon-1 polypeptide, and the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is [D99N, D105N, E134T] interferon-alpha-1-glycopetide, where [D99N, D105 E134T] interferon alfacon-1 glycopeptide is a variant of interferon alfacon-1 polypeptide having (a) an aspartic residue replacing each of the native aspartic acid residues at amino acid positions 99 and 105 in the Infergen amino acid sequence, image 1, (b) a threonine residue replacing the native glutamic acid residue at amino acid position 134 of the Infergen amino acid sequence shown in FIG. 1, and (c) a hydrocarbon moiety covalently attached to the R-group of each of these aspartic and threonine residues.

[00199] In another aspect, the parent polypeptide is an interferon-alfacon-1 polypeptide, and the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is [D99N, E134T] interferon-alpha-1-glycopeptide, where [D99N, El 34T] interferon- alfacon-1 glycopeptide is a variant of interferon-alfacon-1 polypeptide having (a) an aspartic moiety that replaces the native aspartic acid moiety at position 99 of the Infergen amino acid sequence of FIG. 1, (b) a threonine moiety replacing the native glutamine residue at position 134 of the Infergen amino acid sequence depicted in Figure 1, and (c) a hydrocarbon moiety covalently attached to the R group of each of these aspartic and threonine residues.

[00200] In another aspect, the parent polypeptide is an interferon alfacon-1 polypeptide, and a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is [D105N, E134T] interferon-alfacon-1 glycopeptide, where [D105N, E134T] interferon alfacon-1 glycopeptide is a variant of interferon-alfacon-1 polypeptide having (a) an aspartic moiety that replaces the native aspartic acid moiety at amino acid position 105 of the Infergen amino acid sequence of FIG. 1, (b) t a rheonine residue replacing the native glutamic acid residue at position 134 of the Infergen amino acid sequence shown in Figure 1, and (c) a hydrocarbon moiety covalently attached to the R group of each of these aspartic and threonine residues.

[00201] The number of amino acids discussed in the context of amino acid substitutions to obtain hyperglycosylated variants of the parent therapeutic protein is the number of amino acids used to display the amino acid sequence of type 1 interferon in Figure 24. In the context of amino acid substitutions, to obtain protease-resistant variants of the parent therapeutic protein , the number of amino acids used to describe IFN-α variants matches the number of amino acids displayed in figure 1.

[00202] In another aspect, the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant of the parent therapeutic interferon-alpha protein is different from the parent therapeutic interferon-alpha to such an extent that the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant includes (1) a carbohydrate moiety covalently attached to a non-native glycosylation site not found in parental therapeutic interferon-alpha and / and whether (2) the carbohydrate moiety covalently attached to the native glycosylation site but found in the parent therapeutic interferon-alpha is non-glycosylated; and includes at least one mutant protease cleavage site at the site of the native protease cleavage site found in the parent therapeutic protein IFN-α.

IFN-β

[00203] The amino acid sequence of any known IFN-β can be modified to obtain the inventive type 1 interferon receptor synthetic polypeptide synthetic agonist. The term interferon beta ("IFN-β") includes IFN-β polypeptides that are found in nature, but are not limited to these naturally occurring IFN-β polypeptides are; IFN-β1 a, for example Avonex® (Biogen, Inc.), and Rebif® (Serono, SA); IFN-β1b (Vetaseron®; BERLEX); and so on. The amino acid sequence of IFN-β is publicly available; for example, the amino acid sequence of human IFN-β1 is found in GenBank Accession No. NP_002167 and is shown in Figure 24 (SEQ ID NO: **). The sequence of human IFN-β is also presented in figure 3.

[00204] A suitable known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant includes hyperglycolated forms of any parent IFN-β polypeptide. In one aspect, a known protease-resistant or protease-resistant, hyperglycosylated variant of the parent IFN-β polypeptide has an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites not found in the parent polypeptide; and includes at least one mutant protease cleavage site at the site of the native protease cleavage site found in the parent IFN-β polypeptide.

[00205] The number of amino acids discussed in the context of amino acid substitutions for obtaining hyperglycosylated variants of the parent therapeutic protein is the number of amino acids used to display the type 1 interferon amino acid sequences in Figure 24. In the context of amino acid substitutions for obtaining protease-resistant variants of the parent therapeutic protein, the number amino acids used to describe IFN-β variants coincides with the number of amino acids displayed in figure 3.

[00206] In another aspect, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant of parental interferon-beta is different from parental therapeutic interferon-beta to such an extent that the protease-resistant or protease-resistant, hyperglycosylated polypeptide variant includes (1) a carbohydrate moiety covalently attached to a non-native glycosylation site not found in parental therapeutic interferon-beta, and / or (2) a carbohydrate moiety covalently attached glued to the native glycosylation site, but found non-glycosylated in parental therapeutic interferon-beta; and includes at least one mutant protease cleavage site at the site of the native protease cleavage site found in the parent

IFN-β polypeptide.

IFN-tau

[00207] The amino acid sequence of any known IFN-tau can be modified to produce the claimed synthetic type 1 interferon receptor synthetic polypeptide agonist. The term interferon-tau includes IFN-tau polypeptides that are found in nature; but not limited to naturally occurring IFN-tau; Suitable tau interferons include, but are not limited to naturally occurring IFN-tau; Tauferon (®) (Pepgen Corp.); and so on. IFN-tau may include the amino acid sequence as indicated in any one GenBank Accession Nos. PI 5696; P56828; P56832; P56829; P56831; Q29429; Q28595; Q28594; S08072; Q08071; Q08070; Q08053; P56830; P28169; P28172; and P28171. Any protease-resistant or protease-resistant, hyperglycosylated IFN-tau polypeptide variant that retains the desired pharmacological activity can be used in the methods and compositions of the invention.

(00208] Suitable known protease-resistant or protease-resistant, hyperglycosylated polypeptide variants include a protease-resistant or protease-resistant, hyperglycosylated form of any parent IFN-tau polypeptide. In one aspect, a known protease-resistant or protease-resistant, hyperglycosylated The IFN-tau polypeptide has an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that Ianthe include one or more glycosylation sites not found in the parent polypeptide, and comprises at least one mutated protease cleavage site in place of a native protease cleavage site found in the parent INF-tau polypeptide.

IFN-ω

[00209] The amino acid sequence of any known IFN-omega can be modified to produce the claimed synthetic type 1 interferon receptor polypeptide agonist. The term interferon-omega ("IFN-ω") includes IFN-ω polypeptides that are found in nature; and which are not found in nature by IFN-ω polypeptides. Suitable IFN-ω include, but are not limited to, the naturally occurring IFN-ω; recombinant IFN-ω; for example, Biomed 510 (Bio-Medicines); and the like. IFN-ω may include an amino acid sequence as indicated in GenBank Accession No. NP_002168; or AAA70091.

[00210] Suitable known protease-resistant or protease-resistant, hyperglycosylated polypeptide variants include protease-resistant or protease-resistant, hyperglycosylated forms of any parent INF-polypeptide. In one aspect, a known protease-resistant or protease-resistant, hyperglycolized variant of the parent INF-ω polypeptide has an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites not found in the parent polypeptide; and includes at least one mutant protease cleavage site at the site of the native protease cleavage site found in the parent polypeptide.

[00211] In another aspect, the parent polypeptide is an INF-ω1 polypeptide and a known protease-resistant or protease-resistant, hyperglycolized polypeptide variant is a [R99N] INF-ω1 glycopeptide, where [R99N]] INF-ω1 glycopeptide is an INF-ω1 variant having (a) an aspartic moiety that replaces the native arginine moiety at amino acid position 99 in the amino acid sequence INF-ω1; and (b) a carbohydrate moiety covalently attached to the R group of said aspartic moiety; where the option includes at least one mutant protease cleavage site at the site of the native protease cleavage site found in the parent polypeptide.

[00212] In another aspect, the parent polypeptide is an INF-ω1 polypeptide and a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is a [G134N] INF-ω1 glycopeptide, where the [G134N] INF-ω1 glycopeptide is an INF-ω1 variant having (a ) an asparagine residue replacing the native glycine residue at amino acid position 134 in the amino acid sequence INF-ω1, and (b) a carbohydrate moiety covalently attached to the R group of said asparagine residue; where the option includes at least one mutant protease cleavage site at the site of the native protease cleavage site found in the parent polypeptide.

[00213] In another aspect, the parent polypeptide is an INF-ω1 polypeptide and a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is a [G134T] INF-ω1 glycopeptide, where a [G134T] INF-ω1 glycopeptide is an INF-ω1 variant having a) a threonine residue replacing the native glycine residue at amino acid position 134 in the amino acid sequence INF-ω1, and (b) a carbohydrate moiety covalently attached to the R group of said threonine residue; where the option includes at least one mutant protease cleavage site at the site of the native protease cleavage site found in the parent polypeptide.

[00214] In another aspect, the parent polypeptide is an INF-ω1 polypeptide and the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is an [S99N, G134N] INF-ω1 glycopeptide, where [S99N, G134N] INF-ω1 glycopeptide is INF-ω1 having (a) arginine residues replacing the native serine and glycine residues at amino acid positions 99 and 134, respectively, in the amino acid sequence of INF-ω1, and (b) a carbohydrate moiety covalently attached to the R group of each of these aspartic residues; where the option includes at least one mutant protease cleavage site at the site of the native protease cleavage site found in the parent polypeptide.

[00215] In another aspect, the parent polypeptide is an INF-ω1 polypeptide and a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is an [S99N, G134T] INF-ω1 glycopeptide, where [S99N, G134T] INF-ω1 glycopeptide is a variant INF-ω1 having (a) aspartic and threonine residues replacing the native serine and glycine residues at amino acid positions 99 and 134, respectively, in the amino acid sequence of INF-ω1 (as indicated in FIG. 24), and (b) the carbohydrate moiety covalently attached to R-gr PPE each of said asparagine and threonine residues; where the option includes at least one mutant protease cleavage site at the site of the native protease cleavage site found in the parent polypeptide.

[00216] The number of amino acids discussed in the context of amino acid substitutions to obtain hyperglycosylated variants of the parent therapeutic protein is the same as the number of amino acids used to display the amino acid sequence of type 1 interferon shown in Figure 24. In the context of amino acid substitutions to obtain protease-resistant variants of the parent therapeutic protein, the number of amino acids used to describe the IFN-omega variants is the same as the number of amino acids displayed nnyh in Figure 1.

[00217] In another aspect, the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant of the parent therapeutic interferon-omega is different from the parent therapeutic interferon-omega to such an extent that the known protease-resistant or protease-resistant hyperglycosylated polypeptide variant includes (1) carbohydrate moiety covalently attached to a non-native glycosylation site not found in parental therapeutic interferon-omega and / or (2) charcoal the aqueous portion covalently attached to the native glycosylation site, found but not glycosylated in the parental therapeutic interferon-omega; and includes at least one mutant protease cleavage site at the site of the native protease cleavage site found in the parent polypeptide.

Interferon gamma

[00218] Nucleic acid sequences encoding IFN-γ polypeptides can be taken from published databases, for example, GenBank, journal publications and the like. While various mammalian IFN-gamma polypeptides are of interest, human protein must be used to treat human diseases. Sequences encoding human IFN gamma can be found in the Genbank collections, accession numbers XI 3274; V00543; and NM_000619. Corresponding genomic sequences can be found in Genbank, accession numbers J00219; M37265; and V00536. See, for example, Gray et al. (1982), Nature 295: 501, (Genbank XI 3274); and Rinderknecht et al. (1984) J. B.C. 259: 6790. In some embodiments, IFN-γ is glycosylated.

[00219] IFN-γ1b (Actimmune®; human interferon) is a single chain polypeptide comprising 140 amino acids. It can be prepared recombinantly in E. coli and is non-glycosylated. (Rinderknecht et al. 1984, J Biol. Chem. 259: 6790-6797). Recombinant IFN-gamma is discussed in U.S. Patent No. 6,497,871 and is also suitable for use here.

[00220] The term "IFN-gamma" includes any natural IFN-gamma, recombinant IFN-gamma and their derivatives having IFN-γ activity, a characteristic of human IFN-γ. Human IFN-gamma has antiviral and antiproliferative properties that characterize interferons, as well as a number of immunomodulatory activities known from the prior art. Although IFN-gamma is sequence-based as described above, protein production and proteolytic processing can result in variants. The unprocessed sequence, as shown by Gray et a1., As indicated above, includes 166 amino acids (aa). Although initially it was believed that the recombinant IFN-gamma produced in E. Coli consists of 146 amino acids (starting from 20 amino acids), it was subsequently found that native human INF-gamma is cleaved after residue 23 to produce 143 aa protein, or 144 aa, if terminal methionine is present as required for expression in bacteria. During purification, the mature protein can be further cleaved at the C-terminus after the residue at position 162 (reference to the sequence of Gray et al.), Resulting in the formation of a protein of 139 or 140 amino acids if the initial methionine is present, which is required for expression in bacteria . N-terminal methionine is an artifact encoded by tRNA translational "start" signal AUG, which in a special case of expression in E. coli is not processed at all. In other microbiological systems or eukaryotic expression systems, methionine can be removed.

[00221] Any native INF gamma peptides, their modifications and variants, or combinations of one or more peptides can be used as the parent reference polypeptide in accordance with the presented methods and / or compositions. INF gamma peptides of interest include fragments and can be truncated in various ways at the carboxyl end of the corresponding full length sequence. Such fragments continue to exhibit properties characteristic of human gamma interferon, their length is from 24 to 149 amino acids (the number of amino acid residues of the unprocessed polypeptide). Marginal sequences can be replaced by those following amino acid 155 without loss of activity. See, for example, U.S. Patent No. 5,690,925. Native IFN-gamma parts include molecules consisting of various amino acid residues 24-150; 24-151, 24-152; 24-153, 24-155; and 24-157.

[00222] Any known protease-resistant or protease-resistant, hyperglycosylated IFN-gamma polypeptide variant that retains the desired pharmacological activity of the parent IFN-gamma polypeptide can be used in the methods and / or compositions of the invention.

[00223] In another aspect, the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant of the parent therapeutic interferon-gamma is different from the parent therapeutic interferon-gamma so much that the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant includes (1) a carbohydrate moiety covalently attached to a non-native glycosylation site not found in parental therapeutic interferons MA and / or (2) a carbohydrate moiety covalently attached to a native glycosylation site found non-glycosylated in parental therapeutic interferon-gamma; and includes at least one mutant protease cleavage site at the site of the native protease cleavage site found in the parent IFN-γ polypeptide.

[00224] In another aspect, the parent therapeutic protein is interferon-gamma-1b, and the known protease-resistant or protease-resistant, hypeglycosylated polypeptide variant of the parental therapeutic interferon-gamma-1b is a protease-resistant variant of the glycosylated native (wild type) human IFN-γ. Glycosylated native (wild-type) human IFN-γ is described in WO 02/081507.

Erythropoietin

[00225] In some embodiments, a known protease-resistant or protease-resistant hyperglycosylated variant of the polypeptide comprises an erythropoietin amino acid sequence containing at least one non-native glycosylation site compared to the parent erythropoietin polypeptide; and contains at least one mutant protease cleavage site found in the parent EPO polypeptide. Useful erythropoietin polypeptides include those that have the biological activity of human erythropoietin, such as erythropoietin analogues, erythropoietin isoforms, erythropoietin fragments, erythropoietin protein hybrids, combination proteins, and oligomers and multimers of all of the above forms of erythropoietin.

[00226] Specific examples of erythropoietin include, but are not limited to, human erythropoietin (see also Jacobs et al. (1985) Nature 313: 806-810; and Lin et al. (1985) Proc Nati Acad Sci USA 82: 7580-7584 ); erythropoietin polypeptides presented in U.S. Patent Nos. 6,696,056; and 6,585,398; amino acid sequences presented in Gen-Bank under the numbers NP_00790 and CAA26095; epoetin alpha (EPREX (R); ERYPO (R)); new erythropoiesis stimulation protein (hyperglycosylated analogue of the human recombinant erythropoiesis stimulation protein described in European patent application EP640619); human erythropoietin analog - combined proteins with human serum albumin described in International patent application W09966054; mutant forms of erythropoietin described in International Patent Application W09938890; erythropoietin omega, a product of the Ara I registration fragment of the human erythropoietin gene described in US Pat. 5,688,679; modified human glycosylated erythropoietin described in International Patent Application W09911781;

PEG conjugated erythropoietin analogues described in WO 9805363 or US Pat. 5,643,575. Special cell lines selected for expression of endogenous human erythropoietin are described in International Patent Applications WO 9905268 and WO 9412650.

[00227] On the one hand, the known protease-resistant or protease-resistant hyperglycosylated variant of parent erythropoietin retains the hematopoietic activity of natural erythropoietin, as shown by monitoring and measuring the hematocrit of patients.

[00228] On the other hand, the parent polypeptide is EPOGEN® epoetin alpha, and the known protease-resistant or protease-resistant hyperglycosylated version of the polypeptide is a protease-resistant variant of ARANESP® darbepoetin alpha. Insulin.

[00229] According to some reports, a known protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide contains an amino acid sequence of insulin containing at least one non-native glycosylation site compared to the parent insulin polypeptide; and contains at least one mutant protease cleavage site instead of the native protease cleavage site found in the parent insulin polypeptide. Suitable insulin polypeptides include, but are not limited to, proinsulin, preproinsulin and the forms of insulin described in US patents 4992417; 4,992,418; 5,474,978; 5,514,646; 5504188; 5,547,929; 5,650,486; 5,693,609; 5,700,662; 5,747,642; 5,922,675; 5952297; 6034054; and 6,211,144; and published in PCT applications WO 00/121197; WO 09/010645; and WO 90/12814. Insulin analogs include, but are not limited to, superactive insulin analogs, monomeric insulins, and liver-specific insulin analogs. Various forms of insulin include Humalog®; Humalog® Mix 50/50 ^TM ; Humalog® Mix 75/25 ^TM ; Humulin® 50/50; Humulin® 70/30; Humulin® L; Humulin® N; Humulin® R; Humulin® Ultralente; Laiirus® Lente® Iletin® II; Lente® Insulin; Lente® L; Novolin® 70/30; Novolin® L; Novolin® N; Novolin® R; NovoLog ^TM; NPH Iletin® I; NPH-N; Pork NPH Iletin® II; Pork Regular Iletin® II; Regular (concentrated) Iletin® II U-500; Regular Iletin® I; and Velosulin® BR human (buffered).

[00230] Insulin polypeptides suitable for modifications and uses according to the present invention include human insulin analogs, wherein at positions B28 are Asp, Lys, Leu, Val or Ala and positions B29 is Lys or Pro; des (B28-B30) human insulin; des (B27) human insulin; des (B30) human insulin; a human insulin analogue in which position B28 includes Asp and position B29 includes Lys or Pro; a human insulin analogue in which position B28 corresponds to Lys, position B29 corresponds to Lys or Pro; Asp ^B28 human insulin; Lys ^B2S Pro ^B29 human insulin; B29-N-myristoyl-des (B30) human insulin; B29-N ⁸ -palmitoyl-des (B30) human insulin; B29-N ⁸ -myristoyl human insulin; B29-N ⁶ - palmitoyl human insulin; B28-N ⁸ - myristoyl Lys ⁸²⁸ Pro ⁸²⁹ human insulin; B28-N ⁸ - palmitoyl Lys ⁸²⁸ Pro ⁸²⁹ human insulin; B30-N ⁶ - myristoyl -Thr ⁸²⁹ Lys ⁸³⁰ human insulin; B30-N ⁸ -palmitoyl-Tlir ⁸²⁹ Lys ⁸³⁰ human insulin; B29-N ⁸ - (N-palm- [gamma] -glutamine) -des (B30) human insulin; B29-N ⁸ - (N-litho-cholyl- [gamma]) - des (B30) human insulin; B29-N ^e - ([Omega] -carboxyheptadecanoyl) -des (B30) human insulin; and B29-N ⁸ - ([OMEGA] -carboxyheptadecanoyl) human insulin.

[00231] Amino acid sequences of various insulin polypeptides are available in databases such as GenBank, journal articles, patents and patent bulletins, etc. For example, the amino acid sequences of human insulin can be found in GenBank under the following numbers: CAA00714; CAA00713; CAA00712; CAA01254; IHISA and IHISB; 1 HIQA and 1 fflQB; IHITA and IHITB; 1 HLSA and IHLSB; IVKTA and IVKTB.

[00232] In addition, insulin derivatives and protease-resistant or protease-resistant, hyperglycosylated forms can be used as parent polypeptides and as protease-resistant or protease-resistant, hyperglycosylated polypeptide variants, respectively, in the methods and / or compositions of the present invention . Derivatives of insulin include acylated insulin, glycosylated insulin, and the like. Examples of acylated insulin are those described in U.S. Patent No. 5922675, for example, an insulin derivative with Sb-C21 fatty acids (myristic, pentadecyl, palmityl, heptadicylic, stearyl) in the a- or e-amino acids of glycine, phenylalanine or lysine.

Antibodies

[00233] In some embodiments, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant comprises the amino acid sequence of an antibody polypeptide; further comprises at least one non-native glycosylation site compared to the parent antibody polypeptide; then includes at least one mutant protease cleavage site instead of the native protease cleavage site found on the parent antibody protein. Suitable antibodies include, but are not limited to, antibodies of various isotypes (IgGI, IgG3 and IgG4); monoclonal antibodies produced by any means; humanized antibodies; chimeric antibodies; single chain antibodies; antibody fragments such as Fv, F (ab ') ₂ , Fab ¹ , Fab, Facb, and the like, ensuring that the antibody is capable of binding to the antigen. Suitable monoclonal antibodies include antibodies specific to cell surface receptors and functioning as receptor antagonists, including, but not limited to, an antibody to a TGF-P receptor, an antibody to a TNF-a receptor, an antibody to a VEGF receptor (see, for example , US Patent Nos. 6617160, 6448077, and 6365157), an antibody to an epidermal growth factor receptor and the like; antibodies specific for receptor ligands include the following, and not only: anti-TGF-β antibody, anti-TNF-a antibody, anti-VEGF antibody and the like; an antibody to a specific antigen associated with a tumor; CD20 specific antibody; epidermal growth factor receptor specific antibody -2; IgE receptor-binding domain specific antibody; an antibody specific for adhesive molecules (for example, an antibody specific for a subunit (CD 11 a) LFA-1; an antibody specific for a4p7; etc.); and others like that.

Blood factors

[00234] In some embodiments, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant comprises the amino acid sequence of a blood factor polypeptide; and further includes at least one non-native glycosylation site compared to the parent blood factor polypeptide; and contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide. Suitable growth factor polypeptides include, but are not limited to, tissue plasminogen activator (TPA); vila factor; factor VIII; factor IX; β-globin; hemoglobin; and the like. Amino acid sequences of various blood factors are publicly available, for example, in the Gen-Bank database; in journal articles, in patents and patent bulletins, etc.

For example, the amino acid sequence of human TPA is found in GenBank under the numbers P0070, NP_127509, and NP-000921; the amino acid sequence of human Vila factor is found in GenBank under the number KFHU7; the amino acid sequence of human BC is found in GenBank under the numbers P00740 and NP_000124; the amino acid sequence of human factor VIII was found in Gen-Bank under the numbers AAN64380, AAN22513 and P00451.

[00235] In one aspect, the parent polypeptide is ACTIVASE® alteplase and protease-resistant, a polypeptide variant is a protease-resistant variant of TNKase (TM) tenecteplase.

Colony-stimulating factors.

[00236] In some embodiments, the known protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide contains the amino acid sequence of a colony stimulating factor polypeptide, then contains at least one non-native glycosylation site compared to the parent colony stimulating factor polypeptide; and contains at least one mutant protease cleavage site instead of the native protease cleavage site present in the original colony stimulating factor polypeptide. Useful colony stimulating factor polypeptides include, but are not limited to, granulocyte colony stimulating factor (C-SZR), such as NEUPOGEN® filgrastim and NEULASTA pegfilgrastim, granulocyte monocyte colony stimulating factor (GM-CSF), such as LEUKINE stimulating factor -3, stem cell factor (SCF); and others like that.

[00237] Amino acid sequences of various blood factors are publicly available, including in public databases such as GenBank; magazine articles, patents and patent applications, etc. For example, the amino acid sequence of IL-3 is found in US Pat. Nos. 4,877,729 and 4,959,455, and international patent application WO 88/00598; the amino acid sequence of human G-CSF is available in US patents under the number 4810643; under the numbers WO 91/02754 and WO 92/04455 there are amino acid sequences of composite proteins containing IL-3; WO 95/21197, WO 95/21254, and US Pat. No. 6,730,303 describe fusion proteins having a wide range of hematopoietic properties; the amino acid sequences of human G-CSF are found in GenBank under the numbers NP_757374, P09919, FQHUGL, and NP_000750; amino acid sequences of human GM-CSF are found in GenBank under the numbers NP_000749 and P04141; the amino acid sequences of IL-3 are found in GenBank under the numbers AAN66272, AAN66273, and AAN66276; etc.

Growth hormones

[00238] In some embodiments, the known protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide is the amino acid sequence of a growth hormone polypeptide, and, moreover, contains at least one non-native glycosylation site compared to the native growth hormone polypeptide ; and, furthermore, contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide. Useful growth hormone polypeptides include, but are not limited to, growth hormone; human growth hormone; any of the growth hormone variants is available in US Pat. Nos. 6143523, 6136563, 6022711, and 5688666; synthetic proteins containing growth hormone, for example, are found in US Pat. 5,889,144; fragments of growth hormones having activity of growth hormones; growth hormone receptor agonist polypeptide described in US patent number 6387879; etc. Growth hormones also include alternative forms of known growth hormones, for example, alternative forms of human growth hormone (hGH), including naturally derived derivatives, variants and metabolic products, degradation products of mainly biosynthetic hGH, and hGH variants developed by recombinant methods. (see, for example, US patent 6348444).

Growth factors

[00239] In some embodiments, the protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide is an amino acid sequence of a growth factor, and, moreover, contains at least one non-native glycosylation site compared to a native growth factor polypeptide;

and, furthermore, contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide. Used growth factor polypeptides include, but are not limited to, keratinocyte growth factor; acidic fibroblast growth factor, stem cell factor, the main fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor, etc. active fragments of growth factors; synthetic proteins containing growth factors, etc. Amino acid sequences of various growth factors are publicly available, including in public databases such as GenBank; magazine articles, patents and patent applications, etc. For example, the amino acid sequence bFGF is available in GenBank under the numbers AAB20640, AAA57275, A43498, and AAB20639; the amino acid sequence of aFGF is available in GenBank under the numbers AAB29059, CAA46661, and 1605206A; the amino acid sequence of stem cell factor is available in GenBank under the numbers AAN69733, AAN69783, and AAN69797; the amino acid sequence of keratinocyte growth factor is available in GenBank under the numbers 035565, AAL05875, and P21781; the amino acid sequence of hepatocyte growth factor is available in GenBank under the numbers AAA64239, AAB20169 and CAA40802.

Soluble receptors

[00240] In some embodiments, the protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is the amino acid sequence of a soluble receptor polypeptide variant, and furthermore contains at least one non-native glycosylation site compared to a native growth factor polypeptide; and, furthermore, contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide. Used growth factor polypeptides include, but are not limited to, a soluble TNF-α binding receptor; soluble VEGF receptor; soluble interleukin receptor; soluble IL-1 receptor; soluble receptor type II-1; soluble γ / δ T cell receptor; soluble receptor ligand-binding fragments; etc. The soluble receptors used bind a ligand, which under normal physiological conditions binds to them and activates the corresponding membrane-bound receptor or receptor on the cell surface. Thus, the soluble receptors used function as receptor antagonists by binding a ligand that was supposed to bind to the receptor in its native (i.e., membrane bound) form.

[00241] The amino acid sequences of various soluble receptors are publicly available, including in public databases such as GenBank; articles in magazines, patents and patent applications, etc. For example, the amino acid sequences of a soluble VEGF receptor are found in GenBank under the numbers AAC50060 and NP_002010;

soluble VEGF receptors are described in US Pat. Nos. 6383486, 6375929, and 6100071; a soluble IL-4 receptor is described in US Pat. No. 5,599,905; soluble IL-I receptors are described in US Patent Publication 20040023869; etc.

Chemokines

[00242] In some embodiments, the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is the amino acid sequence of a chemokine polypeptide variant, and furthermore contains at least one non-native glycosylation site compared to a native chemokine polypeptide; and, furthermore, contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide. Used chemokine polypeptides include, among many others, IP-10; Mig Groα / IL-8, RANTES; MIP-1a; MIP-1β; MCP-1; PF-4; etc; and also fusion proteins containing chemokines. Amino acid sequences of various soluble receptors are publicly available, including in public databases such as GenBank; articles in magazines, patents and patent applications, etc. For example, the amino acid sequences of IP-10 are described in US patents 6491906, 5935567, 6153600, 5728377, and 5994292; Mig amino acid sequences are described in US Pat. No. 6,491,906, and in Farber (1993) Biochemical and Biophysical Research Communications 192 (1): 223-230; RANTES amino acid sequences are described in US Pat. Nos. 6,709,649, 6,168,784, and 5,965,697; etc.

Angiogenic factors

[00243] In some embodiments, a known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is the amino acid sequence of an angiogenic polypeptide polypeptide variant, and, moreover, contains at least one non-native glycosylation site compared to a native angiogenic factor polypeptide ; and, furthermore, contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide. Used angiogenic polypeptides include, among many others, VEGF vascular endothelial growth factor polypeptides, including VEGF 121, VEGF 165, VEGF-C, VEGF-2, and others; transforming growth factor - beta; major fibroblast growth factor; growth factor secreted by glioma cells; angiogenin; angiogenin -2; etc. Amino acid sequences of various soluble receptors are publicly available, including in public databases such as GenBank; magazine articles, patents and patent applications, etc. For example, the amino acid sequences of VEGF polypeptides are described in US Pat. Nos. 5194596, 5332671, 5240848, 6475796, 6485942, and 6057428; the amino acid sequences of VEGF-2 polypeptides are described in US Pat. Nos. 5,726,152 and 6,608,182; the amino acid sequences of growth factor polypeptides secreted by glioma cells having angiogenic activity are described in US patents 5338840 and 5532343; the amino acid sequences of angiogenin are found in GenBank under the numbers AAA72611, AAA51678, AAA02369, AAL67710, AAL67711, AAL67712, AAL67713, and AAL67714; etc.

Neuroactive Peptides

[00244] In some embodiments, a known protease-resistant or protease-resistant, hyperglycosylated variant of a polypeptide is the amino acid sequence of a variant of a neuroactive peptide, and, moreover, contains at least one non-native glycosylation site compared to a native neuroactive peptide; and, furthermore, contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide. Used angiogenic polypeptides include, among many others, nerve growth factor, bradykinin, hucotinokinin, gastin, secretin, oxytocin, gonadoliberin, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, hormone, releasing growth hormone, bombesin, neurorphin, dynorphin , motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, tryptophan releasing hormone, non-reactive intestinal peptide, sleep peptide, etc.

Other peptides.

[00245] In a broad sense, the composition and methods of the invention involve the use of any known protease-resistant or protease-resistant, hyperglycosylated variant of the polypeptide that contains at least one non-native glycosylation site compared to the native polypeptide; and, furthermore, contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide. Other proteins of interest to pharmacology include, but are not limited to, thrombolytic factor, atrial sodium urethic peptide, bone morphogenesis protein, thrombopoietin, glia fibrillar acid protein, follicle-stimulating hormone, human alpha-1 antitrypsin, growth factor leukemia inhibitory factor, transforming , insulin-like growth factor, luteinizing hormone, macrophage activation factor, tumor necrosis factor, neutrophil chemotaxis factor, nerve growth factor, tissue metal inhibitor teinaz; vasoactive intestinal peptide, angiotropin, fibrin; hirudin; leukemia inhibition factor, etc. Amino acid sequences of various therapeutic proteins are publicly available, including in public databases such as GenBank; magazine articles, patents and patent applications, etc. For example, the amino acid sequence of a tissue plasminogen activator is found in the GenBank database under the numbers P00750, AAA01895, AAA01378, AAB06956, and CAA00642.

[00246] In some embodiments, the known protease-resistant or protease-resistant, hyperglycosylated polypeptide variant is the amino acid sequence of the relaxin variant, and furthermore contains at least one non-native glycosylation site compared to native relaxin; and, furthermore, contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide. The relaxin peptide can be natural and synthetic. Natural biologically active relaxin can be human, mouse or rat, pork, and obtained from other mammals. The term “relaxin” includes human H1 preprorelaxin, prorelaxin, and relaxin; H2 preprorelaxin, prorelaxin, and relaxin; recombinant human relaxin (rhRLX); and H3 preprorelaxin, prorelaxin, and relaxin. H3 relaxin is described in the article. See also Sudo et al. (2003) J Biol Chem. 7: 278 (10): 7855-62. The amino acid sequences of human relaxin are described in the articles. For example, the amino acid sequences of human relaxin are in GenBank under the following numbers: Q3WXF3, human H3 prorelaxin; P04808, human H1 prorelaxin; NP_604390 and NP_005050, human H2 prorelaxin; AAN05956, human relaxin 1 preprotein; NP_008842, human H1 preprorelaxin; etc. Relaxin may contain A and B chains truncated at the N and / or C ends. For example, in H2 relaxin, the A chain can vary from A (1-24) to A (10-24) and the B chain from B (1-33) to B (10-22); in relaxin H1, the A chain can vary from A (1-24) to A (10-24) and the B chain from B (1-32) to B (10-22). Also suitable for various modifications are relaxin analogues with a non-wild type (native) amino acid sequence, which include relaxin analogues described in US Pat. Nos. 5,811,395 and 6,200,953. Other applicable relaxins and analogs are described in US Pat. No. 5,945,402. Other possible polypeptides relaxin include relaxin having substitutions of one or more amino acids in A and / or B chains for other amino acids (including the D-form of natural amino acids), including, but not limited to, methionine substitution in placing B24 on norleucine (Nie), Valine (Val), alanine (A1a), glycine (Gly), serine (Ser) or homoserin (HomoSer). Other possible relaxin polypeptides include relaxin having amino acid substitutions in the B / C and C / A compounds of prorelaxin, the modifications of which facilitate cleavage of the C chain from prorelaxin; and a relaxin variant having a non-native C-peptide as described in US Pat. No. 5,759,807.

Protease-resistant or protease-resistant, hyperglycosylated variants of the parent cytokine polypeptide polypeptide.

[00247] In some embodiments, a known protease-resistant or protease-resistant, hyperglycosylated variant of the polypeptide is a variant of the natural therapeutic protein, which is a cytokine. In some embodiments, a known protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide contains one or more amino acid substitutions compared to the unmodified natural cytokine, located in positions: 2-181 (IFN-α2b variants), 233-289 (IFN -β variants), 290-311 (IFN-γ variants), 362-400 (GM-CSF variants), 631-662 (G-CSF variants), 850-895 (hGH variants), 940-977 (EPO variants) 978-988 (IFN-α variants) and 989-1302 (IFN-β variants); and, moreover, are amino acid sequences that differ from the native ones in that they have one or more glycosylation sites that are not present in the native polypeptide. Typical amino acid substitutions leading to the formation of a glycosylation site are illustrated in FIGS. 23-30. In some embodiments, a known protease-resistant or protease-resistant, hyper-glycosylated version of the polypeptide is a structural homolog of a protein having the amino acid sequence as in the following SEQ ID NOs: 2-181 (IFN-α2b variants), 233-289 (IFN -β variants), 290-311 (IFN-γ variants), 362-400 (GM-CSF variants), 631-662 (G-CSF variants), 850-895 (hGH variants), 940-977 (EPO variants) 978-988 (IFN-α variants), and 989-1302 (IFN-β variants); and, moreover, is an amino acid sequence that differs from the native one in that it has one or more glycosylation sites that are not in the native polypeptide.

[00248] In some embodiments, a known protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide contains one or more amino acid substitutions compared to an unmodified natural cytokine, as shown in the amino acid sequences of any of these SEQ ID numbers: 87, 89, 90, 93, 96, 101, 103, 107, 124, 979, 980, 983, 984, 986, and 987; and, moreover, contains an amino acid sequence that differs from the native one in that it has one or more glycosylation sites that are not in the native polypeptide.

[00249] In some embodiments, a known protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide is a cytokine modified based on 3-dimensional structural homology with any of SEQ ID NOs: 87, 89, 90, 93. 96, 101 103, 107, 124, 979, 980, 983, 984, 986, and 987; where this option is an amino acid sequence that differs from the native one in that it has one or more glycosylation sites that are not in the native polypeptide.

[00250] In some embodiments, the known protease-resistant or protease-resistant, hyperglycosylated version of the polypeptide is selected from the following protease-resistant or protease-resistant, hyperglycosylated versions of the polypeptide: interleukin-10 (IL-10), interferon beta (IFNR), interferon alpha-2a (IFN-α2a), interferon alpha -2b (IFN-α2b), interferon gamma (IFN-γ), granulocyte colony stimulating factor (G-CSF), leukemia inhibition factor (LIF), human growth hormone (hGH) ciliary neurotrophic fact RA (CNTF), leptin, oncostatin M, interleukin-6 (IL-6), interleukin-12 (IL-12), erythropoietin (EPO), granulocyte-macrophage colony stimulating (GM-CSF), interleukin-2 (IL-2 ), interleukin-3 (IL-3), interleukin -4 (IL-4), interleukin -5 (IL-5), interleukin-13 (IL-13), Flt3 ligand and stem cell factor (SCF). In specific embodiments of the invention, a known protease-resistant or protease-resistant, hyperglycosylated variant of the polypeptide is selected from protease-resistant or protease-resistant, hyperglycosylated variants of IFNβ, IFN-α2a, IFN-α2b, IFN-γ, G-CSF, hGH, EPO and GM-CSF. In specific embodiments of the invention, a known protease-resistant variant of the cytokine is interferon.

[00251] A known hyperglycosylated version of a natural cytokine that is resistant to proteases or resistant to proteases has increased proteolysis resistance compared to an unmodified native cytokine. In some embodiments, a known cytokine-resistant protease-resistant variant of the cytokine is interferon. In some embodiments, a known hyperglycosylated protease-resistant or protease-resistant variant of interferon is interferon alpha-2a (IFN-α2a). In some embodiments, a known hyperglycosylated protease-resistant or protease-resistant variant of interferon is interferon alpha-2b (IFN-α2 b). In some embodiments, a known hyperglycosylated protease-resistant or protease-resistant variant of interferon is IFNβ. In some embodiments, a known hyperglycosylated protease-resistant or protease-resistant variant of interferon is IFNγ. In some embodiments, the known hyperglycosylated protease-resistant or protease-resistant cytokine variant is an interferon variant, and is an amino acid sequence defined as SEQ ID NO: 232, or as shown in FIG. 9 or as shown in FIG. 24 .

Variants of the IFN-α polypeptide.

[00252] In some embodiments, a known hyperglycosylated protease-resistant or protease-resistant variant polypeptide comprises one or more mutations shown in Table 1 and below, where the amino acid numbering corresponds to the amino acid numbering in Figure 1; and, moreover, contains an amino acid sequence that differs from the amino acid sequence of the parent polypeptide in that it has one or more glycosylation sites.

Table 1 IFN-α example SEQ ID NO Mutation (s) one 983 K121Q / P109A 2 987 E159H / Y89H 3 124 E159Q four 90 E58H 5 89 E58Q 6 979 E41H / Y89H / N45D 7 103 L117I 8 986 R125H / M111V 9 96 E107H 10 101 E113H eleven 87 E41Q 12 107 R125Q 13 985 L117V / A139G fourteen 980 E41Q / D94G fifteen 93 E78H 16 984 K133Q / K121Q / P109A / G102R

[00253] On the one hand, the parent polypeptide is IFN-α2a or IFN-α2b, and the known hyperglycosylated protease-resistant or protease-resistant variant of the polypeptide has one or more amino acid substitutions in the amino acid sequence of IFN-α2a, illustrated in Figure 1 , or the sequence IFN-α 2b illustrated in FIG. 2, corresponding to the substitutions: L by V at position 3; L on I at position 3; P on S at position 4; P on A at position 4; R on H at position 12; R on Q at position 12; R on H at position 13; R on Q at position 13; M on V at position 16; M on I at position 16; R on H at position 22; R on Q at position 22; R or K on H at position 23; R or K on Q at position 23; F on I at position 27; F on V at position 27; L on V at position 30; L on I at position 30; K on Q at position 31; K on T at position 31; R on H at position 33; R on Q at position 33; E on Q at position 41; E on H at position 41; K on Q at position 49; K on T at position 49; E on Q at position 58; E on H at position 58; K on Q at position 70; K on T at position 70; E on Q at position 78; E on H at position 78; K on Q 'at position 83; K on T at position 83; Y on H at position 89; Y on I at position 89; E on Q - at position 96; E on H at position 96; E on Q at position 107; E on H at position 107; P on S at position 109; P on A at position 109; L on V at position 110; L on I at position 110; M on V at position 111; M on I at position 111; E on Q at position 113; E on H at position 113; L on V at position 117; L on I at position 117; R on H at position 120; R on Q at position 120; K on Q at position 121; K on T at position 121; R on H at position 125; R on Q at position 125; L on V at position 128; L on I at position 128; K on Q at position 131; K on T at position 131; E on Q at position 132; E on H at position 132; K on Q at position 133; K on T at position 133; K on Q at position 134; K on T at position 134; Y on H at position 135; Y on I at position 135; P on S at position 137; P on A at position 137; M on V at position 148; M on I at position 148; R on H at position 149; R on Q at position 149; E on Q at position 159; E on H at position 159; L on V at position 161; L on I at position 161; R on H at position 162; R on Q at position 162; K on Q at position 164; K on T at position 164; E on Q at position 165; and E on H at position 165, where residue 1 corresponds to residue 1 in the finished IFN-α2a protein, as shown in Figure 1 or where residue 1 corresponds to residue 1 in the mature form of IFN-α2b protein, as shown in Figure 2; and, moreover, contains an amino acid sequence that differs from the amino acid sequence of the parent polypeptide in that it has one or more glycosylation sites.

[00254] On the other hand, the parent peptide is IFN-a2a or IFN-α2b, and the known hyperglycosylated protease-resistant or protease-resistant variant polypeptide has one or more amino acid substitutions in the amino acid sequence of IFN-a2a, as shown in FIG. 1, or IFN-α2b, as shown in FIG. 2, corresponding to substitutions: F by V at position 27; R on H at position 33;

E on Q at position 41; E on H at position 41; E on Q at position 58; E on H at position 58; E on Q at position 78; E on H at position 78; Y on H at position 89; E on Q at position 107; E on H at position 107; P on A at position 109; L on V at position 110; M on V at position 111; E on Q at position 113; E on H at position 113; L on V at position 117; L on I at position 117; K on Q at position 121; K on T at position 121; R on H at position 125; R on Q at position 125; K on Q at position 133; K on T at position 133; E on Q at position 159 and E on H at position 159, where residue 1 corresponds to residue 1 in the finished IFN-α2a protein, as shown in Figure 1, or where residue 1 corresponds to residue 1 in the mature form of IFN-α2b protein, as shown in FIG. 2; and, furthermore, contains an amino acid sequence different from the amino acid sequence of the parent polypeptide in that it has one or more glycosylation sites not found in the parent polypeptide.

[00255] On the other hand, the parent polypeptide is IFN-α2a or IFN-α2b, and the known hyperglycosylated protease-resistant or protease-resistant variant polypeptide has one or more double amino acid substitutions in the amino acid sequence of IFN-α2a, as shown in FIG. .1, or IFN-α2b, as shown in FIG. 2, corresponding to the substitutions:

[00256] D at N at position 2 and P at S at position 4;

[00257] D at N at position 2 and P at T at position 4;

[00258] L on N at position 3 and Q on S at position 5;

[00259] L on N at position 3 and Q on T at position 5;

[00260] P at N at position 4 and T at S at position 6;

[00261] P on N at position 4 and T on T at position 6;

[00262] Q at N at position 5 and H at S at position 7;

[00263] Q on N at position 5 and H on T at position 7;

[00264] T on N at position 6 and S on S at position 8;

[00265] T on N at position 6 and S on T at position 8;

[00266] H on N at position 7 and L on S at position 9;

[00267] H to N at position 7 and L to T at position 9;

[00268] S on N at position 8 and G on S at position 10;

[00269] S on N at position 8 and G on T at position 10;

[00270] L on N at position 9 and S on S at position 11;

[00271] L on N at position 9 and S on T at position 11;

[00272] M at N at position 21 and K at S at position 23;

[00273] M at N at position 21 and K at T at position 23;

[00274] R at N at position 22 and I at S at position 24;

[00275] R at N at position 22 and I at T at position 24;

[00276] R or K at N at position 23 and S at S at position 25;

[00277] R or K at N at position 23 and S at T at position 25;

[00278] I to N at position 24 and L to S at position 26;

[00279] I on N at position 24 and L on T at position 26;

[00280] S on N at position 25 and F on S at position 27;

[00281] S on N at position 25 and F on T at position 27;

[00282] L on N at position 26 and S on S at position 28;

[00283] L on N at position 26 and S on T at position 28;

[00284] S on N at position 28 and L on S at position 30;

00285] S on N at position 28 and L on T at position 30;

[00286] L on N at position 30 and D on S at position 32;

[00287] L on N at position 30 and D on T at position 32;

[00288] K at N at position 31 and R at S at position 33;

[00289] K at N at position 31 and R at T at position 33;

[00290] D at N at position 32 and H at S at position 34;

[00291] D at N at position 32 and H at T at position 34;

00292] R at N at position 33 and D at S at position 35;

[00293] R at N at position 33 and D at T at position 35;

[00294] H on N at position 34 and F on S at position 36;

[00295] H on N at position 34 and F on T at position 36;

[00296] D at N at position 35 and G at S at position 37;

[00297] D on N at position 35 and G on T at position 37;

[00298] F on N at position 36 and F on S at position 38;

[00299] F on N at position 36 and F on T at position 38;

[00300] G at N at position 37 and P at S at position 39;

[00301] G on N at position 37 and P on T at position 39;

[00302] F on N at position 38 and Q on S at position 40;

[00303] F on N at position 38 and Q on T at position 40;

[00304] P at N at position 39 and E at S at position 41;

[00305] P at N at position 39 and E at T at position 41;

[00306] Q on N at position 40 and E on S at position 42;

[00307] Q on N at position 40 and E on T at position 42;

[00308] E on N at position 41 and F on S at position 43;

[00309] E on N at position 41 and F on T at position 43;

[00310] E at N at position 42 and G at S at position 44;

[00311] E at N at position 42 and G at T at position 44;

[00312] F at N at position 43 and N at S at position 45;

[00313] F on N at position 43 and N on T at position 45;

[00314] G at N at position 44 and Q at S at position 46;

[00315] G at N at position 44 and Q at T at position 46;

[00316] N to N at position 45 and F to S at position 47;

[00317] N on N at position 45 and F on T at position 47;

[00318] Q at N at position 46 and Q at S at position 48;

[00319] Q on N at position 46 and Q on T at position 48;

[00320] F on N at position 47 and K on S at position 49;

[00321] F on N at position 47 and K on T at position 49;

[00322] Q on N at position 48 and A on S at position 50;

[00323] Q on N at position 48 and A on T at position 50;

[00324] K at N at position 49 and E at S at position 51;

[00325] K on N at position 49 and E on T at position 51;

[00326] A at N at position 50 and T at S at position 52;

[00327] And on N at position 50 and T on T at position 52;

[00328] S on N at position 68 and K on S at position 70;

[00329] S on N at position 68 and K on T at position 70;

[00330] K at N at position d and S at S at position 72;

[00331] K on N at position 70 and S on T at position 72;

[00332] A at N at position 75 and D at S at position 77;

[00333] A at N at position 75 and D at T at position 77;

[00334] D at N at position 77 and T at S at position 79;

[00335] D on N at position 77 and T on T at position 79;

[00336] I to N at position 100 and G to S at position 102;

[00337] I to N at position 100 and G to T at position 102;

[00338] Q on N at position 101 and V on S at position 103;

[00339] Q on N at position 101 and V on T at position 103;

[00340] G on N at position 102 and G on S at position 104;

[00341] G on N at position 102 and G on T at position 104;

[00342] V on N at position 103 and V on S at position 105;

[00343] V on N at position 103 and V on T at position 105;

[00344] G on N at position 104 and T on S at position 106;

[00345] G on N at position 104 and T on T at position 106;

[00346] V on N at position 105 and E on S at position 107;

[00347] V on N at position 105 and E on T at position 107;

[00348] T at N at position 106 and T at S at position 108;

[00349] T on N at position 106 and T on T at position 108;

[00350] E at N at position 107 and P at S at position 109;

[00351] E at N at position 107 and P at T at position 109;

[00352] T on N at position 108 and I on S at position 110;

[00353] T on N at position 108 and I on T at position 110;

[00354] K at N at position 134 and S at S at position 136;

[00355] K at N at position 134 and S at T at position 136;

[00356] S on N at position 154 and N on S at position 156;

[00357] S on N at position 154 and N on T at position 156;

[00358] T on N at position 155 and L on S at position 157;

[00359] T on N at position 155 and L on T at position 157;

[00360] N on N at position 156 and Q on S at position 158;

[00361] N on N at position 156 and Q on T at position 158;

[00362] L on N at position 157 and E on S at position 159;

[00363] L on N at position 157 and E on T at position 159;

[00364] Q on N at position 158 and S on S at position 160;

[00365] Q on N at position 158 and S on T at position 160;

[00366] E on N at position 159 and L on S at position 161;

[00367] E on N at position 159 and L on T at position 161;

[00368] S on N at position 160 and R on S at position 162;

[00369] S on N at position 160 and R on T at position 162;

[00370] L on N at position 161 and S on S at position 163;

[00371] L on N at position 161 and S on T at position 163;

[00372] R at N at position 162 and K at S at position 164;

[00373] R on N at position 162 and K on T at position 164;

[00374] S on N at position 163 and E on S at position 165;

[00375] S on N at position 163 and E on T at position 165, where residue 1 corresponds to residue 1 in the finished IFN-α2a protein, as shown in FIG. 1, or where residue 1 corresponds to residue 1 in the mature form of IFN-β protein α2b, as shown in FIG. 2; and, furthermore, contains an amino acid sequence different from the amino acid sequence of the parent polypeptide in that it has one or more glycosylation sites not found in the parent polypeptide.

[00376] On the other hand, the parent polypeptide is IFN-α2a or IFN-α2b, and the known hyperglycosylated protease-resistant or protease-resistant variant polypeptide has one or more double amino acid substitutions in the amino acid sequence of IFN-α2a, as shown in FIG. .1, or IFN-α2b, as shown in FIG. 2, corresponding to the substitutions:

[00377] Q at N at position 5 and H at S at position 7;

[00378] P at N at position 39 and E at S at position 41;

[00379] P on N at position 39 and E on T at position 41;

[00380] Q at N at position 40 and E at S at position 42;

[00381] Q on N at position 40 and E on T at position 42;

[00382] E at N at position 41 and F at S at position 43;

[00383] E on N at position 41 and F on T at position 43;

[00384] F at N at position 43 and N at S at position 45;

[00385] G on N at position 44 and Q on T at position 46;

[00386] N on N at position 45 and F on S at position 47;

[00387] N to N at position 45 and F to T at position 47;

[00388] Q at N at position 46 and Q at S at position 48;

[00389] F on N at position 47 and K on S at position 49;

[00390] F on N at position 47 and K on T at position 49;

[00391] I to N at position 00 and G to S at position 102;

[00392] I on N at position 100 and G on T at position 102;

[00393] V on N at position 105 and E on S at position 107;

[00394] V on N at position 105 and E on T at position 107;

[00395] T at N at position 106 and T at S at position 108;

[00396] T on N at position 106 and T on T at position 108;

[00397] E at N at position 107 and P at S at position 109;

[00398] E at N at position 107 and P at T at position 109;

[00399] L on N at position 157 and E on 8 at position 159;

[00400] L on N at position 157 and E on T at position 159;

[00401] E on N at position 159 and L on S at position 161; and

[00402] E on N at position 159 and L on T at position 161, where residue 1 corresponds to residue 1 in the finished IFN-α2a protein, as shown in Figure 1, or where residue 1 corresponds to residue 1 in the mature form of IFN-β protein α2b, as shown in FIG. 2; and, furthermore, contains an amino acid sequence different from the amino acid sequence of the parent polypeptide in that it has one or more glycosylation sites not found in the parent polypeptide.

[00403] In some embodiments, a known hyperglycosylated, protease-resistant or protease-resistant cytokine variant is IFN-α2b, IFN-α2a, or IFN-2 with a variant containing one or more single amino acid substitutions, corresponding substitutions: N on D at position 45; D on G at position 94; G on R at position 102; And on G at position 139; or any combination of them, where the numbering of amino acids is such as shown in Figure 1.

[00404] In some embodiments, a known hyperglycosylated, protease-resistant or protease-resistant cytokine variant is IFN-α2b, IFN-α2a, or IFN-2c containing one or more single amino acid substitutions in any of SEQ ID Nos. I, 182, 185 or 232 (that is, in any of the sequences shown in FIGS. 2, 1, 11 and 9, respectively), corresponding to the following substitutions: L by V at position 3; L on I at position 3; P on S at position 4; P on S at position 4; P on A at position 4; R on H at position 12; R on Q at position 12; R on H at position 13; R on Q at position 13; M on V at position 16; M on I at position 16; R on H at position 22; R on Q at position 22; R or K on H at position 23; R or K on Q at position 23; F on I at position 27; F on V at position 27; L on V at position 30; L on I at position 30; K on Q at position 31; K on T at position 31; R on H at position 33; R on Q at position 33; E on Q at position 41; E on H at position 41; K on Q at position 49; K on T at position 49; E on Q at position 58; E on H at position 58; K on Q at position 70; K on T at position 70; E on Q at position 78; E on H at position 78; K on Q at position 83; K on T at position 83; Y on H at position 89; Y on I at position 89; E on Q at position 96; E on H at position 96; E on Q at position 107; E on H at position 107; P on S at position 109; P on A at position 109; L on V at position 110; L on I at position 110; M on V at position 111; M on I at position 111; E on Q at position 113; E on H at position 113; L on V at position 117; L on I at position 117; R on H at position 120; R on Q at position 120; K on Q at position 121; K on T at position 121; R on H at position 125; R on Q at position 125; L on V at position 128; L on I at position 128; K on Q at position 131; K on T at position 131; E on Q at position 132; E on H at position 132; K on Q at position 133; K on T at position 133; K on Q at position 134; K on T at position 134; Y on H at position 135; Y on I at position 135; P on S at position 137; P on A at position 137; M on V at position 148; M on I at position 148; R on H at position 149; R on Q at position 149; E on Q at position 159; E on H at position 159; L on V at position 161; L on I at position 161; R on H at position 162; R on Q at position 162; K on Q at position 164; K on T at position 164; E on Q at position 165; or E on H at position 165; or any combination of them, where residue 1 corresponds to residue 1 in the mature form of IFN-α2b protein or IFN-α2a protein shown in SEQ ID NOS: 1 or 182 (or as given in FIGS. 2 and 1, respectively); and furthermore, contains an amino acid sequence that differs from the amino acid sequence of the parent polypeptide in that it has one or more glycosylation sites not found in the parent polypeptide.

[00405] In some embodiments, a known hyperglycosylated, protease-resistant or protease-resistant cytokine variant is IFN-α2b, IFN-α2a, or IFN-2c containing one or more single amino acid substitutions in any of SEQ ID Nos. 1, 182, 185 or 232 (that is, in any of the sequences shown in Figs. 2,1, 11 and 9, respectively), corresponding to the following substitutions: L by V at position 3; L on I at position 3; P on S at position 4; P on A at position 4; R on H at position 12; R on Q at position 12; R on H at position 13; R on Q at position 13; M on V at position 16; M on I at position 16; R on H at position 22; R on Q at position 22; R or K on H at position 23; R or K on Q at position 23; F on I at position 27; F on V at position 27; L on V at position 30; L on I at position 30; K on Q at position 31; K on T at position 31; R on H at position 33; R on Q at position 33; E on Q at position 41; E on H at position 41; K on Q at position 49; K on T at position 49; E on Q at position 58; E on And at position 58; K on Q at position 70; K on T at position 70; E on Q at position 78; E on H at position 78; K on Q at position 83; K on T at position 83; Y on H at position 89; Y on I at position 89; E on Q at position 96; E on H at position 96; E on Q at position 107; E on H at position 107; P on S at position 109: P on A at position 109: L on V at position 110: L on I at position 110: M on V at position 121; K on T at position 121; R on H at position 125; R on Q at position 125; L on V at position 128; L on I at position 128; K on Q at position 131; K on T at position 131; E on Q at position 132; E on H at position 132; K on Q at position 133; K on T at position 133; K on Q at position 134; K on T at position 134; Y on H at position 135; Y on I at position 135; P on S at position 137; P on A at position 137; M on V at position 148; M on I at position 148; R on H at position 149; R on Q at position 149; E on Q at position 159; E on H at position 159; L on V at position 161; L on I at position 161; R on H at position 162; R on Q at position 162; K on Q at position 164; K on T at position 164; E on Q at position 165; E on H at position 165; N on D at position 45; D on G at position 94; G on R at position 102; or A on G at position 139; or any combination of them, where residue 1 corresponds to residue 1 in the mature form of the IFN-α2b protein or IFN-α2a protein shown in SEQ ID NOS: 1 or 182; and furthermore, contains an amino acid sequence that differs from the amino acid sequence of the parent polypeptide in that it has one or more glycosylation sites not found in the parent polypeptide.

[00406] In some embodiments, any of the above described protease-resistant or hyperglycosylated IFN-α2a protease-resistant proteases is the [D99N] IFN-α2a glyco-peptide, where the [D99N] IFN-α2a glycopeptide is an IFN variant -α2a having (a) an asparagine residue instead of aspartate at position 99 of the amino acid sequence of IFN-α2a (where this amino acid at position is as shown in FIG. 24; and corresponds to D71 in the sequence given in FIG. 1); and (b) a hydrocarbon moiety covalently bonded to the R group of said aspartic moiety. In some embodiments, IFN-α2a contains lysine instead of arginine at position 50 of the amino acid sequence of IFN-α2b shown in FIG. 24 (corresponding to position 23 in the IFN-α2b sequence shown in FIG. 2).

[00407] In some embodiments, any of the above described protease resistant or hyperglycosylated IFN-α2a resistant proteases is the glyco-peptide [D99N, D105N] IFN-α2a, where the glycopeptide [D99N, D105N] IFN- α2a is an IFN-α2a variant having (a) an asparagine residue instead of aspartate at positions 99 and 105 of the amino acid sequence of IFN-α2a (where these amino acid positions are as shown in FIG. 24; and where D99 and D 105 in FIG. 24 correspond D71 and D77, respectively, in the sequence given in FIGS. 1 and 2); and (b) a hydrocarbon moiety covalently bonded to the R group of each of said asparagine residues. In some embodiments, IFN-α2a contains lysine instead of arginine at position 50 of the amino acid sequence IFN-α2b shown in FIG. 24 (corresponding to arginine 23 in the IFN-α2a sequence shown in FIG. 2).

[00408] In some embodiments, any of the above options for a hyperglycosylated, protease resistant or IFN-α2b resistant protease is the [D99N] IFN-α2b glycopeptide, where the [D99N] IFN-α2b glycopeptide is an IFN-α2b variant having (a) an asparagine residue instead of aspartate at position 99 of the amino acid sequence of IFN-α2b (where this amino acid position is as shown in FIG. 24; and corresponds to D71 in the sequence given in FIG. 1); and (b) a hydrocarbon moiety covalently bonded to the R group of said aspartic moiety.

[00409] In some embodiments, any of the above options for hyperglycosylated IFN-α2b resistant to proteases or resistant to proteases is the glycopeptide [D99N, D105N] IFN-α2b, where the glycopeptide [D99N, D105N] IFN-α2b is an IFN-α2b variant having (a) an asparagine residue instead of aspartate at positions 99 and 105 of the amino acid sequence of IFN-α2b shown in FIG. 24 (where these amino acid positions are as shown in FIG. 24; and where D99 and D 105 on 24 correspond to D71 and D77, respectively, in sequence Given in Figures 1 and 2); and (b) a hydrocarbon moiety covalently bonded to the R group of each of said asparagine residues.

[00410] On the other hand, any of the above options for hyper-glycosylated, protease-resistant or protease-resistant IFN-α2a or IFN-α2b, contains one or more pseudo-wild types of mutations. In specific embodiments of the invention, any of the aforementioned variants of a hyperglycosylated IFN-α2a polypeptide that is protease resistant or protease resistant contains one or more pseudo-wild type mutations in one or more amino acid residues 9, 10, 17, 20, 24, 25 , 35, 37, 41, 52, 54, 56, 57, 58, 60, 63, 64, 65, 76, 89, and 90 as shown in FIG. 1, where the mutation (s) are insertions, deletions and substitutions native amino acids. In other embodiments, any of the aforementioned variants of a hyperglycosylated, protease resistant or protease resistant IFN-α2b contains one or more pseudo-wild types of mutations in one or more amino acid residues 9, 10, 17, 20, 24, 25, 35 , 37, 41, 52, 54, 56, 57, 58, 60, 63, 64, 65, 76, 89, and 90 as shown in FIG. 1, where the mutation (s) are insertions, deletions and substitutions of native amino acids .

[00411] Typical pseudo-wild type substitutions are one or more mutations in the amino acid sequence of IFN-α2a described in Figure 1, or in the amino acid sequence of IFN-α2b described in Figure 2, corresponding to the following substitutions: P by A at position 4; Q on A at position 5, T on A at position 6; L on A at position 9, LG on A at position 10; L on A at position 17; Q on A at position 20; I to A at position 24, S to A at position 25; D on A at position 35, G on A at position 37; G on A at position 39; E on A at position 41; E on A at position 42 E on A at position 51; T on A at position 52, P on A at position 54; V on A at position 55 L on A at position 56; H on A at position 57, E on A at position 58; I on A at position 60, 1 on A at position 63; F on A at position 64, N on A at position 65; W to A at position 76; D to A at position 77; E at A at position 78 L at A at position 81; Y at A at position 85; Y at A at position 89; Q at A at position 90 G at A at position 104; L on A at position 110 and S on A at position 115 and E on A at position 146.

[00412] On the other hand, any of the aforementioned hyperglycosylated protease-resistant or protease-resistant variants of the IFN-α2a or IFN-α2b polypeptide contains one or more pseudo-wild mutation types, in specific embodiments, any of the aforementioned protease-resistant or protease-resistant variants of the IFN-α2a polypeptide contain one or more pseudo-wild type mutations in one or more amino acid residues 4, 5, 6, 9, 10, 17, 20, 24, 25, 35, 37, 39, 41, 42, 51 , 52, 54, 56, 57, 58, 60, 63, 64, 65, 76, 77, 78, 81, 85, 89, 90, 104, 110, 115 and 146 as shown in figure 1, where the mutation (s) represent the insertion, deletion and replacement of native amino acids. In other specific embodiments of the invention, the protease-resistant or protease-resistant variants of the IFN-α2b polypeptide contains one or more pseudo-wild type mutations in one or more amino acid residues 4, 5, 6, 9, 10, 17, 20, 24, 25, 35, 37, 39, 41, 42, 51, 52, 54, 56, 57, 58, 60, 63, 64, 65, 76, 77, 78, 81, 85, 89, 90, 104, 110, 115 and 146, as shown in FIG. 1, where the mutation (s) are insertions, deletions and substitutions of native amino acids.

[00413] Typical pseudo-wild type substitutions are one or more mutations in the amino acid sequence of IFN-α2a described in Figure 1, or in the amino acid sequence of IFN-α2b described in Figure 2, corresponding to the following substitutions: P by A at position 4; Q on A at position 5; T on A at position 6; L on A at position 9; LG on A at position 10; L on A at position 17; Q on A at position 20; I to A at position 24; S on A at position 25; D on A at position 35; G on A at position 37; G on A at position 39; E on A at position 41; B to A at position 42; E on A at position 51; T on A at position 52; P on A at position 54; V on A at position 55; L on A at position 56; H on A at position 57; E on A at position 58; I to A at position 60; I to A at position 63; F on A at position 64; N on A at position 65; W to A at position 76; D on A at position 77; E on A at position 78; L on A at position 81; Y on A at position 85; Y on A at position 89; Q on A at position 90; G on A at position 104; L on A at position 110; S on A at position 115 and E on A at position 146.

[00414] In some embodiments, a known hyperglycosylated protease resistant or protease resistant polypeptide variant is a native cytokine variant having antiviral activity. In some embodiments, a known hyperglycosylated protease-resistant or protease-resistant antiviral cytokine (i.e., protease-resistant or protease-resistant hyperglycosylated version of the IFN-α2a, IFN-α2b, IFN-γ polypeptide) has at least 10%, at least 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% at least about 90%, or up to 100% counter usnoy corresponding activity of unmodified (native) cytokine (i.e., as compared to the native cytokine IFN-α2a, IFN-α2b, or IFN-γ).

[00415] Antiviral activity is readily determined by various known methods. For example, the antiviral activity of IFN-α2a was tested in vitro as follows. An interferon-sensitive HeLa cell culture (its ATCC number is CCL-2) was incubated in vitro with IFN-α2a; subsequently, cells were incubated with encephalomyocarditis virus (EMCV). Antiviral activity was detected by the presence of a cytopathic effect (SRV); or by measuring the amount of EMCV mRNA in extracts of infected cells by reverse transcription-polymerase chain reaction (RT-PCR).

[00416] The determination may be quantitative. For example, in some embodiments, antiviral activity was determined by reverse transcription of a quantitative polymerase chain reaction (RT-qPCR). For example, confluent cells (cells with ATCC number CCL-2) were seeded with a density of are 2 × 10 ⁴ cells per well in culture medium (for example, DMEM with 5% calf serum). Cells were then incubated with IFN-α2b at a concentration of 500 U / ml for 24 hours at 37 ° C. After a 24-hour incubation with IFN-α2b, the cells were treated with EMCV (MOI = 100). After incubation with the virus for 16 hours or when the virus-induced CPE is near its maximum in control cells not incubated with IFN-α2b, the number of EMCV granules in each well was determined by RT-PCR by determining the amount of EMCV mRNA in cell lysates. RNA was purified from cell lysates. See, for example, U.S. Patent Publication No. 2004/0132977.

[00417] In some embodiments, a known hyperglycosylated protease-resistant or protease-resistant variant polypeptide effectively reduces the severity of viral diseases. The severity of the course of viral diseases can be assessed by measuring the level or titer of the virus in the blood. Determination methods, among many others, include quantitative polymerase chain reaction (PCR) and branched DNA test (bDNA test). A quantitative method for determining the titer of HCV virus has been developed. Many such methods are commercially available, including polymerase chain reaction (PCR) (Amplicor HCV Monitor (TM), Roche Molecular Systems, New Jersey); and a DNA branching test (bDNA test) amplification signal (Quantiplex (TM) HCV RNA Assay (bDNA), Chiron Corp., EmeryvIUIle, California). See also, for example, Gretch et all (1995) Ann. Intern. Med. 123: 321-329. Also of interest is the nucleic acid test (NAT) developed by Gen-Probe Inc. (San Diego) and Chiron Corporation sold by Chiron Corporation under the trademark Procleix®, tests determine the presence of HIV-1 and HCV simultaneously. See, for example, Vargo et al. (2002) Transfusion 42: 876-885.

[00418] In some embodiments, a known hyperglycosylated protease-resistant or protease-resistant variant of an antiviral cytokine (ie, IFN-α2a paolipeptide, IFN-α2b polypeptide and IFN-γ polypeptide) has antiproliferative activity compared to unmodified (parent ) therapeutic cytokine.

[00419] Antiproliferative activity can be measured by any known method. For example, antiproliferative activity is measured by determining cell proliferation in the presence of a protease-resistant antiviral cytokine variant, and cell proliferation can be measured in any convenient way. Cell proliferation can be measured by a test involving H-3-thymidine; the inclusion of an analogue of thymidine - BrdU; cleavage of tetrazolium salts; the formation of stained complexes with DNA; etc. One example of a convenient determination of cell proliferation is the CellTiter 96®Aquous Non-Radioactive Cell Proliferation Assay (Promega). The CellTiter 96®Aqueous assay method is a colorimetric method for determining the number of viable cells in proliferation or chemosensitivity tests. CellTiter 96®AQueous Assay consists of solutions of tetrazolium (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, internal salt; MTS) and eleconon conjugate reagent (phenazine metosulfate; PMS). MTS is restored by cells to formazan product, soluble in the culture medium. The absorption wavelength of formazan is 490 nm, which can be measured immediately in 96 wells at no additional cost. The recovery process of MTS cells into a formazan water-soluble product is carried out by dehydrogenases of viable cells. The amount of formazan formed is measured by absorbance at 490 nm and is directly proportional to the number of living cells in the culture.

[00420] In some embodiments, a known hyperlycosylated protease-resistant or protease-resistant variant of an antiviral cytokine (ie, a hyperglycosylated protease-resistant variant of the IFN-α2a polypeptide, IFN-α2b polypeptide and IFN-γ polypeptide) binds to the interferon receptor , but has less antiviral activity compared to the native unmodified therapeutic cytokine or has less antiproliferative activity compared to skim therapeutic cytokine.

[00421] In some embodiments, a known hyperglycosylated protease-resistant or protease-resistant variant of an antiviral cytokine (ie, protease-resistant variant of the IFN-α2a polypeptide, IFN-α2b polypeptide and IFN-γ polypeptide) contains two or more mutations, i.e. protease-resistant antiviral cytokines contain 2, 3, 4, 5, 6, 7, 8, 9, or 10 single amino acid substitutions compared to the corresponding native cytokines. In some embodiments, a known hyperglycosylated version of an antiviral cytokine is an IFN-α2a polypeptide. In other embodiments, a known hyper-glycosylated version of an antiviral cytokine is an IFN-α2a polypeptide. In other embodiments, a known hyperglycosylated version of an antiviral cytokine is an IFN-γ polypeptide.

[00422] In some embodiments, a known hyperglycosylated protease-resistant or protease-resistant cytokine variant has the amino acid sequence as given in any of SEQ ID NOs: 2-181, where the arginine at position 23 is replaced by lysine; and, among other things, contains at least one or more glycosylation sites compared to a parent polypeptide that does not contain glycosylation sites. In other embodiments, the known hyperglycosylated protease-resistant or protease-resistant cytokine variant is more resistant to protease cleavage than the unmodified (parental) cytokine, and the protease-resistant or hyperglycosylated protease-resistant cytokine variant contains one or more amino acid substitutions in one or more positions of the cytokine corresponding to the structurally correlated altered position of amino acids in 3-mer s structures IFN-α2a, IFN-α2b, IFN-α2c, or consensus IFN-α, as shown in Figure 9. In some embodiments, proteolysis resistance is measured by incubating a variant polypeptide in vitro, as described above. In other embodiments, proteolysis resistance is measured by incubating a variant polypeptide in vitro or in vivo with blood (meaning human blood). In other embodiments, proteolysis resistance is measured by incubating an in vitro variant of a polypeptide with blood serum (meaning human serum) as described above.

[00423] In some embodiments, any of the protease-resistant or protease-resistant hyperglycosylated variants of the IFN-α2b polypeptide described above has increased stability compared to an unmodified (native) cytokine, where stability is measured by assessing the remaining biological activity of the protein to suppress virus replication or inhibit cell proliferation in certain cells after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum as described above.

[00424] In some embodiments, any of the above protease-resistant or protease-resistant hyperglycosylated variants of the IFN-α2a polypeptide has increased biological activity compared to an unmodified native cytokine, where biological activity is evaluated as the ability of a protein to suppress virus replication or inhibit cell proliferation in certain cells after incubation with a mixture of proteases, with individual proteases, with a blood or serum lysate oh, as described above.

[00425] In some embodiments, any of the protease-resistant or protease-resistant hyperglycosylated variants of the IFN-α2a polypeptide described above has increased stability compared to unmodified native cytokine, where stability is measured by evaluating the remaining biological activity of the protein to inhibit virus replication or inhibit cell proliferation in certain cells after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, as written above.

[00426] In some embodiments, any of the protease-resistant or protease-resistant hyperglycosylated variants of the IFN-α2a polypeptide described above has increased biological activity compared to an unmodified (native) cytokine, where biological activity is evaluated as the ability of a protein to suppress virus replication or inhibit cell proliferation in certain cells after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, as described above.

[00427] In some embodiments, any of the protease-resistant or protease-resistant hyperglycosylated variants of the IFN-α2c polypeptide described above has increased stability compared to the unmodified (native) cytokine, where stability is measured by evaluating the remaining biological activity of the protein to suppress virus replication or inhibit cell proliferation in certain cells after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, as described above.

[00428] In some embodiments, any of the protease-resistant or protease-resistant hyperglycosylated variants of the IFN-α2c polypeptide described above has a higher biological activity compared to unmodified (parental) cytokine after incubation with either stand-alone proteases or protease mixtures or a lysate blood or serum as described above.

3D structural homologs

[00429] In some embodiments, the hyperglycosylated, protease-resistant variant polypeptide is a modified cytokine variant. In some embodiments, the hyperglycosylated, protease-resistant variant of the cytokine is a modified interferon. In some embodiments, any of the hyperglycosylated, protease-resistant variants of cytokines that are structural homologues of IFN-α2b described above contain one or more amino acid substitutions at positions corresponding to 3-dimensional structurally similar modified positions in the 3-dimensional structure of modified IFN-α α2b, IFN-α2a, IFN-α2c, or consensus IFN-α, as shown in Fig.9. In some embodiments, the structural homolog has increased resistance to proteolysis compared to the unmodified (natural) analogue, and resistance to proteolysis was evaluated after incubation with a protease in vitro, or with blood or blood serum, as described above.

[00430] In some examples, a hyperglycosylated, protease-resistant cytokine variant is a structural homolog of an IFN-α cytokine. In some embodiments, a hyperglycosylated, protease-resistant variant IFN-α is a structural homolog of IFN-α2b. In some of these examples, the IFN-α cytokine is selected from the variants IFN-α2a, IFN-α2c, IFN-αc, IFN-αd, IFN-α 5, IFN-α 6, IFN-α 4, IFN-α b, IFN- α 1, IFN-α J, IFN-α H, IFN-α F, IFN-α 8 and consensus IFN-α. Thus, in some embodiments, a known hyperglycosylated, protease-resistant, IFN-α variant comprises one or more amino acid substitutions at 1 or more target positions in the amino acid sequence of IFN-α2a, IFN-α2c, IFN-αc, IFN- αd, IFN-α 5, IFN-α 6, IFN-α 4, IFN-α b, IFN-α 1, IFN-α J, IFN-α H, IFN-α F, IFN-α 8, or consensus IFN -α corresponding to a structurally related modified amino acid position in the 3-dimensional structure of the modified IFN-α 2b proteins, as described above. Amino acid substitutions lead to greater resistance to proteases, as shown by incubation with proteases or blood lysates, serum, compared with unmodified (native) IFN-α, i.e. compared to the native IFN-α2a or IFN-α2b polypeptide.

[00431] In some embodiments, the hyperglycosylated, protease-resistant IFN-α variant contains 1 or more amino acid substitutions at one or more amino acid target positions in the amino acid sequence given in Figure 1 (or SEQ ID NO: 182), corresponding to the structurally correlated altered amino acid position in the 3-dimensional structure of the aforementioned IFN-α2b variant, where the replacement (s) lead to greater resistance to proteases, as shown after incubation with proteases or serum (As described above) when compared with unmodified native IFN-α2a.

[00432] In some embodiments, the hyperglycosylated protease-resistant IFN-α variant contains 1 or more single amino acid substitutions at one or more target positions in SEQ ID NO: 182 (or the amino acid sequence shown in FIG. 1) corresponding to the following amino acid position: 41, 58, 78, 107, 117, 125, 133 and 159; and further, different from the parent polypeptide by the presence of 1 or more glycosylation sites not found in the parent polypeptide.

[00433] In other embodiments, the protease resistant hyperglycosylated IFN-α variant is a modified IFN-α cytokine containing 1 or more amino acid substitutions at one or more amino acid target positions in the amino acid sequence given in FIG. 10 (or SEQ ID NO: 183) corresponding to structurally correlated altered amino acid positions in the 3-dimensional structure of the aforementioned IFN-α2b variant, where the replacement (s) leads to greater resistance to proteases, as shown after and kubatsii with or serum proteases (as described above) when compared with unmodified native nym IFN-α 2c. In some embodiments of these inventions, the modified IFN-α c is selected from proteins containing 1 or more amino acid substitutions in SEQ ID NO: 183 (as shown in FIG. 10) corresponding to any of these: 41, 59, 79, 108, 118, 126, 134 and 160; and, in addition, differs from the parent polypeptide by the presence of 1 or more glycosylation sites that are not present in the native polypeptide.

[00434] In other embodiments, the protease resistant hyperglycosylated IFN-α variant is a modified IFN-α2c cytokine containing 1 or more amino acid substitutions at one or more amino acid target positions in the amino acid sequence given in FIG. 11 ( or SEQ ID NO: 185) corresponding to a structurally correlated altered amino acid position in the 3-dimensional structure of the aforementioned IFN-α2b variant, where the replacement (s) lead to greater resistance to proteases, as shown after cubations with proteases or serum (as described above) compared to unchanged native IFN-α2c. In some embodiments of these inventions, the modified IFN-α2c is selected from proteins containing 1 or more amino acid substitutions in SEQ ID NO: 185 (as shown in FIG. 11) corresponding to any of these: 41, 58, 78, 107, 117 , 125, 133, and 159, or any of these: 27, 33, 41, 59, 79, 90, 108, 110, 111, 112, 114, 118, 122,126, 134, and 160; and, in addition, differs from the parent polypeptide by the presence of 1 or more glycosylation sites that are not present in the native polypeptide.

[00435] In some embodiments, any of the hyperglycosylated protease-resistant IFN-α2c variants described above has increased stability compared to the unmodified (parental) cytokine, where stability is measured by evaluating the remaining biological activity of a protein to inhibit virus replication or inhibit cellular proliferation in certain cells after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, as described above. In other embodiments, any of the hyperglycosylated variants of IFN-α2c polypeptide resistant to proteases described above has increased biological activity compared to unmodified (parental) cytokine after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum as described above.

[00436] In other embodiments, the protease resistant hyperglycosylated IFN-α variant is a modified IFN-αd cytokine containing one or more amino acid substitutions at 1 or more target positions in the amino acid sequence given in FIG. 12 (or SEQ ID NO: 186) corresponding to a structurally correlated altered amino acid position in the 3-dimensional structure of the aforementioned IFN-α2b variant, where the replacement (s) leads to greater resistance to proteases, as shown after incubation with proteases or serum (as described above) compared with unchanged IFN-αd. In some embodiments of these inventions, the modified IFN-αd is selected from proteins containing one or more amino acid substitutions in SEQ ID NO: 186 (as shown in FIG. 12) corresponding to any of these: 41, 59, 79, 108, 118 , 126, 134, and 160, or any of these: 27, 33,41,59,79,90, 108,110,111, 112, 114, 118, 122, 126, 134, and 160; and, in addition, differs from the parent polypeptide by the presence of one or more glycosylation sites that are not present in the native polypeptide.

[00437] In some embodiments, any of the hyperglycosylated protease-resistant IFN-αd variants described above has increased stability compared to the unmodified (parental) cytokine, where stability is measured by evaluating the remaining biological activity of the protein after incubation with the protease mixture, with individual proteases, with a blood lysate or serum, as described above. In other embodiments, any of the hyperglycosylated protease-resistant IFN-αd polypeptide variants described above has increased biological activity compared to unmodified (parental) cytokine after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum as described above.

[00438] In other examples, the hyperglycosylated protease resistant IFN-α variant is a modified IFN-α 5 cytokine containing one or more amino acid substitutions at one or more target positions in SEQ ID NO: 187 (as given in FIG. 13) corresponding structurally correlated altered amino acid position in the 3-dimensional structure of the aforementioned IFN-α2b variant, where the replacement (s) lead to greater resistance to the action of proteases, as shown after incubation with proteases or blood serum (as described above) compared to nei modified native IFN-α5. In some of these examples, the modified IFN-α5 is selected from proteins containing one or more amino acid substitutions in SEQ ID NO: 187 (as shown in FIG. 13) corresponding to any of these: 41, 59, 79, 108, 118, 126 , 134 and 160, or any of these: 27, 33, 41, 59, 79, 90, 108, 110, 111, 112, 114, 118, 122, 126, 134, and 160; and, in addition, differs from the parent polypeptide by the presence of one or more glycosylation sites that are not present in the native polypeptide.

[00439] In some examples, any of the hyperglycosylated IFN-α5 protease-resistant variants described above has increased stability compared to an unmodified (native) cytokine, where stability is measured by evaluating the remaining biological activity of a protein after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, as described above. In other embodiments, any of the hyperglycosylated protease resistant IFN-α 5 polypeptide variants described above has increased biological activity compared to unmodified (parental) cytokine after incubation with a mixture of proteases, with individual proteases, with a blood lysate, or serum as described above.

[00440] In other examples, the hyperglycosylated protease resistant IFN-α variant is a modified IFN-α 6 cytokine containing one or more amino acid substitutions at one or more target positions in SEQ ID NO: 188 (as given in FIG. 14) corresponding structurally correlated altered amino acid position in the 3-dimensional structure of the aforementioned IFN-α2b variant, where the replacement (s) lead to greater resistance to proteases, as shown after incubation with proteases or blood serum (as described above) compared to nei altered native IFN-α6. In some of these examples, the modified IFN-α 5 is selected from proteins containing one or more amino acid substitutions in SEQ ID NO: 188 (as shown in FIG. 14) corresponding to any of these: 41, 59, 79, 108, 118, 126, 134 and 160, or any of these: 27, 33, 41, 59, 79, 90, 108, 110, 111, 112, 114, 118, 122, 126, 134, and 160; and, in addition, differs from the parent polypeptide by the presence of 1 or more glycosylation sites that are not present in the native polypeptide.

[00441] In some embodiments, any of the hyperglycosylated protease-resistant IFN-α 6 variants described above is more stable than the unmodified parent cytokine, where stability is measured by evaluating the remaining biological activity of the protein after incubation with the protease mixture, c individual proteases, with a blood lysate or serum, as described above. In other examples, any of the hyperglycosylated protease-resistant IFN-α6 polypeptide variants described above has increased biological activity compared to unmodified (native) cytokine, after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, as described above.

[00442] In other examples, the hyperglycosylated protease resistant IFN-α variant is a modified IFN-α4 cytokine containing 1 or more amino acid substitutions at one or more target positions in SEQ ID NO: 189 (as given in FIG. 15 ) corresponding to a structurally correlated altered amino acid position in the 3-dimensional structure of the aforementioned IFN-α2b variant, where the replacement (s) leads to greater resistance to proteases, as shown after incubation with proteases or blood serum (as described above) compared to unchanged native IFN-α 4. In some of these examples, the modified IFN-α 4 is selected from proteins containing 1 or more amino acid substitutions in SEQ ID NO: 188 (as shown in FIG. 14) corresponding to any of these: 41, 59 , 79, 108, 118, 126, 134, and 160, or any of these: 27, 33, 41, 59, 79, 90, 108, 110, 111, 112, 114, 118, 122, 126, 134, and 160 ; and, in addition, differs from the parent polypeptide by the presence of 1 or more glycosylation sites that are not present in the native polypeptide.

[00443] In some embodiments, any of the hyperglycosylated protease-resistant IFN-α 4 variants described above has enhanced stability compared to an unmodified native cytokine, where stability is measured by evaluating the remaining biological activity of a protein after incubation with a protease mixture, c individual proteases, with a blood lysate or serum, as described above. In other embodiments of the invention, any of the hyperglycosylated variants of IFN-α 4 protease resistant polypeptide described above has increased biological activity compared to unmodified native cytokine, after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, as described above.

[00444] In other embodiments, the protease resistant hyperglycosylated IFN-α variant is a modified IFN-α4b cytokine containing 1 or more amino acid substitutions at one or more target positions in SEQ ID NO: 190 (as given in FIG. .16), corresponding to the structurally correlated altered amino acid position in the 3-dimensional structure of the aforementioned IFN-α4b variant, where the replacement (s) lead to greater resistance to proteases, as shown after incubation with proteases or blood serum (as described above) when compared with unmodified native IFN-α4b. In some of these embodiments, the modified IFN-α4b is selected from proteins containing 1 or more amino acid substitutions in SEQ ID NO: 190 (as shown in FIG. 16) corresponding to any of these: 41, 59, 79, 108, 118 , 126, 134, and 160, or any of these: 27, 33, 41, 59, 79, 90, 108, 110, 111, 112, 114, 118, 122, 126, 134, and 160; and, in addition, differs from the parent polypeptide by the presence of 1 or more glycosylation sites that are not present in the native polypeptide.

[00445] In some embodiments, any of the hyperglycosylated protease-resistant IFN-α4b variants described above has increased stability compared to an unmodified native cytokine, where stability is measured by evaluating the remaining biological activity of the protein after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, as described above. In other embodiments, any of the hyperglycosylated protease resistant IFN-α4b polypeptide variants described above has increased biological activity compared to an unmodified native cytokine, after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, such as described above.

[00446] In other embodiments, the protease-resistant hyperglycosylated IFN-α variant is a modified IFN-α 1 cytokine containing 1 or more amino acid substitutions at one or more target positions in SEQ ID NO: 191 (as given in 17), corresponding to a structurally correlated altered amino acid position in the 3-dimensional structure of the aforementioned IFN-α 2b variant, where the replacement (s) lead to greater resistance to proteases, as shown after incubation with proteases or blood serum (as described o above) as compared to unaltered native IFN-α 1. In some of these embodiments, the modified IFN-α 1 is selected from proteins containing 1 or more amino acid substitutions in SEQ ID NO: 191 (as shown in FIG. 17), corresponding to any of these: 41, 59, 79, 108, 118, 126, 134 and 160, or any of these: 27, 33, 41, 59, 79, 90, 108, 110, 111, 112, 114, 118, 122, 126, 134, and 160; and, in addition, differs from the parent polypeptide by the presence of 1 or more glycosylation sites that are not present in the native polypeptide.

[00447] In some embodiments, any of the hyperglycosylated protease-resistant IFN-α 1 variants described above has increased stability compared to an unmodified native cytokine, where stability is measured by evaluating the remaining biological activity of a protein after incubation with a protease mixture, c individual proteases, with a blood lysate or serum, as described above. In other embodiments, any of the hyperglycosylated protease resistant IFN-α1 polypeptide variants described above has increased biological activity compared to unmodified native cytokine after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, such as described above.

[00448] In other embodiments, the protease resistant hyperglycosylated IFN-α variant is a modified IFN-αJ cytokine containing 1 or more amino acid substitutions at one or more target positions in SEQ ID NO: 192 (as given in FIG. .18) corresponding to the structurally correlated altered amino acid position in the 3-dimensional structure of the aforementioned IFN-α 2b variant, where the replacement (s) lead to greater resistance to proteases, as shown after incubation with proteases or blood serum (as described above), as compared with native unmodified IFN-αJ. In some of these embodiments, the modified IFN-α 1 is selected from proteins containing 1 or more amino acid substitutions in SEQ ID NO: 192 (as shown in FIG. 18) corresponding to any of these: 41, 59, 79, 108, 118, 126, 134, and 160, or any of these: 27, 33, 41, 59, 79, 90, 108, 110, 111, 112, 114, 118, 122, 126, 134, and 160; and, in addition, differs from the parent polypeptide by the presence of 1 or more glycosylation sites that are not present in the native polypeptide.

[00449] In some embodiments, any of the hyperglycosylated protease-resistant IFN-αJ variants described above has increased stability compared to an unmodified native cytokine, where stability is measured by evaluating the remaining biological activity of a protein after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, as described above. In other embodiments, any of the hyperglycosylated protease resistant IFN-αJ polypeptide variants described above has increased biological activity compared to an unmodified native cytokine, after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, such as described above.

[00450] In other embodiments, the protease resistant hyperglycosylated IFN-α variant is a modified IFN-αH cytokine containing 1 or more amino acid substitutions at one or more target positions in SEQ ID NO: 193 (as shown in FIG. .19) corresponding to the structurally correlated altered amino acid position in the 3-dimensional structure of the aforementioned IFN-α2b variant, where the replacement (s) lead to greater resistance to proteases, as shown after incubation with proteases or blood serum (as described higher) compared to unchanged native IFN-αH. In some of these embodiments, the modified IFN-αH is selected from proteins containing 1 or more amino acid substitutions in SEQ ID NO: 193 (as shown in FIG. 19) corresponding to any of these: 41, 59, 79, 108, 118 , 126, 134, and 160, or any of these: 27, 33, 41, 59, 79, 90, 108, 110, 111, 112, 114, 118, 122, 126, 134, and 160; and, in addition, differs from the parent polypeptide by the presence of 1 or more glycosylation sites that are not present in the native polypeptide.

[00451] In some embodiments, any of the hyperglycosylated protease-resistant IFN-αH variants described above has increased stability compared to an unmodified native cytokine, where stability is measured by evaluating the remaining biological activity of a protein after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, as described above. In other embodiments, any of the hyperglycosylated protease resistant IFN-αH polypeptide variants described above has increased biological activity compared to an unmodified native cytokine after incubation with a protease mixture, with individual proteases, with a blood lysate or serum, such as described above.

[00452] In other embodiments, the protease resistant hyperglycosylated IFN-α variant is a modified IFN-αF cytokine containing 1 or more amino acid substitutions at one or more target positions in SEQ ID NO: 194 (as shown in FIG. .20) corresponding structurally correlated altered amino acid position in the 3-dimensional structure of the aforementioned IFN-α2b variant, where the substitution (s) lead to greater resistance to proteases, as shown after incubation with proteases or blood serum (as described higher) compared to unchanged native IFN-αF. In some of these examples, the modified IFN-αF is selected from proteins containing 1 or more amino acid substitutions in SEQ ID NO: 194 (as shown in FIG. 20) corresponding to any of these: 41, 59, 79, 108, 118, 126 , 134 and 160, or any of these: 27, 33, 41, 59, 79, 90, 108, 110, 111, 112, 114, 118, 122, 126, 134, and 160; and, in addition, differs from the parent polypeptide by the presence of 1 or more glycosylation sites that are not present in the native polypeptide.

[00453] In some embodiments, any of the hyperglycosylated protease-resistant IFN-αF variants described above has increased stability compared to an unmodified native cytokine, where stability is measured by evaluating the remaining biological activity of the protein after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, as described above. In other embodiments, any of the hyperglycosylated protease resistant IFN-αF polypeptide variants described above has increased biological activity compared to an unmodified native cytokine after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, such as described above.

[00454] In other embodiments, the protease resistant hyperglycosylated IFN-α variant is a modified IFN-α8 cytokine containing 1 or more amino acid substitutions at one or more target positions in SEQ ID NO: 195 (as shown in FIG. .21) corresponding to a structurally correlated altered amino acid position in the 3-dimensional structure of the aforementioned IFN-α2b variant, where the replacement (s) lead to greater resistance to proteases, as shown after incubation with proteases or blood serum (as described yshe) compared to the unmodified native IFN-α8. In some of these examples, the modified IFN-αF is selected from proteins containing 1 or more amino acid substitutions in SEQ ID NO: 195 (as shown in FIG. 21) corresponding to any of these: 41, 59, 79, 108, 118, 126 , 134 and 160, or any of these: 27, 33, 41, 59, 79, 90, 108, 110, 111, 112, 114, 118, 122, 126, 134, and 160; and, in addition, differs from the parent polypeptide by the presence of 1 or more glycosylation sites that are not present in the native polypeptide.

[00455] In some embodiments, any of the hyperglycosylated protease-resistant IFN-α8 variants described above has enhanced stability compared to an unmodified native cytokine, where stability is measured by evaluating the remaining biological activity of a protein after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, as described above. In other embodiments, any of the hyperglycosylated protease resistant IFN-α8 polypeptide variants described above has increased biological activity compared to an unmodified native cytokine after incubation with a mixture of proteases, with individual proteases, with a blood lysate or serum, such as described above.

Variants of the consensus IFN-α polypeptide.

[00456] In other embodiments, the protease resistant hyperglycosylated IFN-α variant is a modified consensus IFN-α cytokine containing 1 or more amino acid substitutions at one or more target positions in SEQ ID NO: 232 (as given in Fig. 9), corresponding to the structurally correlated altered amino acid position in the 3-dimensional structure of the aforementioned IFN-α2b variant, where the replacement (s) lead to greater resistance to proteases, as shown after incubation with proteases or blood serum (as described above) compared to unchanged native IFN-α. In some of these examples, the modified IFN-α is selected from proteins containing 1 or more amino acid substitutions in SEQ ID NO: 232 (as shown in FIG. 9) corresponding to any of these: 41, 59, 79, 108, 118, 126 , 134 and 160, or any of these: 27, 33, 41, 59, 79, 90, 108, 110, 111, 112, 114, 118, 122, 126, 134, and 160; and, in addition, differs from the parent polypeptide by the presence of 1 or more glycosylation sites that are not present in the native polypeptide.

[00457] In some embodiments, any of the above consensus IFN-α variants is the [D99N] interferon alfacon-1 glycopeptide, where the [D99N] interferon alfacon-1 glycopeptide is a variant of interferon alfacon-1 having (a) asparagine instead of aspartate (native) at amino acid position 99 of the amino acid sequence of Infergen (interferon alfacon-1) illustrated in FIG. 24 (where the amino acid position is as given in FIG. 24; and where D99 in FIG. 24 corresponds to D71 in FIG. 9); and (b) a hydrocarbon moiety covalently bonded to the R group of said aspartic moiety.

[00458] In some embodiments, any of the above consensus IFN-α variants is the [D99N, D105N] interferon alfacon-1 glycopeptide, where the [D99N, D105N] interferon alfacon-1 glycopeptide is a variant of interferon alfacon-1 having (a ) asparagine instead of aspartate (native) at amino acid position 99 and position 105 of the Infergen (interferon alfacon-1) amino acid sequence illustrated in FIG. 24 (where the amino acid position is as given in FIG. 24; and where D99 and D105 in FIG. 24 correspond to D71 and D77, respectively Fig. 9); and (b) a hydrocarbon moiety covalently bonded to the R group of said aspartic moieties.

[00459] In some embodiments, any of the above consensus IFN-α variants is a glycopeptide [D99N, D105N, E134N] interferon alfacon-1 ([D99N, D105N, E134N] interferon alfacon-1 glycopeptide), where the glycopeptide [D99N, D105N, E134N] interferon alfacon-1 is a variant of interferon alfacon-1 having (a) asparagine instead of aspartate (native) at amino acid position 99 and position 105, and instead of glutamate (native) at position 134 of the amino acid sequence of Infergen (interferon alfacon-1 ) illustrated in FIG. 24 (where amino acid e position is as given in FIG. 24; and where D99, D105 and E134 in FIG. 24 correspond to D71, D77 and E106, respectively, in FIG. 9); and (b) the hydrocarbon moiety is covalently linked to the R group of said aspartic moieties.

[00460] In some embodiments, any of the above consensus IFN-α variants is the [D99N, E134N] interferon alfacon-1 glycopeptide, where the [D99N, E134N] interferon alfacon-1 glycopeptide is an interferon alfacon-1 variant having (a ) asparagine instead of aspartate (native) at amino acid position 99, and aspragin instead of glutamate (native) at position 134 of the Infergen (interferon alfacon-1) amino acid sequence illustrated in FIG. 24 (where the amino acid position is as given in FIG. 24; and where is D99 and E134 in FIG. 24 correspond to D71 and E106, respectively, in FIG. 9); and (b) a hydrocarbon moiety covalently bonded to the R group of said aspartic moieties.

[00461] In some embodiments, any of the above consensus IFN-α variants is the [D105N, E134N] interferon alfacon-1 glycopeptide, where the [D105N, E134N] interferon alfacon-1 glycopeptide is an interferon alfacon-1 variant having (a ) asparagine instead of aspartate (native) at amino acid position 105, and aspragin instead of glutamate (native) at position 134 of the Infergen (interferon alfacon-1) amino acid sequence illustrated in FIG. 24 (where the amino acid position is as given in FIG. 24; and where D 105 and E134 in FIG. 24 correspond to D77 and E106, respectively, in FIG. 9); and (b) the hydrocarbon moiety is covalently linked to the R group of said aspartic moieties.

[00462] In some embodiments, any of the above consensus IFN-α variants is the [D99N, D105N, E134T] interferon alfacon-1 glycopeptide, where the [D99N, D105N, E134T] glycopeptide interferon alfacon-1 is an interferon alfacon-1 variant having (a) asparagine instead of aspartate (native) at amino acid position 99 and position 105 of the Infergen (interferon alfacon-1) amino acid sequence illustrated in FIG. 24 (where the amino acid position is as given in FIG. 24; (b) threonine instead of the original (native ) glutamate at position 134 of the Infergen (interferon alfacon-1) amino acid sequence illustrated in FIG. 24, (where the amino acid position is as given in FIG. 24; and where D99, D105 and E134 in FIG. 24 correspond to D71, D77 and E106, respectively, in Fig. 9); and (c) a hydrocarbon moiety covalently bonded to the R group of each of said asparagine and threonine residues.

[00463] In some embodiments, any of the above consensus IFN-α variants is the [D99N, E134T] interferon alfacon-1 glycopeptide, where the [D99N, E134T] interferon alfacon-1 glycopeptide is an interferon alfacon-1 variant having (a ) asparagine instead of aspartate (native) at amino acid position 99 of the amino acid sequence of Infergen (interferon alfacon-1) illustrated in Fig. 24 (where the amino acid position is as shown in Fig. 24; (b) threonine instead of the original (native) glutamate in provisions and the 134 amino acid sequence of Infergen (interferon alfacon-1) illustrated in FIG. 24 (where the amino acid position is as given in FIG. 24; and where D99 and E134 in FIG. 24 correspond to D71 and E106, respectively, in FIG. 9 ); and (c) a hydrocarbon moiety covalently bonded to the R group of each of said asparagine and threonine residues.

[00464] In some embodiments, any of the above consensus IFN-α variants is the [D105N, E134T] interferon alfacon-1 glycopeptide, where the [D105N, E134T] interferon alfacon-1 glycopeptide is an interferon alfacon-1 variant having (a ) asparagine instead of aspartate (native) at amino acid position 105 of the amino acid sequence of Infergen (interferon alfacon-1), illustrated in Fig.24 (where the amino acid position is as given in Fig.24); (b) threonine instead of the initial (native) glutamate at position 134 of the Infergen (interferon alfacon-1) amino acid sequence illustrated in FIG. 24 (where the amino acid position is as given in FIG. 24; and where D105 and E134 in FIG. 24 correspond to D77 and E106, respectively, in FIG. 9); and (c) a hydrocarbon moiety covalently bonded to the R group of each of said asparagine and threonine residues.

Hybrid Type 1 Interferon Receptor Polypeptide Agonists

[00465] In this context, a "hybrid type 1 interferon receptor polypeptide agonist" is a polypeptide having an amino acid sequence containing individual subsequences corresponding in amino acid composition and the number of subsequences of different natural (native) type 1 interferon receptor agonist polypeptides, wherein the amino acid sequence This polypetid agonist is different from that of any of the native type 1 interferon receptor polypeptide agonists. In some embodiments, a polypeptide variant consists of separate subsequences isolated from IFN-α2b, IFN-α14, IFN-β1, and IFN-ω, the amino acid sequence of this variant polypetid agonist is different from the amino acid sequences of IFN-α2b, IFN-α14, IFN-βl, and IFN-ω). In other embodiments, a polypeptide variant consists of individual subsequences isolated from IFN-α2b, IFN-αl4, IFN-β1, consensus IFN-α-ingergen® and IFN-ω, and the amino acid sequence of this variant polypetid agonist is different from the amino acid sequences IFN-α2b, IFN-α14, IFN-β1, consensus IFN-α - Infergen® and IFN-ω).

[00466] Suitable protease-resistant or protease-resistant, hyperglycosylated polypeptide variants include protease-resistant or protease-resistant, hyperglycosylated forms of any parent polypeptide interferon receptor agonists of hybrid type 1. On the one hand, a protease-resistant or protease-resistant, variant hybrid type 1 parental interferon receptor polypeptide agonist has an amino acid sequence different from amino acid tnoj parent polypeptide sequence that comprises one or more glycosylation sites that are not contained in the native polypeptide; and contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00467] In one aspect, the parent hybrid Type I interferon receptor polypeptide agonist is the [D99N] IFN-α2a glycopeptide, where the [D99N] IFN-α2a glycopeptide is an IFN-α2a variant having an aspartic residue instead of native aspartate at position 99 of the IFN amino acid sequence -α2a; and a hyperglycosylated, protease-resistant or protease-resistant variant of the parent polypeptide is a [D99N, D105N] IFN-α2a glycopeptide, protease-resistant, where the protease-resistant glycopeptide [D99N, D105N] IFN-α2a is an IFN-α2a variant having (a) asparagine instead of aspartate (native) at amino acid position 99 and position 105 of the amino acid sequence IFN-α2a illustrated in FIG. 24 (where the amino acid position is as given in FIG. 24; and where D99 and D105 in FIG. 24 correspond D71 and D77, respectively in FIG. 9); and (b) a hydrocarbon moiety covalently attached to the R group of said asparagine residues. It should be understood that the amino acid sequence of IFN-α2a is the same as the amino acid sequence of IFN-α2b shown in Figure 24, representing that the sequence of IFN-α2a has a lysine residue at the arginine residue at amino acid position 50 in the IFN-α2b sequence shown in Figure 24 (corresponding to R50 of the IFN-α2b sequence shown in Figure 2).

[00468] In another aspect, the parent hybrid type 1 interferon receptor polypeptide agonist is the [D99N] IFN-α2b glycopeptide, where the [D99N] IFN-α2b glycopeptide is an IFN-α2b variant having an aspartic moiety instead of native aspartate at position 99 of the IFN amino acid sequence -α2b; and the protease-resistant or protease-resistant, hyperglycosylated version of the parent polypeptide is the [D99N, D105N] IFN-α2b glycopeptide resistant to proteases, where the protease-resistant glycopeptide [D99N, D105N] IFN-α2b is an IFN-α2b variant a) asparagine instead of aspartate (native) at amino acid position 99 and position 105 of the amino acid sequence of IFN-α2b illustrated in Fig.24 (where the amino acid position is as indicated in Fig.24; and where D99 and D105 in Fig.24 correspond to D71 and D77 respectively on phi .2); and (b) a hydrocarbon moiety covalently linked to the R group of said aspartic moieties; and containing at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00469] On the other hand, the parent hybrid type 1 interferon receptor polypeptide agonist is an interferon alfacon-1 polypeptide; and the protease-resistant or protease-resistant hyperglycosylated version of the parent polypeptide polypeptide is the [D99N] interferon alfacone-1 glycopeptide, where the [D99N] interferon alfacone-1 glycopeptide is a variant of the interferon alfacon-1 polypeptide in which (a) asparagine instead of aspartate ) at position 99 of the amino acid sequence of Infergen illustrated in FIG. 24 (where D99 corresponds to D71 of the consensus IFN-α shown in FIG. 9); and (b) a hydrocarbon moiety covalently attached to the R group of an aspartic moiety; and which contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00470] On the other hand, the parent hybrid Type I interferon receptor polypeptide agonist is an interferon alfacon-1 polypeptide; and the hyperglycosylated protease-resistant or protease-resistant polypeptide variant of the parent polypeptide is the [D99N, D105N] interferon alfacon-1 glycopeptide, where the [D99N, D105N] interferon alfacon-1 glycopeptide is a variant of the interferon alfacon-1 polypeptide, which ) asparagine instead of aspartate (native) at position 99 and position 105 of the Infergen amino acid sequence illustrated in FIG. 24 (where D99 and D105 correspond to D71 and D77 of the consensus IFN- [alpha] shown in FIG. 9); and (b) a hydrocarbon moiety covalently linked to the R group of said aspartic moieties; and which contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00471] On the other hand, the parent hybrid Type I interferon receptor polypeptide agonist is an interferon alfacon-1 polypeptide; and a hyperglycosylated protease-resistant or protease-resistant variant polypeptide of the parent polypeptide is the [D99N, D105N, -E134N] interferon alfacon-1 glycopeptide, where the [D99N, D105N, E134N] interferon alfacon-1 interferon alfacon-1 polypeptide is a variant in which (a) asparagine instead of aspartate (native) at position 99 and position 105 and asparagine instead of glutamate at position 134 of the Infergen amino acid sequence illustrated in FIG. 24 (where D99 and D105 and E134 correspond to D71, D77 and E106, respectively, of the consensus IFN-α shown in FIG. 9); and (b) a hydrocarbon moiety covalently linked to the R group of said aspartic moieties; and which contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00472] On the other hand, the parent hybrid Type I interferon receptor polypeptide agonist is an interferon alfacon-1 polypeptide; and a hyperglycosylated, protease-resistant or protease-resistant polypeptide variant of the parent polypeptide is the [D99N, E134N] interferon alfacon-1 glycopeptide, where the [D99N, E134N] interferon alfacon-1 glycopeptide is a variant of the interferon alfacon-1 polypeptide, a) asparagine instead of aspartate (native) at position 99 and position 134 of the Infergen amino acid sequence illustrated in FIG. 24 (where D99 and E134 correspond to D71 and E106, respectively, of the consensus IFN-α shown in Fig.9); and (b) a hydrocarbon moiety covalently linked to the R group of said aspartic moieties; and which contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00473] On the other hand, the parent hybrid Type I interferon receptor polypeptide agonist is an interferon alfacon-1 polypeptide; and the protease-resistant or protease-resistant hyperglycosylated polypeptide variant of the parent polypeptide is the [D105N, E134N] interferon alfacon-1 glycopeptide, where the [D105N, E134N] interferon alfacon-1 glycopeptide is a variant of the interferon alfacon-1 polypeptide, which ) asparagine instead of aspartate (native) at position 105 and position 134 of the Infergen amino acid sequence illustrated in FIG. 24 (where D105 and E134 correspond to D77 and E106, respectively, of the consensus IFN-α shown in and Fig. 9); and (b) a hydrocarbon moiety covalently bonded to the R group of said asparagin residues; and which contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00474] On the other hand, a parent hybrid type 1 interferon receptor polypeptide agonist is an interferon alfacon-1 polypeptide; and the protease-resistant or protease-resistant hyperglycosylated polypeptide variant of the parent polypeptide is the [D99N, D105N, E134T] interferon alfacon-1 glycopeptide, where the [D99N, D105N, E134T] interferon alfacon-1 interferon alfacon-1 polypeptide is a variant in which (a) asparagine instead of aspartate (native) at position 99 and position 105 of the amino acid sequence of Infergen, illustrated in Fig.24; (b) threonine instead of native glutamate at position 134 of the Infergen amino acid sequence illustrated in FIG. 24 (where D99, D105 and E134 correspond to D71, D77 and E106, respectively, of the consensus IFN-α shown in FIG. 9); and (c) a hydrocarbon moiety covalently attached to the R group of said asparagine and threonine residues; and which contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00475] On the other hand, the parent hybrid Type I interferon receptor polypeptide agonist is an interferon alfacon-1 polypeptide; and the protease-resistant or protease-resistant hyperglycosylated polypeptide variant of the parent polypeptide is the [D99N, E134T] interferon alfacon-1 glycopeptide, where the [D99N, E134T] interferon alfacon-1 glycopeptide is a variant of the interferon alfacon-1 interferon polypeptide, (a ) asparagine instead of aspartate (native) at position 99 of the Infergen amino acid sequence illustrated in FIG. 24; (b) threonine instead of native glutamate at position 134 of the amino acid sequence of Infergen illustrated in FIG. 24 (where D99 and E134 of the Infergen sequence illustrated in FIG. 24 correspond to D71 and E106, respectively, of the consensus IFN-α shown in FIG. 9 ); and (c) a hydrocarbon moiety covalently attached to the R group of said asparagine and threonine residues; and which contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00476] On the other hand, the parent hybrid type I interferon receptor polypeptide agonist is an interferon alfacon-1 polypeptide; and the protease-resistant or protease-resistant hyperglycosylated polypeptide variant of the parent polypeptide is the [D105N, E134T] interferon alfacon-1 glycopeptide, where the [D105N, E134T] interferon alfacon-1 glycopeptide is a variant of the interferon alfacon-1 polypeptide, which ) asparagine instead of aspartate (native) at position 105 of the Infergen amino acid sequence illustrated in FIG. 24; (b) threonine instead of native glutamate at position 134 of the amino acid sequence of Infergen illustrated in FIG. 24 (where D105 and E134 of the Infergen sequence illustrated in FIG. 24 correspond to D77 and E106, respectively, of the consensus IFN-α shown in FIG. 9 ); and (c) a hydrocarbon moiety covalently attached to the R group of said asparagine and threonine residues; and which contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00477] On the other hand, the parent hybrid Type I interferon receptor polypeptide agonist contains the "majority" amino acid sequence of the type 1 consensus interferon shown in FIG. 24; and the protease-resistant or protease-resistant hyperglycosylated polypeptide variant of the parent polypeptide is a [D99N] "major" consensus interferon type 1 glycopeptide, where a [D99N] majority consensus interferon type 1 glycopeptide contains a "majority consensus 1 interferon amino acid sequence 24, and has (a) asparagine instead of aspartate (native) at position 99 of the "majority" amino acid sequence and (b) hydrocarbon covalently bonded to the R group of said aspartic moiety; and contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00478] In another aspect, the parent hybrid Type I interferon receptor polypeptide agonist has the "majority" amino acid sequence of the type 1 consensus interferon shown in FIG. 24; and the protease-resistant or protease-resistant hyperglycosylated polypeptide variant of the parent polypeptide is [D99N, D105N] the "major" consensus interferon type I glycopeptide, where [D99N, D105N] the "consumable" consensus interferon 1 glyco-peptide contains the consensus interferon 1 the amino acid sequence of the consensus interferon type 1, shown in Fig, and contains (a) asparagine instead of aspartate (native) at positions 99 and 105 of the "majority" amino acid sequence and (b) evodorodnuyu portion, covalently attached to the R-group of each of said asparagine residues; and contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00479] In another aspect, the parent hybrid Type I interferon receptor polypeptide agonist has a "majority" amino acid sequence of the type 1 consensus interferon shown in FIG. 24; and the protease-resistant or protease-resistant hyperglycosylated polypeptide variant of the parent polypeptide is [D99N, D105N, E134N] the "majority" type 1 consensus interferon glycopeptide, where [D99N, D105N, E134N] the "majority" type consensus interferon 1 the majority "amino acid sequence of the consensus interferon type 1 shown in Fig. 24 and has (a) asparagine instead of aspartate (native) at positions 99 and 105 of the" majority "amino acid sequence and a paragin instead of glutamate at position 134 in the "majority" of the amino acid sequence and (b) hydrocarbon moiety covalently attached to the R-group of each of said asparagine residues; and contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00480] In another aspect, the parent hybrid type 1 interferon receptor polypeptide agonist has a "majority" amino acid sequence of the type 1 consensus interferon shown in FIG. 24; and a protease-resistant or protease-resistant, hyperglycosylated polypeptide variant of the parent polypeptide is [D99N, E134N] the "major" glycopeptide of the consensus interferon type 1, where [D99N, E134N] the "major" glycopeptide of the consensus "major" amino acid interferon 1 the consensus interferon type 1 sequence shown in FIG. 24 and having (a) asparagine instead of aspartate (native) at position 99 of the "majority" amino acid sequence and asparagine in the position of glutamate at position 134 in the "majority" amino acid sequence and (b) the hydrocarbon moiety covalently linked to the R-group of each of said asparagine residues; and contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00481] On the other hand, the parent hybrid Type 1 interferon receptor polypeptide agonist contains the "majority" amino acid sequence of the type 1 consensus interferon shown in FIG. 24; and protease-resistant or protease-resistant, the hyperglycosylated variant of the parent polypeptide is [D105N, E134N] the "majority" glycopeptide of the consensus interferon type I, where [D105N, E134N] the "major" glycopeptide of the consensus interferon type 1 M contains the amino acid sequence of the consensus interferon type I shown in Fig.24, and having (a) an asparagine residue instead of native aspartic acid at positions 105 and 134, respectively, in the "majority" amino acid sequences (where the amino acid positions are indicated in Figure 24), and (b) a hydrocarbon moiety covalently linked to the R group of each of said asparagine residues; and contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00482] On the other hand, the parent hybrid type 1 interferon receptor polypeptide agonist has a "majority" amino acid sequence of the type 1 consensus interferon shown in FIG. 24; and a protease-resistant or protease-resistant, hyperglycosylated version of the parent polypeptide is [D99N, D105N, E134T] the "major" type I consensus interferon glycopeptide, where [D99N, D105N, E134T] the "majority" type consensus interferon glycopeptide 1 the majority "amino acid sequence of the consensus interferon type I shown in Fig. 24 and having (a) an asparagine residue instead of native aspartic acid at positions 99 and 105 of the" majority "amino acid sequence (b) the threonine residue instead of the initial (native) glutamate at position 134 of the "majority" amino acid sequence and (c) the hydrocarbon moiety covalently linked to the R-group of each of the mentioned asparagine and threonine residues; and contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00483] On the other hand, the parent hybrid type I interferon receptor polypeptide agonist has a "majority" amino acid sequence of the consensus interferon type I shown in FIG. 24; and a protease-resistant or protease-resistant, hyperglycosylated variant of the parent polypeptide is [D99N, E134T] the "major" glycopeptide of the consensus interferon type I, where [D99N, E134T] the "major" glycopeptide of the consensus interferon type I contains the "major consensus interferon amino acid sequence I" consensus interferon type I, shown in Fig.24, and having (a) an asparagine residue instead of native aspartic acid at position 99 of the "majority" amino acid sequence, (b) current of the native threonine instead of glutamate at position 134 "majority" amino acid sequence, and (c) uglevodorodnyyu portion, covalently attached to the R-group of each of said asparagine and threonine residues; and contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00484] On the other hand, the parent hybrid Type I interferon receptor polypeptide agonist has a "majority" amino acid sequence of the type I consensus interferon shown in FIG. 24; and a protease-resistant or protease-resistant, hyperglycosylated variant of the parent peptide is [D105N, E134T] the "majority" glycopeptide of the consensus interferon type 1, where [D99N, E134T] the "major" glycopeptide of the consensus interferon type 1 contains the major consensus interferon type 1 amino acid consensus interferon type 1, shown in Fig, and having (a) an asparagine residue instead of native aspartate at position 105 of the "majority" amino acid sequence, (b) a threoni residue instead of native glutamate at position 134 "majority" amino acid sequence, and (c) a carbohydrate moiety covalently attached to the R-group of each of said asparagine and threonine residues; and contains at least one mutant protease cleavage site instead of the native protease cleavage site of the parent polypeptide.

[00485] The numbering of amino acid substitutions (discussed in the context of creating hyperglycosylated variants of the parent therapeutic protein) used to describe the hyperglycosylated variants of the parent hybrid type 1 interferon receptor polypeptide agonists is the same as the amino acid numbering used in FIG. 24 to display the amino acid sequence of type 1 interferon.

[00486] On the other hand, a protease-resistant or protease-resistant, hyperglycosylated variant of the parent polypeptide differs from the parent hybrid type 1 interferon receptor polypeptide agonist in that the protease-resistant or protease-resistant, hyperglycosylated variant of the parent polypeptide contains 1 .) a hydrocarbon moiety covalently linked to a non-native glycosylation site that is not present in the parent therapeutic polypeptide agonist hybrid type 1 interferon receptor, and 2) a hydrocarbon moiety covalently linked to native glycosylation site, available in a parent polypeptide agonist therapeutic hybrid type 1 interferon receptor, but not glycosylated.

[00487] In some embodiments, any of the hyperglycosylated, protease-resistant protease-resistant variants of consensus IFN-α is more stable than the unmodified (parental) cytokine, where stability is measured by the amount of remaining biological activity after incubation with a mixture of proteases, individual protases, blood lysate, or serum as described above. In other embodiments, any of the hyperglycosylated protease-resistant consensus IFN-α variants described above has increased biological activity compared to unmodified (parental) cytokine after incubation with a protease mixture, individual protases, blood lysate or serum as described above .

Polypeptide Options IFN-β

In some embodiments of the invention, the protease-resistant or protease-resistant, hyperglycosylated cytokine variant is an IFN-β variant. In some embodiments of the invention, the protease-resistant or protease-resistant, hyperglycosylated IFN-β variant comprises one or more single amino acid substitutions in SEQ ID NO: 196 (or the amino acid sequence shown in FIG. 3 below) corresponding to the substitution of one or more: M on V at position 1, M on I at position 1, M on T at position 1, M on Q at position 1, M on A at position 1, L on V at position 5, L on I at position 5, L on T at position 5, L at Q at position 5, L at H at position 5, L at A at position 5, F at I at position 8, F at V at position and 8, L on V at position 9, L on I at position 9, L on T at position 9, L on Q at position 9, L on H at position 9, L on A at position 9, R on H at position 11, R on Q at position 11, F on I at position 15, F on V at position 15, K on Q at position 19, K on T at position 19, K on S at position 19, K on H at position 19 , W to S at position 22, W to H at position 22, N to H at position 25, N to S at position 25, N to Q at position 25, R to H at position 27, R to Q at position 27, L to V at position 28, L to I at position 28, L to T at position 28, L to Q at position 28, L to H at position 28, L to A at position 28, E to Q at position 29, Eon H at position 29, Y on H at position 30, Y on I at position 30, L on V at position 32, L on I at position 32, L on T at position 32, L on Q at position 32, L on H at position 32, L at A at position 32, K at Q at position 33, K at T at position 33, K at S at position 33, K at H at position 33, R at H at position 35, R at Q at position 35, M on V at position 36, M on I at position 36, M on T at position 36, M on Q at position 36, M on A at position 36, D on Q at position 39, D on H at position 39, D to G at position 39, E to Q at position 42, E to H at position 42, K to Q at position 45, K to T at position 45, K to S at position 45, K on H at position 45, L on V at position 47, L on I at position 47, L on T at position 47, L by, Q at position 47, L on H at position 47, L on A at position 47, K on Q at position 52, K on T at position 52, K on S at position 52, K on H at position 52, F on I at position 67, F on V at position 67, R on H at position 71 , R on Q at position 71, D on Q at position 73, D on H at position 73, D on G at position 73, E on Q at position 81, E on H at position 81, E on Q at position 85, E on H at position 85, Y on H at position 92, Y on I at position 92, K on Q at position 99, K on T at position 99, K on S at position 99, K on H at position AI 99, E on Q at position 103, E on H at position 103, E on Q at position 104, E on H at position 104, K on Q at position 105, K on T at position 105, K on S at position 105, K on H at position 105, E on Q on, position 107, E on H at position 107, K on Q at position 108, K on T at position 108, K on S at position 108, K on H at position 108, E on Q at position 109, E on H at position 109, D on Q at position 110, D on H at position 110, D on G at position 110, F on I at position 111, F on V at position 111 , R to H at position 113, R to Q at position 113, L to V at position 116, L to I at position 116, L to T at position 116, L to Q at position 116 , L on H at position 116, L on A at position 116, L on V at position 120, L on I at position 120, L on T at position 120, L on Q at position 120, L on H at position 120, L on A at position 120, K on Q at position 123, K on T at position 123, K on S at position 123, K on H at position 123, R on H at position 124, R on Q at position 124, R on N at position 128, R on Q at position 128, L on V at position 130, L on I at position 130, L on T at position 130, L on Q at position 130, L on H at position 130, L on And at position 130, K on Q at position 134, K on T at position 134, K on S at position 134, K on H at position 134, K on Q at position 136, K to T at position 136, K to S at position 136, K to H at position 136, E to Q at position 137, E to H at position 137, Y to H at position 138, Y to I at position 138, R to H at position 152, R at Q at position 152, Y at H at position 155, Y at I at position 155, R at And at position 159, R at Q at position 159, Y at H at position 163, Y at I at position 163, R to H at position 165, R to Q at position 165, M to D at position 1, M to E at position 1, M to K at position 1, M to N at position 1, M to R at position 1, M on S in position 1, L on D at position 5, L on E at position 5, L on K at position 5, L on N at position 5, L on R at and 5, L to S at position 5, L to D at position 6, L to E at position 6, L to K at position 6, L to N at position 6, L to R at position 6, L to S at position 6, L to Q at position 6, L to T at position 6, F to E at position 8, F to K at position 8, F to R at position 8, F to D at position 8, L to D at position 9 , L to E at position 9, L to K at position 9, L to N at position 9, L to R at position 9, L to S at position 9, Q to D at position 10, Q to E at position 10, Q to K at position 10, Q to N at position 10, Q to R at position 10, Q to S at position 10, Q to T at position 10, S to D at position 12, S to E at position 12, S K to polo 12, S on R at position 12, S on D at position 13, S on B at position 13, S on K at position 13, S on R at position 13, S on N at position 13, S on Q at position 13, S to T at position 13, N to D at position 14, N to E at position 14, N to K at position 14, N to Q at position 14, N to R at position 14, N to S at position 14 , N to T at position 14, F to D at position 15, F to E at position 15, F to K at position 15, F to R at position 15, Q to D at position 16, Q to E at position 16, Q to K at position 16, Q to N at position 16, Q to R at position 16, Q to S at position 16, Q to T at position 16, C to D at position 17, C to E at AI 17, C to K at position 17, C to N at position 17, C to Q at position 17, C to R at position 17, C to S at position 17, C to T at position 17, L to N at position 20, L to Q at position 20, L to R at position 20, L to S at position 20, L to T at position 20, L to D at position 20, L to E at position 20, L to K at position 20 , W to D at position 22, W to E at position 22, W to K at position 22, W to R at position 22, Q to D at position 23, Q to E at position 23, Q to K at position 23, Q on R at position 23, L on D at position 24, L on E at position 24, L on K at position 24, L on R at position 24, W on D at position 79, W on E at position and 79, W on K at position 79, W on R at position 79, N on D at position 80, N on E at position 80, N on K at position 80, N on R at position 80, T on D at position 82, T to E at position 82, T to K at position 82, T to R at position 82, I to D at position 83, I to E at position 83, I to K at position 83, I to R at position 83 , I to N at position 83, I to Q at position 83, I to S at position 83, I to T at position 83, N to D at position 86, N to E at position 86, N to K at position 86, N to R at position 86, N to Q at position 86, N to S at position 86, N to T at position 86, L to D at position 87, L to E at position 87, L to K at position 87, L to R at position 87, L to N at position 87, L to Q at position 87, L to S at position 87, L to T at position 87, A to D at position 89, A to E at position 89 , A to K at position 89, A to R at position 89, N to D at position 90, N to E at position 90, N to K at position 90, N to Q at position 90, N to R at position 90, N at S at position 90, N at T at position 90, V at D at position 91, V at E at position 91, V at K at position 91, V at N at position 91, V at Q at position 91, V to R at position 91, V to S at position 91, V to T at position 91, Q to D at position 94, Q to E at position 94, Q to Q at position 94, Q to N at position 94, Q to R at position 94, Q to S at position 94, Q to T at position 94, I to D at position 95, I to E at position 95, I to K at position 95, I to N at position 95, I to Q at position 95, I to R at position 95, I to S at position 95, I to T at position 95, N to D at position 97, N to E at position 97, N to K at position 97, N to N at position 97, N on Q at position 97, N on R at position 97, N on S at position 97, N on T at position 97, L on D at position 98, L on E at position 98, L on K at position 98, L to N at position 98, L to Q at position 98, L to R at position 98, L to S at position 98, L to T at position 98, V to D at position 101, V and E at position 101, V at K at position 101, V at N at position 101, V at Q at position 101, V at R at position 101, V at S at position 101, V at T at position 101, M at C at position 1, L at C at position 6, Q at C at position 10, S at C at position 13, Q at C at position 16, L at C at position 17, V at C at position 101, L at C at position 98, N at C at position 97, Q at C at position 94, V at C at position 91, or N at C at position 90, where residue 1 corresponds to residue 1 of the mature IFN-β cytokine, presented further in SEQ ID NO: 196; and further includes an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites that are not present in the parent polypeptide.

3-D structural homologs

In other embodiments of the invention, the hyperglycosylated protease-resistant variant of interferon is a modified IFN-β cytokine comprising one or more amino acid substitutions at one or more target positions in SEQ ID NO: 196 (as shown below in FIG. 3) corresponding to a structurally linked a modified amino acid position in the three-dimensional structure of the above IFN-α2b polypeptide variant, where the replacement (s) leads to greater resistance to proteases, as measured by incubation with a protease or blood lysate Or by incubation with serum (as described above), as compared to the unmodified IFN-β. In some of these embodiments of the invention, the modified IFN-β is selected from proteins comprising one or more single amino acid substitutions at one or more target positions in SEQ ID NO: 196 (as shown below in FIG. 3) corresponding to any amino acid positions: 39, 42 , 45, 47, 52, 67, 71, 73, 81, 107, 108, 109, 110, 111, 113, 116, 120, 123, 124, 128,130, 134, 136, 137, 163 and 165, where the mutations include insertions, deletions and substitutions of the native amino acid residue (s); wherein the variant further comprises an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites that are not present in the parent polypeptide.

In other embodiments of the invention, the hyperglycosylated, protease-resistant variant of interferon is a modified IFN-β cytokine comprising one or more amino acid substitutions, where the substitutions are selected from amino acid substitutions in SEQ ID NO: 196 (as shown below in FIG. 3), corresponding to: D to Q at position 39, D to H at position 39, D to G at position 39, E to Q at position 42, E to H at position 42, K to Q at position 45, K to T at position 45, K on S at position 45, K on H at position 45, L on V at position 47, L on I at position 47, L on T at position 47, L and Q at position 47, L at H at position 47, L at A at position 47, K at Q at position 52, K at T at position 52, K at S at position 52, K at H at position 52, F at I at 67, F at V at 67, R at H at 71, R at Q at 71, D at H at 73, D at G at 73, D at Q at 73, E at Q at position 81, E at H at position 81, E at Q at position 107, E at H at position 107, K at Q at position 108, K at T at position 108, K at S at position 108, K at H at position 108, E on Q at position 109, E on H at position 109, D on Q at position 110, D on H at position 110, D on G at position 110, F on I in polo position 111, F on V at position 111, R on H at position 113, R on Q at position 113, L on V at position 116, L on I at position 116, L on T at position 116, L on Q at position 116, L on H at position 116, L on A at position 116, L on V at position 120, L on I at position 120, L on T at position 120, L on Q at position 120, L on H at position 120 , L on A at position 120, K on Q at position 123, K on T at position 123, K on S at position 123, K on H at position 123, R on H at position 124 “R on Q at position 124, R to H at position 128, R to Q at position 128, L to V at position 130, L to I at position 130, L to T at position 130, L to Q at position 130, L on H at position 130, L on A at position 130, K on Q at position 134, K on T at position 134, K on S at position 134, K on H at position 134, K on Q at position 136 , K to T at position 136, K to S at position 136, K to H at position 136, E to Q at position 137, E to H at position 137, Y to And at position 163, Y to I at position 163, R on H at position 165, or R on Q at position 165, where the first amino acid from the list is substituted by the second at the indicated position; wherein the variant further comprises an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites that are not present in the parent polypeptide.

In some embodiments of the invention, any hyperglycosylated protease-resistant IFN-R variants have increased stability compared to the unmodified (parental) cytokine, where stability is assessed by measuring residual biological activity after incubation with either a mixture of proteases, or with individual proteases, or with a blood lysate , or blood serum as described above. In other embodiments of the invention, any hyperglycosylated protease-resistant IFN-β variants have increased biological activity compared to the unmodified (parental) cytokine after incubation or with a mixture of proteases, or with individual proteases, or with a blood lysate, or with blood serum as described above .

In other embodiments of the invention, the hyperglycosylated protease-resistant variant of interferon is a modified IFN-β1 cytokine comprising one or more amino acid substitutions at one or more target positions in SEQ ID NO: 197 (as shown in FIG. 22) corresponding to a structurally-linked modified the amino acid position in the three-dimensional structure of the above IFN-α2b polypeptide variant, where the replacement (s) leads to greater resistance to proteases, as measured by incubation with a protease or blood lysate, or incubation with blood serum (as described above), compared with unmodified IFN-β1. In some of these embodiments of the invention, the modified IFN-β1 is selected from proteins comprising one or more single amino acid substitutions at one or more target positions in SEQ ID NO: 197 (as shown in FIG. 22) corresponding to any amino acid positions: 39, 42 , 45, 47, 52, 67, 71, 73, 81, 107, 108, 109, 110, 111, 113, 116, 120, 123, 124, 128, 130, 134, 136, 137, 163 and 165, where mutations include insertions, deletions and substitutions of the native amino acid residue (s); wherein the variant further comprises an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites that are not present in the parent polypeptide.

In some embodiments of the invention, any hyperglycosylated protease-resistant IFN-β1 variants described above have increased stability compared to the unmodified (parental) cytokine, where stability is assessed by measuring the residual biological activity after incubation with either a mixture of proteases or individual proteases, or with a blood lysate or blood serum as described above. In other embodiments of the invention, any hyperglycosylated protease-resistant IFN-β1 variants described above have increased biological activity compared to unmodified (parental) cytokines after incubation or with a mixture of proteases, or with individual proteases, or with a blood lysate or with blood serum as described above.

In other embodiments of the invention, the protease-resistant variant of interferon is a modified IFN-β2a cytokine comprising one or more amino acid substitutions at one or more target positions in SEQ ID NO: 198 (as shown in FIG. 23) corresponding to a structurally linked modified amino acid the position in the three-dimensional structure of the above described IFN-α2b polypeptide variant, where the replacement (s) leads to greater protease resistance, as measured by incubation with a protease or blood lysate, or by incubation with serum blood (as described above), compared with unmodified IFN-β2a. In some of these embodiments of the invention, the modified IFN-β2a is selected from proteins comprising one or more single amino acid substitutions at one or more target positions in SEQ ID NO: 198 (as shown in FIG. 23) corresponding to any amino acid positions: 39, 42 , 45, 47, 52, 67, 71, 73, 81, 107, 108, 109, 110, 111, 113, 116, 120, 123, 124, 128, 130, 134, 136, 137, 163 and 165, where mutations include insertions, deletions and substitutions of the native amino acid residue (s); where the options further include an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites that are not present in the parent polypeptide.

In some embodiments of the invention, any hyperglycosylated protease-resistant IFN-β2a variants described above have increased stability compared to unmodified (parental) cytokines, where stability is assessed by measuring residual biological activity after incubation with either a mixture of proteases or individual proteases, or with blood lysate, or with blood serum as described above. In other embodiments of the invention, any hyperglycosylated protease-resistant IFN-β2a variants described above have increased biological activity compared to unmodified (parental) cytokine after incubation or with a mixture of proteases, or with individual proteases, or with a blood lysate, or with blood serum as described above.

In another aspect, the present invention relates to the cytokine structural homolog of any of the protease-resistant IFN-β variants described above, wherein the homolog comprises one or more amino acid substitutions at positions corresponding to three-dimensional structural-like modified positions in the three-dimensional structure of the modified IFN-β. In many embodiments of the invention, the homolog has increased proteolysis resistance compared to its unmodified cytokine copy, where proteolysis resistance is determined by in vitro mixing with a protease, incubation with blood or incubation with blood serum. In many embodiments of the invention, the cytokine is an IFN-β cytokine.

In another aspect, the present invention relates to a modified IFN-β cytokine (e.g., a hyperglycosylated protease-resistant IFN-p variant) comprising one or more amino acid substitutions at one or more target positions in SEQ ID NO: 196 (the amino acid sequence is shown in FIG. .23), corresponding to the structurally linked modified amino acid position in the three-dimensional structure of any of the modified IFN-β cytokines described above, where substitutions lead to greater protease resistance, which This can be achieved by incubation with a protease or a blood lysate, or by incubation with a blood serum, as compared with unmodified IFN-β.

In some embodiments of the invention, any of the hyperglycosylated protease-resistant IFN-β variants described above are more stable than the unmodified (parental) cytokine, where stability is assessed by measuring the residual biological activity with respect to inhibiting virus replication or stimulating cell proliferation in the corresponding cells after incubation or with a mixture of proteases, or with individual proteases, or with a blood lysate, or with blood serum as described above.

In some embodiments of the invention, any of the hyperglycosylated protease-resistant IFN-β variants described above have increased biological activity compared to the unmodified (parental) cytokine, where the activity is evaluated by the ability to inhibit virus replication in the corresponding cells or to inhibit cell proliferation in the corresponding cells after incubation or with a mixture of proteases, or with individual proteases, or with a blood lysate, or with blood serum, as described above.

In some embodiments of the invention, the hyperglycosylated, protease-resistant variant of IFN-β (“modified cytokine IFN-β”) is selected from the group of proteins comprising one or more single amino acid substitutions in SEQ ID NO: 196, as shown below in FIG. 3, corresponding to replacement: M by V in position 1, M by I in position 1, M by T in position 1, M by Q in position 1, M by A in position 1, L by V in position 5, L by I in position 5 , L on T at position 5, L on Q at position 5, L on H at position 5, L on A at position 5, F on I at position 8, F on V at position 8, L on V at AI 9, L on I at position 9, L on T at position 9, L on Q at position 9, L on H at position 9, L on A at position 9, R on H at position 11, R on Q at position 11, F on I at position 15, F on V at position 15, K on Q at position 19, K on T at position 19, K on S at position 19, K on H at position 19, W on S at position 22 , W on H at position 22, N on H at position 25, N on S at position 25, N on Q at position 25, R at H position 27, R at Q position 27, L at V at position 28, L at I at position 28, L at T at position 28, L at Q at position 28, L at H at position 28, L at A at position 28, E at Q at position 29, E at H at position 29, Y at N in pos 30, Y on I at position 30, L on V at position 32, L on I at position 32, L on T at position 32, L on Q at position 32, L on H at position 32, L on A at position 32, K on Q at position 33, K on T at position 33, K on S at position 33, K on H at position 33, R on H at position 35, R on Q at position 35, M on V at position 36 , M on I at position 36, M on T at position 36, M on Q at position 36, M on A at position 36, D on Q at position 39, D on H at position 39, D on G at position 39, E at Q at position 42, E at H at position 42, K at Q at position 45, K at T at position 45, K at S at position 45, K at H at position 45, L at V at position 47, L on I at position 47, L on T at position 47, L on Q at position 47, L on H at position 47, L on A at position 47, K on Q at position 52, K on T at position 52, K on S at position 52, K on H at position 52, F on I at position 67, F on V at position 67, R on H at position 71, R on Q at position 71, D on Q at position 73 , D to H at position 73, D to G at position 73, E to Q at position 81, E to H at position 81, E to Q at position 85, E to H at position 85, Y to H at position 92, Y on I at position 92, K on Q at position 99, K on T at position 99, K on S at position 99, K on H at position 99, E on Q at position 103, E on H at position 103, E on Q at position 104, E on H at position 104, K on Q at position 105, K on T at position 105, K on S at position 105, K on H at position 105, E on Q at position 107, E on H at position 107, K on Q at position 108, K on T at position 108, K on S at position 108, K on H at position 108, E on Q at position 109, E on H at position 109, D on Q at position 110, D on H at position 110, D on G at position 110, F on I at position 111, F on V at position 111, R on H at position 113, R on Q at position 113 , L on V at position 116, L on I at position 116, L on T at position 116, L on Q at position 116, L on H at position 116, L on A at position 116, L on V at position 120, L on I at position 120, L on T at position 120, L on Q at position 120, L on H at position 120, L on A at position 120, K on Q at position 123, K on T at position 123, K on S at position 123, K on H at position 123, R on H at position 124, R on Q at position 124, R on H at position 128, R on Q at position 128 , L on V at position 130, L on I at position 130, L on T at position 130, L on Q at position 130, L on H at position 130, L on A at position 130, K on Q at position 134, K on T at position 134, K on S at position 134, K on H at position 134, K on Q at position 136, K on T at position 136, K on S at p position 136, K on H at position 136, E on Q at position 137, E on H at position 137, Y on H at position 138, Y on I at position 138, R on H at position 152, R on Q at position 152, Y on H at position 155, Y on I at position 155, R on H at position 159, R on Q at position 159, Y on H at position 163, Y on I at position 163, R on H at position 165 , R to Q at position 165, M to D at position 1, M to E at position 1, M to K at position 1, M to N at position 1, M to R at position 1, M to S at position 1, L to D at position 5, L to E at position 5, L to K at position 5, L to N at position 5, L to R at position 5, L to S at position 5, L to D position 6, L to E at position 6, L to K at position 6, L to N at position 6, L to R at position 6, L to S at position 6, L to Q at position 6, L to T at position 6, F to E at position 8, F to K at position 8, F to R at position 8, F to D at position 8, L to D at position 9, L to E at position 9, L to K at position 9 , L to N at position 9, L to R at position 9, L to S at position 9, Q to D at position 10, Q to E at position 10, Q to K at position 10, Q to N at position 10, Q to R at position 10, Q to S at position 10, Q to T at position 10, S to D at position 12, S to E at position 12, S to K at position 12, S to R at position 12, S on D at position 13, S on E at position 13, S on K at position 13, S on R at position 13, S on N at position 13, S on Q at position 13, S on T at position 13, N on D at position 14, N at E at position 14, N at K at position 14, N at Q at position 14, N at R at position 14, N at S at position 14, N at T at position 14, F at D at position 15, F to E at position 15, F to K at position 15, F to R at position 15, Q to D at position 16, Q to E at position 16, Q to K at position 16, Q to N at position 16, Q to R at position 16, Q to S at position 16, Q to T at position 16, C to D at position 17, C to E at position 17, C to K at position 17, C n and N at position 17, C on Q at position 17, C at R at position 17, C at S at position 17, C at T at position 17, L at N at position 20, L at Q at position 20, L at R at position 20, L at S at position 20, L at T at position 20, L at D at position 20, L at E at position 20, L at K at position 20, W at D at position 22, W at E at position 22, W to K at position 22, W to R at position 22, Q to D at position 23, Q to E at position 23, Q to K at position 23, Q to R at position 23, L to D at position 24, L to E at position 24, L to K at position 24, L to R at position 24, W to D at position 79, W to E at position 79, W to K at position 79, W to R to the floor 79, N on D at position 80, N on E at position 80, N on K at position 80, N on R at position 80, T on D at position 82, T on E at position 82, T on K at position 82, T on R at position 82.1 on D at position 83.1 on E at position 83.1 on K at position 83.1 on R at position 83.1 on N at position 83.1 on Q at position 83 , 1 on S at position 83.1 on T at position 83, N on D at position 86, N on E at position 86, N on K at position 86, N on R at position 86, N on Q at position 86, N to S at position 86, N to T at position 86, L to D at position 87, L to E at position 87, L to K at position 87, L to R at position 87, L to N at position 87, L to Q at position 87, L to S at position 87, L to T at position 87, A to D at position 89, A to E at position 89, A to K at position 89, A to R at position 89 , N on D at position 90, N on E at position 90, N on K at position 90, N on Q at position 90, N on R at position 90, N on S at position 90, N on T at position 90, V on D at position 91, V on E at position 91, V on K at position 91, V on N at position 91, V on Q at position 91, V on R at position 91, V on S at position 91, V to T at position 91, Q to D at position 94, Q to E at position 94, Q to Q at position 94, Q to N at position 94, Q to R at position 94, Q to S at position 94, Q on T at position 94.1 on D at position 95.1 on E at position 95.1 on K at position 95.1 on N at position 95.1 on Q at position 95.1 on R at position 95.1 on S at position 95.1 on T at position 95, N on D at position 97, N on E at position 97, N on K at position 97, N on N at position 97, N on Q at position 97, N on R at position 97, N at S at position 97, N at T at position 97, L at D at position 98, L at E at position 98, L at K at position 98, L at N at position 98, L at Q at position 98, L to R at position 98, L to S at position 98, L to T at position 98, V to D at position 101, V to E at position 101, V to K at position 101, V to N at position 101, V at Q at position 101, V at R at position 101, V at S at position 101, V at T at position 101, M at C at position 1, L at C at position 6, Q at C at position 10, S at C at position 13, Q at C at position 16, L at C at position 17, V at C at position 101, L at C at position 98, N at C at position 97, Q at C at position 94, V to C at position 91, or N to C at position 90, or a combination of such substitutions, where residue 1 corresponds to residue 1 of the mature IFN-β cytokine, which is further presented in SEQ ID NO: 196 (as shown further in FIG. 3); wherein the variant further comprises an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites that are not present in the parent polypeptide.

In other embodiments of the invention, the hyperglycosylated, protease-resistant variant of IFN-J3 (“modified cytokine IFN-β”) is selected from the group of proteins comprising one or more single amino acid substitutions in SEQ ID NO: 196, as shown in FIG. 3, corresponding to the substitution : D on Q at position 39, D on H at position 39, D on G at position 39, E on Q at position 42, E on H at position 42, K on Q at position 45, K on T at position 45, K to S at position 45, K to H at position 45, L to V at position 47, L to I at position 47, L to T at position 47, L to Q at position 47, L to H at position and 47, L on A at position 47, K on Q at position 52, K on T at position 52, K on S at position 52, K on H at position 52, F on I at position 67, F on V at position 67, R on H at position 71, R on Q at position 71, D on H at position 73, D on G at position 73, D on Q at position 73, E on Q at position 81, E on H at position 81 , E on Q at position 107, E on And at position 107, K on Q at position 108, K on T at position 108, K on S at position 108, K on H at position 108, E on Q at position 109, E on H at position 109, D on Q at position 110, D on H at position 110, D on G at position 110, F on I at position 111, F on V at position 111, R on H at position 113, R on Q at position 113, L on V at position 116, L on I at position 116, L on T at position 116, L on Q at position 116, L on H at position 116, L on A at position 116, L on V at position 120, L on I at position 120, L on T at position 120, L on Q at position 120, L on H at position 120, L on A at position 120, K on Q at position 123, K on T at position 123, K on S at position 123, K on H at position 123, R on H at position 124, R on Q at position 124, R on H at position 128, R on Q at position 128 , L to V at position 130, L to I at position 130, L to T at position 130, L to Q at position 130, L to H at position 130, L to A to polo When 130, K on Q at position 134, K on T at position 134, K on S at position 134, K on H at position 134, K on Q at position 136, K on T at position 136, K on S at position 136, K on H at position 136, E on Q at position 137, E on H at position 137, Y on H at position 163, Y on I at position 1631, R on H at position 165, or R on Q at position 165, or any combination of such substitutions, where the first amino acid from the list is substituted by the second at the indicated position; wherein the variant further comprises an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites that are not present in the parent polypeptide.

In some embodiments of the invention, the hyperglycosylated, protease-resistant variant of IFN-β (“modified cytokine IFN-β”) is selected from the group of proteins comprising one or more single amino acid substitutions in SEQ ID NO: 196, as shown below in FIG. 3, corresponding to replacement: M by V in position 1, M by I in position 1, M by T in position 1, M by Q in position 1, M by A in position 1, L by V in position 5, L by I in position 5 , L on T at position 5, L on Q at position 5, L on H at position 5, L on A at position 5, F on I at position 8, F on V at position 8, L on V at AI 9, L on I at position 9, L on T at position 9, L on Q at position 9, L on And at position 9, L on A at position 9, R on H at position 11, R on Q at position 11, F on I at position 15, F on V at position 15, K on Q at position 19, K on T at position 19, K on S at position 19, K on H at position 19, W on S at position 22 , W on H at position 22, N on H at position 25, N on S at position 25, N on Q at position 25, R at H position 27, R at Q position 27, L at V at position 28, L at I at position 28, L at T at position 28, L at Q at position 28, L at H at position 28, L at A at position 28, E at Q at position 29, E at H at position 29, Y at N in pos 30, Y on I at position 30, L on V at position 32, L on I at position 32, L on T at position 32, L on Q at position 32, L on H at position 32, L on A at position 32, K on Q at position 33, K on T at position 33, K on S at position 33, K on H at position 33, R on H at position 35, R on Q at position 35, M on V at position 36 , M on I at position 36, M on T at position 36, M on Q at position 36, M on A at position 36, D on Q at position 39, D on H at position 39, D on G at position 39, E at Q at position 42, E at H at position 42, K at Q at position 45, K at T at position 45, K at S at position 45, K at H at position 45, L at V at position 47, L on I at position 47, L on T at position 47, L by, Q at position 47, L on H at position 47, L on A at position 47, K on Q at position 52, K on T at position 52, K on S at position 52, K on H at position 52, F on I at position 67, F on V at position 67, R on H at position 71, R on Q at position 71, D on Q at position 73, D on H at position 73, D on G at position 73, E on Q at position 81, E on H at position 81, E on Q at position 85, E on H at position 85, Y on H at position 92 , Y on I at position 92, K on O at position 99, K on T at position 99, K on S at position 99, K on H at position 99, E on Q at position 103, E on H at position 103, E on Q at position 104, E on H at position 104, K on Q at position 105, K on T at position 105, K on S at position 105, K on H at position 105, E on Q at position 107, E on H at position 107, K on Q at position 108, K on T at position 108, K on S at position 108, K on H at position 108, E on Q at position 109, E on H at position 109, D on Q at position 110, D on H at position 110, D on G at position 110, F on I at position 111, F on V at position 111, R on H at position 113, R on Q at position 113 , L to Vat position 116, L to I at position 116, L to T at position 116, L to Q at position 116, L to H at position 116, L to A to position 116, L on V at position 120, L on I at position 120, L on T at position 120, L on Q at position 120, L on H at position 120, L on A at position 120, K on Q at position 123, K on T at position 123, K on S at position 123, K on H at position 123, R on H at position 124, R on Q at position 124, R on H at position 128, R on Q at position 128 , L on V at position 130, L on I at position 130, L on T at position 130, L on Q at position 130, L on H at position 130, L on A at position 130, K on Q at position 134, K on T at position 134, K on S at position 134, K on H at position 134, K on Q at position 136, K on T at position 136, K on S at AI 136, K on H at position 136, E on Q at position 137, E on H at position 137, Y on H at position 138, Y on I at position 138, R on H at position 152, R on Q at position 152, Y on H at position 155, Y on I at position 155, R on H at position 159, R on Q at position 159, Y on H at position 163, Y on I at position 163, R on H at position 165 , R to Q at position 165, M to D at position 1, M to E at position 1, M to K at position 1, M to N at position 1, M to R at position 1, M to S at position 1, L to D at position 5, L to E at position 5, L to K at position 5, L to N at position 5, L to R at position 5, L to S at position 5, L to D in polo 6, L on E at position 6, L on K at position 6, L on N at position 6, L on R at position 6, L on S at position 6, L on Q at position 6, L on T at position 6, F to E at position 8, F to K at position 8, F to R at position 8, F to D at position 8, L to D at position 9, L to E at position 9, L to K at position 9 , L to N at position 9, L to R at position 9, L to S at position 9, Q to D at position 10, Q to E at position 10, Q to K at position 10, Q to N at position 10, Q to R at position 10, Q to S at position 10, Q to T at position 10, S to D at position 12, S to E at position 12, S to K at position 12, S to R at position 12, S on d in position 13, S on E at position 13, S on K at position 13, S on R at position 13, S on N at position 13, S on Q at position 13, S on T at position 13, N on D at position 14, N to E at position 14, N to K at position 14, N to Q at position 14, N to R at position 14, N to S at position 14, N to T at position 14, F to D at position 15 , F to E at position 15, F to K at position 15, F to R at position 15, Q to D at position 16, Q to E at position 16, Q to K at position 16, Q to N at position 16, Q to R at position 16, Q to S at position 16, Q to T at position 16, C to D at position 17, C to E at position 17, C to K at position 17, C to N at position 17, C on Q at position 17, C on R at position 17, C on S at position 17, C on T at position 17, L on N at position 20, L on Q at position 20, L on R at position 20, L to S at position 20, L to T at position 20, L to D at position 20, L to E at position 20, L to K at position 20, W to D at position 22, W to E at position 22 , W to K at position 22, W to R at position 22, Q to D at position 23, Q to E at position 23, Q to K at position 23, Q to R at position 23, L to D at position 24, L to E at position 24, L to K at position 24, L to R at position 24, W to D at position 79, W to E at position 79, W to K at position 79, W to R to the floor 79, N on D at position 80, N on E at position 80, N on K at position 80, N on R at position 80, T on D at position 82, T on E at position 82, T on K at position 82, T on R at position 82.1 on D at position 83.1 on E at position 83.1 on K at position 83.1 on R at position 83.1 on N at position 83.1 on Q at position 83 , 1 on S at position 83.1 on T at position 83, N on D at position 86, N on E at position 86, N on K at position 86, N on R at position 86, N on Q at position 86, N to S at position 86, N to T at position 86, L to D at position 87, L to E at position 87, L to K at position 87, L to R at position 87, L to N at position 87, L to Q at position 87, L to S at position 87, L to T at position 87, A to D at position 89, A to E at position 89, A to K at position 89, A to R at position 89 , N on D at position 90, N on E at position 90, N on K at position 90, N on Q at position 90, N on R at position 90, N on S at position 90, N on T at position 90, V on D at position 91, V on E at position 91, V on K at position 91, V on N at position 91, V on Q at position 91, V on R at position 91, V on S at position 91, V to T at position 91, Q to D at position 94, Q to E at position 94, Q to Q at position 94, Q to N at position 94, Q to R at position 94, Q to S at position 94, Q on T at position 94.1 on D at position 95.1 on E at position 95.1 on K at position 95.1 by N at position 95.1 on Q at position 95.1 on R at position 95.1 on S at position 95.1 on T at position 95, And on D at position 97, N at E at position 97, N at K at position 97, N at N at position 97, N at Q at position 97, N at R at position 97, N at S at position 97, N at T at position 97, L at D at position 98, L at E at position 98, L at K at position 98, L at N at position 98, L at Q at position 98, L to R at position 98, L to S at position 98, L to T at position 98, V to D at position 101, V to E at position 101, V to K at position 101, V to N at position 101, V on Q at position 101, V on R at position 101, V on S at position 101, V at T at position 101, M at C at position 1, L at C at position 6, Q at C at position 10, S at C at position 13, Q at C at position 16, L at C at position 17, V at C at position 101, L at C at position 98, N at C at position 97, Q at C at position 94, V on C at position 91, N on C at position 90, D on Q at position 39, D on H at position 39, D on G at position 39, E on Q at position 42, E on H at position 42 , K to Q at position 45, K to T at position 45, K to S at position 45, K to H at position 45, L to V at position 47, L to I at position 47, L at T at position 47, L at Q at position 47, L at And at position 47, L at A at position 47, K at Q at position 52, K at T at position 52, K at S at position 52, K on H at position 52, F on I at position 67, F on V at position 67, R on H at position 71, R on Q at position 71, D on H at position 73, D on G at position 73, D on Q at position 73, E at Q at position 81, E at H at position 81, E at Q at position 107, E at H at position 107, K at Q at position 108, K at T at position 108, K at S at position 108, K on H at position 108, E on Q at position 109, E on H at position 109, D on Q at position 110, D on H at position 110, D on G to the floor When 110, F on I at position 111, F on V at position 111, R on H at position 113, R on Q at position 113, L on V at position 116, L on I at position 116, L on T at position 116, L on Q at position 116, L on H at position 116, L on A at position 116, L on V at position 120, L on I at position 120, L on T at position 120, L on Q at position 120 , L on H at position 120, L on A at position 120, K on Q at position 123, K on T at position 123, K on S at position 123, K on H at position 123, R on H at position 124, R to Q at position 124, R to H at position 128, R to Q at position 128, L to V at position 130, L to I at position 130, L to T at position 130, L on Q at position 130, L on H at position 130, L on A at position 130, K on Q at position 134, K on T at position 134, K on S at position 134, K on H at position 134 , K on Q at position 136, K on T at position 136, K on S at position 136, K on H at position 136, E on Q at position 137, E on H at position 137, Y on H at position 163, Y on I at position 163, R on H at position 165, or R on Q at position 165, or any combination of such substitutions, where the first amino acid in the list is replaced by the second at the indicated position; wherein the variant further comprises an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites that are not present in the parent polypeptide.

In a preferred embodiment of the invention, the hyperglycosylated, protease-resistant variant of IFN-β (“modified IFN-β cytokine”) is selected from the group consisting of a modified IFN-β comprising the amino acid sequence as set forth in any SEQ ID Nos. 234-289, and 989-1302; wherein the variant further comprises an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites that are not present in the parent polypeptide.

In a preferred embodiment of the invention, the hyperglycosylated, protease-resistant variant of IFN-β (“modified IFN-β cytokine”) includes one or more amino acid substitutions, as shown in Table 2 (IFN-β) below; wherein the variant further comprises an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites that are not present in the parent polypeptide.

In some embodiments of the invention, any of the above protease-resistant or protease-resistant, hyperglycosylated IFN-β variants is an IFN-β1a variant and this variant is a [S99N] IFN-β1a glycopeptide, where the [S99N] IFN-β1a glycopeptide is an IFN-β1a variant having (a) an asparagine residue substituted with a native serine residue at the 99 amino acid position in the amino acid sequence of IFN-β1a (where the amino acid position of S99 is shown further in Fig. 24; and corresponds to S74 in the amino acid pos the IFN-β assay presented further in FIG. 3); and (b) a carbohydrate moiety covalently linked to the R group of said asparagine residue.

In some embodiments of the invention, any of the protease-resistant or protease-resistant, hyperglycosylated IFN-β variants described above is an IFN-β1a variant and this variant is a glycopeptide [S99N, E134N] IFN-β1a, where the glycopeptide [S99N, E134N] IFN -β1a is an IFN-β1a variant having (a) an asparagine residue substituted for each of the native serine and glutamic acid residues at 99 and 134 amino acid positions, respectively, in the amino acid sequence of IFN-β1a (where amino acid positions S99 and E134 are further shown in FIG. 24; and correspond to S74 and E109, respectively, in the amino acid sequence of IFN-β shown in FIG. 3); and (b) a carbohydrate moiety covalently linked to the R group of each of these asparagine residues.

In some embodiments of the invention, any of the protease-resistant or protease-resistant, hyperglycosylated IFN-β variants described above is an IFN-β1a variant and this variant is a glycopeptide [S99N, E134N, F136T] IFN-β1a, where the glycopeptide [S99N, E134N , F136T] IFN-β1a is a variant of IFN-β1a having (a) residues of asparagine, asparagine and threonine substituted for residues of native serine, glutamic acid and phenylalanine at amino acid positions 99, 134 and 136, respectively, in the amino acid sequence of IFN-β1a ( where am nokislotnye position S99, E134 and F136 are set forth in Figure 24, and correspond to S74, Fill and E109, respectively, in the amino acid sequence of IFN-β, shown hereinafter in Figure 3); and (b) a carbohydrate moiety covalently linked to the R group of each of these asparagine residues.

In some embodiments of the invention, any of the above protease-resistant or protease-resistant, hyperglycosylated IFN-β variants is an IFN-β1a variant and this variant is a [E134N] IFN-β1a glycopeptide, where the [E134N] IFN-β1a glycopeptide is an IFN-β1a variant having (a) an asparagine residue substituted with a native glutamic acid residue at 134 amino acid positions in the amino acid sequence of IFN-β1a (where amino acid positions are presented further in FIG. 24); and (b) a carbohydrate moiety covalently linked to the R group of said asparagine residue.

In some embodiments of the invention, any of the above protease-resistant or protease-resistant, hyperglycosylated IFN-β variants is an IFN-β1a variant and this variant is a glycopeptide [E134N, F136T] IFN-β1a, where the glycopeptide [E134N, F136T] IFN β-1a is a variant of IFN-β1a having (a) residues of asparagine and threonine substituted for residues of native glutamic acid and phenylalanine at 134 and 136 amino acid positions respectively in the amino acid sequence of IFN-β1a (where the amino acid positions are further shown in FIG. 24); and (b) a carbohydrate moiety covalently linked to the R group of each of these asparagine residues.

In some embodiments of the invention, any of the above protease-resistant or protease-resistant, hyperglycosylated IFN-β variants is an IFN-β1a variant and this variant is a [E134T] IFN-β1a glycopeptide, where [E134T] IFN-β1a glycopeptide is an IFN-β1a variant having (a) a threonine residue substituted with a native glutamic acid residue at position 134 in the amino acid sequence of IFN-β1a (where amino acid positions are presented further in FIG. 24); and (b) a carbohydrate moiety covalently linked to the R group of said threonine residue.

In some embodiments of the invention, any of the protease-resistant or protease-resistant, hyperglycosylated IFN-β variants described above is an IFN-β1a variant, and this variant is a glycopeptide [S99N, E134T] IFN-β1a, where the glycopeptide [S99N, E134T] IFN-β1a is a variant of IFN-β1a having (a) residues of asparagine and threonine substituted for residues of native serine and glutamic acid at amino acid positions 99 and 134, respectively, in the amino acid sequence of IFN-β1a (where amino acid positions are represented Further in Figure 24); and (b) a carbohydrate moiety covalently linked to the R group of each of these asparagine residues.

In some embodiments of the invention, any of the above protease-resistant or protease-resistant, hyperglycosylated IFN-β variants is an IFN-β1b variant, and this variant is a glycopeptide [S99N] IFN-β1b, where the glycopeptide [S99N]

IFN-β1b is an IFN-β1b variant having (a) an asparagine residue substituted with a native serine residue at the 99 amino acid position in the amino acid sequence of IFN-β1b (where the amino acid positions are shown further in Fig.24); and (b) a carbohydrate moiety covalently linked to the R group of said asparagine residue.

In some embodiments of the invention, any of the above protease-resistant or protease-resistant, hyperglycosylated IFN-β variants is an IFN-β1b variant, and this variant is a glycopeptide [S99N, E134N] IFN-β1b, where the glycopeptide [S99N, E134N] IFN-β1b is an IFN-β1b variant having (a) an asparagine residue substituted with a native serine residue and a glutamic acid residue at positions 99 and 134, respectively, in the amino acid sequence of IFN-β1b (where the amino acid positions are presented further in FIG. 24); and (b) a carbohydrate moiety covalently linked to the R group of each of these asparagine residues.

In some embodiments of the invention, any of the above protease-resistant or protease-resistant, hyperglycosylated IFN-β variants is an IFN-β1b variant, and this variant is a glycopeptide [S99N, E134N, F136T] IFN-β1b, where the glycopeptide [S99N, E134N, F136T] IFN-β1b is a variant of IFN-β1b having (a) residues of asparagine, asparagine and threonine substituted with residues of native serine, glutamic acid and phenylalanine at positions 99, 134 and 136, respectively, in the amino acid sequence of IFN-β1b ( where amino acid n Proposition shown below in Figure 24); and (b) a carbohydrate moiety covalently linked to the R group of each of these asparagine residues.

In some embodiments of the invention, any of the above protease-resistant or protease-resistant, hyperglycosylated IFN-β variants is an IFN-β1b variant, and this variant is a [E134N] IFN-β1b glycopeptide, where the [E134N] IFN-β1b glycopeptide is an IFN-β1b variant having (a) an asparagine residue substituted with a native glutamic acid residue at amino acid position 134 in the amino acid sequence of IFN-β1b (where amino acid positions are presented further in FIG. 24); and (b) a carbohydrate moiety covalently linked to the R group of said asparagine residue.

In some embodiments of the invention, any of the above protease-resistant or protease-resistant, hyperglycosylated IFN-β variants is an IFN-β1b variant, and this variant is a glycopeptide [E134N, F136T] IFN-β1b, where the glycopeptide [E134N, F136T] IFN-β1b is an IFN-β1b variant having (a) residues of asparagine and threonine substituted with residues of native glutamic acid and phenylalanine at amino acid positions 134 and 136, respectively, in the amino acid sequence of IFN-β1b (where the amino acid positions are further shown in FIG. 24); and (b) a carbohydrate moiety covalently linked to the R group of said asparagine residue.

In some embodiments of the invention, any of the above protease-resistant or protease-resistant, hyperglycosylated IFN-β variants is an IFN-β1b variant, and this variant is a [E134T] IFN-β1b glycopeptide, where the [E134T] IFN-β1b glycopeptide is an IFN-β1b variant having (a) a threonine residue substituted with a native glutamic acid residue at amino acid position 134 in the amino acid sequence of IFN-β1b (where amino acid positions are presented further in FIG. 24); and (b) a carbohydrate moiety covalently linked to the R group of said asparagine residue.

In some embodiments of the invention, any of the protease-resistant or protease-resistant, hyperglycosylated IFN-β variants described above is an IFN-β1b variant, and this variant is a glycopeptide [S99N, E134T] IFN-β1b, where the glycopeptide [S99N, E134T] IFN-β1b is a variant of IFN-β1b having (a) residues of asparagine and threonine substituted for residues of native serine and glutamic acid at positions 99 and 134, respectively, in the amino acid sequence of IFN-β1b (where amino acid positions are presented further in Fig.24) ; and (b) a carbohydrate moiety covalently linked to the R group of each of these asparagine and threonine residues.

Polypeptide variants of IFN-γ

In other embodiments of the invention, the hyperglycosylated protease-resistant variant of interferon is a modified IFN-γ cytokine comprising one or more amino acid substitutions at one or more target positions in SEQ ID NO: 199 (as shown in FIG. 4) corresponding to a structurally-linked modified the amino acid position in the three-dimensional structure of the above described IFN-α2b polypeptide variant, where the replacement (s) leads to greater resistance to proteases, as measured by incubation with a protease or with a blood lysate, or incubation with blood serum (as described above), compared with unmodified IFN-γ. In some of these embodiments of the invention, the modified IFN-γ is selected from proteins comprising one or more single amino acid substitutions at one or more target positions in SEQ ID NO: 199 (as shown in FIG. 4) corresponding to any amino acid positions: 33, 37 , 40, 41, 42, 58, 66, 64, 65, and 66, where the mutations include insertions, deletions, and substitutions of the native amino acid residue (s). In preferred embodiments of the invention, the substitutions are selected from amino acid substitutions in SEQ ID NO: 199 shown in Table 3 below, where the first amino acid in the list is substituted with the second amino acid at the indicated position; and where the variant further includes an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites that are not present in the parent polypeptide.

In other embodiments of the invention, the modified IFN-γ comprises an amino acid sequence corresponding to any SEQ ID NOS: 290-311, and further includes one or more glycosylation sites that are not present in the parent polypeptide.

In some embodiments of the invention, any of the protease-resistant or protease-resistant, hyperglycosylated IFN-γ variants described above is the [S99T] IFN-gamma glycopeptide, where the [S99T] IFN-gamma peptide is a variant of a mature, native IFN-gamma having (a) a threonine residue substituted with a native serine residue at amino acid position 99 in the amino acid sequence of IFN-gamma shown in Fig.31 (corresponding to S102 in the amino acid sequence of IFN-γ shown below in Fig.4); and (b) a carbohydrate moiety covalently linked to the R group of an asparagine residue at the 97 amino acid position in the amino acid sequence (a); and comprising at least one mutated protease cleavage site at the site of the native protease cleavage site present in the parent IFN-γ polypeptide.

Since the glycosylation site formed by N97, Y98, T99 in the variant [S99T] IFN-gamma differs from the glycosylation site formed by N97, Y98, S99 in the native IFN-gamma, the glycosylation site N97, Y98, T99 qualifies as a non-native glycosylation site that is not present in the parent polypeptide. Furthermore, as described in WO 02/081507, substitution of S99T in the amino acid sequence of native IFN-gamma provides greater glycosylation efficiency at the N97, Y98, T99 glycation site of the [S99T] IFN-gamma variant compared to glycosylation at the N97 glycosylation site, Y98, S99 in the variant [S99T] IFN-gamma. Thus, [S99T] IFN-gamma qualifies as a hyperglycosylated polypeptide variant of the parent IFN-gamma polypeptide (where the amino acid positions N97, Y98, S99 in the amino acid sequence of IFN-gamma shown in Fig. 31 correspond to N100, Y102 and S102 in the amino acid sequence IFN gamma shown in FIG. 4).

In some embodiments of the invention, any of the above protease-resistant or protease-resistant, hyperglycosylated IFN-γ variants is the [E38N] IFN-gamma glycopeptide, where the [E38N] IFN-gamma peptide is a variant of a mature, native IFN-gamma having (a) an asparagine residue substituted with a native glutamic acid residue at amino acid position 38 in the amino acid sequence of IFN-gamma shown in Fig. 31 (where amino acid E38 in the amino acid sequence of IFN-γ represented by e in Figure 31, corresponds to E41 in the amino acid sequence of IFN-γ, shown hereinafter in Figure 4); and (b) a carbohydrate moiety covalently linked to the R group of the aspartic moiety at 38 amino acid positions in amino acid sequence (a); and comprising at least one mutated protease cleavage site at the site of the native protease cleavage site present in the parent IFN-γ polypeptide.

In some embodiments of the invention, any of the protease-resistant or protease-resistant, hyperglycosylated IFN-γ variants described above is the [E38N, S99T] IFN gamma peptide, where the [E38N, S99T] IFN gamma peptide is a variant of the mature, native IFN -gamma having (a) residues of asparagine and threonine substituted for residues of native glutamic acid and serine at amino acid positions 38 and 99 in the amino acid sequence of IFN-gamma shown in Fig. 31 (where amino acids E38 and S99 are in the amino acid sequence telnosti IFN-γ, shown hereinafter in Figure 31 correspond to E41 and S102, respectively, in the amino acid sequence of IFN-γ, shown hereinafter in Figure 4); and (b) a carbohydrate moiety covalently linked to the R group of the aspartic residue at each amino acid position 38 and 97 in the amino acid sequence (a); and comprising at least one mutated protease cleavage site at the site of the native protease cleavage site present in the parent IFN-γ polypeptide.

In some embodiments of the invention, any of the protease-resistant or protease-resistant, hyperglycosylated IFN-γ variants described above is the [E38N, S40T] IFN gamma peptide, where the [E38N, S40T] IFN gamma peptide is a variant of the mature, native IFN -gamma having (a) residues of asparagine and threonine substituted for residues of native glutamic acid and serine at amino acid positions 38 and 40 in the amino acid sequence of IFN-gamma shown in Fig. 31 (where amino acids E38 and S99 are in the amino acid sequence IFN-γ shown in FIG. 31 further corresponds to E41 and S42, respectively, in the amino acid sequence of IFN-γ shown in FIG. 4); and (b) a carbohydrate moiety covalently linked to the R group of an asparagine residue at amino acid position 38 in amino acid sequence (a); and comprising at least one mutated protease cleavage site at the site of the native protease cleavage site present in the parent IFN-γ polypeptide.

In some embodiments of the invention, any of the protease-resistant or protease-resistant, hyperglycosylated IFN-γ variants described above is a glycopeptide [E38N, S40T, S99T]

IFN-gamma, where the glycopeptide [E38N, S40T, S99T] IFN-gamma is a variant of the mature, native IFN-gamma having (a) residues of asparagine, asparagine and threonine substituted for residues of native glutamic acid, serine and serine at amino acid positions 38, 40 and 99, respectively, in the amino acid sequence of the IFN-gamma shown in Fig. 31 (where amino acids E38, S40 and S99 in the amino acid sequence of IFN-γ, shown below in Fig. 31, correspond to E41, S43 and S102, respectively, in the amino acid sequence IFN-γ represented by d Lee in Figure 4); and (b) a carbohydrate moiety covalently linked to the R group of an asparagine residue at amino acid position 38 in amino acid sequence (a), and optionally further having (c) a carbohydrate moiety covalently linked to an R group of an asparagine residue at amino acid position 97 at amino acid position sequence (a); and comprising at least one mutated protease cleavage site at the site of the native protease cleavage site present in the parent IFN-γ polypeptide.

In some embodiments of the invention, any hyperglycosylated protease-resistant IFN-γ variants described above have increased stability compared to the unmodified (parental) cytokine, where stability is assessed by measuring residual biological activity after incubation with either a mixture of proteases or individual proteases, or with blood lysate, or with blood serum as described above. In other embodiments of the invention, any of the protease-resistant IFN-γ variants described above have increased biological activity compared to an unmodified (parental) cytokine, after incubation or with a mixture of proteases, or with individual proteases, or with a blood lysate, or with blood serum as described above.

Polypeptide variants of erythropoietin

In other embodiments of the invention, the hyperglycosylated protease-resistant variant of the cytokine is a modified erythropoietin cytokine comprising one or more amino acid substitutions at one or more target positions in SEQ ID NO: 201 (as shown further in FIG. 7) corresponding to a structurally linked modified amino acid position in the three-dimensional structure of the above described IFN-α2b polypeptide variant, where the replacement (s) leads to greater resistance to proteases, as measured by incubation with a protease or with a lysate ohm blood, or by incubation with blood serum (as described above), compared with unmodified erythropoietin. In some of these embodiments of the invention, the modified erythropoietin is selected from proteins comprising one or more single amino acid substitutions at one or more target positions in SEQ ID NO: 201 (as shown further in FIG. 7) corresponding to any amino acid position: 34, 45, 48, 49, 52, 53, 55, 72, 75, 76, 123, 129, 130, 131, 162 and 165, where the mutations include insertions, deletions and substitutions of the native amino acid residue (s). In preferred embodiments of the invention, the substitutions are selected from amino acid substitutions in SEQ ID NO: 201 shown in Table 4 below, where the first amino acid in the list is substituted with the second amino acid at the indicated position; and where the variant further includes an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites that are not present in the parent polypeptide.

In other embodiments of the invention, the modified erythropoietin comprises an amino acid sequence corresponding to any SEQ ID NO8: 940-977, and further includes one or more glycosylation sites that are not present in the parent polypeptide.

Polypeptide Options GM-CSF

In other embodiments of the invention, the hyperglycosylated protease-resistant variant of the cytokine is a modified GM-CSF cytokine comprising one or more amino acid substitutions at one or more target positions in SEQ ID NO: 202 (as shown further in FIG. 8) corresponding to a structurally linked modified amino acid position in the three-dimensional structure of the erythropoietin polypeptide variant described above, where replacement (s) leads to greater resistance to proteases, as measured by incubation with a protease or with a lysate ohm blood, or by incubation with blood serum (as described above), compared with unmodified GM-CSF. In some of these embodiments of the invention, the modified GM-CSF is selected from proteins comprising one or more single amino acid substitutions at one or more target positions in SEQ ID NO: 202 (as shown below in FIG. 8) corresponding to any amino acid positions: 38, 41, 45, 46, 48, 49, 51, 60, 63, 67, 92, 93, 119, 120, and 124, where mutations include insertions, deletions, and substitutions of the native amino acid residue (s). In preferred embodiments of the invention, the substitutions are selected from the amino acid substitutions in SEQ ID NO: 202 shown in Table 5 below, where the first amino acid in the list is substituted with the second amino acid at that position; wherein the variant further comprises an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites that are not present in the parent polypeptide.

In other embodiments of the invention, the modified GM-CSF includes an amino acid sequence corresponding to any SEQ ID N0s: 362-400, and further includes one or more glycosylation sites that are not present in the parent polypeptide.

G-CSF polypeptide variants

In other embodiments of the invention, the hyperglycosylated protease-resistant variant of the cytokine is a modified G-CSF cytokine comprising one or more amino acid substitutions at one or more target positions in SEQ ID NO: 210 (as shown below in FIG. 5) corresponding to a structurally linked a modified amino acid position in the three-dimensional structure of the IFN-α2b polypeptide variant described above, where the replacement (s) leads to greater protease resistance, as measured by incubation with a protease or with a blood lysate, and whether by incubation with blood serum (as described above), compared with unmodified G-CSF. In some of these embodiments of the invention, the modified G-CSF is selected from proteins comprising one or more single amino acid substitutions at one or more target positions in SEQ ID NO: 210 (as shown below in FIG. 5) corresponding to any amino acid positions: 61, 63, 68, 72, 86, 96, 100, 101, 131, 133, 135, 147, 169, 172 and 177, where the mutations include insertions, deletions and substitutions of the native amino acid residue (s). In preferred embodiments of the invention, the substitutions are selected from amino acid substitutions in SEQ ID NO: 210 shown in Table 6 below, where the first amino acid in the list is substituted with the second amino acid at that position; wherein the variant further comprises an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites that are not present in the parent polypeptide.

In other embodiments of the invention, the modified G-CSF includes an amino acid sequence corresponding to any SEQ ID NOs: 631-662, and further includes one or more glycosylation sites that are not present in the parent polypeptide.

Polypeptide variants of human growth hormone

In other embodiments of the invention, the hyperglycosylated protease-resistant variant of the cytokine is a modified human growth hormone cytokine (hGH) comprising one or more amino acid substitutions at one or more target positions in SEQ ID NO: 216 (as shown below in FIG. 6) corresponding to a structurally linked modified amino acid position in the three-dimensional structure of the above-described G-CSF polypeptide variant, where the replacement (s) leads to greater protease resistance, as measured by incubation with prot an ease or a blood lysate, or an incubation with blood serum (as described above), compared with unmodified hGH. In some of these embodiments of the invention, the modified hGH is selected from proteins comprising one or more single amino acid substitutions at one or more target positions in SEQ ID NO: 216 (as shown below in FIG. 6) corresponding to any amino acid positions: 56, 59, 64, 65, 66, 88, 92, 94, 101, 129, 130, 133, 134, 140, 143, 145, 146, 147, 183 and 186, where mutations include insertions, deletions and substitutions of the native amino acid residue (s) . In preferred embodiments of the invention, the substitutions are selected from amino acid substitutions in SEQ ID NO: 216, presented below in table 7, where the first amino acid from the list is substituted with the second amino acid at the indicated position; wherein the variant further comprises an amino acid sequence that differs from the amino acid sequence of the parent polypeptide to such an extent that the variant includes one or more glycosylation sites that are not present in the parent polypeptide.

In other embodiments of the invention, the modified hGH includes an amino acid sequence corresponding to any SEQ ID NOs: 850-895, and further includes one or more glycosylation sites that are not present in the parent polypeptide.

In other embodiments of the invention, the hyperglycosylated protease-resistant variant is a modified cytokine that is more resistant to proteolysis than the corresponding unmodified (parental) cytokine, where the modified cytokine includes one or more amino acid substitutions at one or more cytokine target positions corresponding to a structurally linked modified amino acid position in the three-dimensional structure of the above described IFN-β polypeptide variant. Amino acid (s) replacement (s) leads to greater resistance to proteolysis compared to unmodified (parental) cytokine. Increased resistance to proteolysis is assessed by incubation with a protease or a blood lysate, or by incubation with blood serum (as described above) compared with unmodified hGH.

Additional modifications

A typical, protease-resistant or protease-resistant, hyper-glycosylated polypeptide variant has an amino acid sequence that is substantially similar to the amino acid sequence of the parent polypeptide. For example, a hyperglycosylated polypeptide variant has an amino acid sequence that differs by at least one amino acid and can differ by at least two but not more than 10 amino acids compared to the amino acid sequence of the parent polypeptide. Sequence changes can be substitutions, insertions, or deletions. Mutations that systematically introduce alanine or other residues can be scanned using the amino acid code. Specific amino acid substitutions include conservative and non-conservative substitutions. Conservative amino acid substitutions typically include substitutions within the following groups: (glycine, alanine); (valine, isoleucine, leucine); (aspartic acid, glutamic acid); (aspargin, glutamine); (series, threonine); (lysine, arginine); or (phenylalanine, tyrosine).

Additional modifications to the inserts, which may or may not change the primary amino acid sequence of the parent therapeutic protein, include chemical derivatives of the polypeptides, for example, acetylated or carboxylated; changes in the amino acid sequence that makes the protein sensitive to polyethylene glycolation (PEG glycolation); and the like. A hyperglycosylated, protease-resistant polypeptide variant of a protein can be modified with one or more polyethylene glycol groups. In one embodiment, the invention contemplates the use of polypeptide variants from one or more unnatural PEG glycolated sites that are introduced to produce PEG derivatives of polypeptides with reduced serum clearance. Also used are sequences that have phosphorylated amino acid residues, for example, phosphotyrosine, phosphoserine or phosphotreonine.

Can also be used in combination with the polypeptides presented in the invention, polypeptides whose properties have been modified using conventional chemical technologies, which are more resistant to proteolytic degradation, have optimized solubility, or are more suitable for use as a therapeutic agent. For example, the peptide framework may be cyclized to increase its stability (see, for example, Friedler et al. 2000, J Biol. Chem. 275: 23783-23789). Analogs may be used that include residues other than the occurring L-amino acids, for example, D-amino acids or non-naturally occurring amino acids. Protein can be pegylated to increase its stability.

Modifications are interesting in that they may or may not change the primary amino acid sequence of the polypeptides, including chemical derivatization of the polypeptides, for example, acetylation or carboxylation; changes in amino acid sequences that make the protein sensitive to PEG glycolation (by adding a polyethylene glycol group); and the like. In one embodiment of the invention, it is contemplated to use variants of a synthetic type I interferon receptor agonist, in which hyperglycosylated, protease-resistant polypeptide variants further include one or more non-natural pegylation sites that are designed to produce PEG-derivatized polypeptides with reduced serum clearance. Thus, the invention includes a pegylated synthetic type I interferon receptor agonist polypeptide. It also includes glycosylation modifications, for example, those that can be obtained by modifying glycosylated samples of a polypeptide during its synthesis and processing or in subsequent processes in stages; for example, by exposing the polypeptide to enzymes that carry out a glycosylation reaction, such as mammalian glycosylation or deglycosylation enzymes. The invention contemplates the use of any PEGylated hyperglycosylated, PEGylated protease-resistant and PEGylated protease-resistant hyperglycosylated polypeptide variants. It also encompasses sequences that have phosphorylated amino acid residues, for example, phosphotyrosine, phosphoserine or phosphotreonine.

Protein Fusion

In certain embodiments of the invention, the hyperglycosylated, protease-resistant polypeptide variant further includes a heterologous polypeptide (eg, partner) necessary for the formation of fusion proteins. Suitable fusion partners include peptides and polypeptides that enhance in vivo stability (for example, lengthening serum half-lives); simplifying purification, for example, (His) n, for example, 6His, and the like; providing secretion of the fused protein from the cell; preserving epitopes, e.g., GST, hemagglutinin (HA; e.g. CYPYDVPD YA; SEQ ID NO: 1304), FLAG (e.g. DYKDDDDK; SEQ ID NO: 1305), c-myc (e.g. CEQKLISEEDL; SEQ ID NO: 1306 ) and the like; providing a signal for detection, for example, enzymes that form readily detectable products (for example, [beta] -galatosidase, luciferase) or a protein that is used for detection, for example, green fluorescent protein, etc .; providing multimerization, for example, multimerization of domains such as immunoglobulin Fc fragment and the like.

A fusion protein may be included in an amino acid sequence that is designed to secrete the fusion protein from the cell. The skilled person knows signal sequences for secretion. Secretion signals that are suitable for use in bacteria include, but are not limited to, the secretion signal of Brown lipoprotein E. coli, S. Marcescens, E. amylosora, M. morganii, and P. mirabilis; TraT protein of E. coli and Salmonella; penicillinase protein (RepR) of B. licheniformis and B. cereus and S. aureus; pullulanase proteins Klebsiella pneumoniae and Klebsiella aerogenese; lipoproteins of E. coli lpp-28, Pal, RpIA, RpIB, OsmB, NIpB and Og117; chitobase protein V. harseyi; [beta] -1,4-endogluconase protein Pseudomonas solanaceamm. Pal and Pep proteins of H. influenzae; Oprl protein of P. aeruginosa; MalX and AmiA proteins of S. pneumoniae; 34 kda antigen and TpmA protein Treponema pallidum; P37 Mycoplasma hyorhinis protein; neutral protease of Bacillus amyloliquefaciens; and 17 kda Rickettsia rickettsii antigen. Secretion signal sequences suitable for use in yeast are known in the art and can be used. See, for example, U.S. Patent No. 5,712,113.

In certain applications of the invention, a signal peptide from IFN- [alpha] is used. In other applications, signal peptides from IFN-β [beta] are used. Example 2 shows the use of a synthetic type I interferon receptor polypeptide agonist, including IFN- [alpha] 14 or IFN- [beta] -signal peptide. Such signal peptides provide secretion from mammalian cells.

In individual applications, a hyperglycosylated, protease-resistant polypeptide variant includes a fusion partner and a protease cleavage site that is positioned between the fusion partner and the remainder of the polypeptide variant.

Proteolytic cleavage sites are known to those skilled in the art; there are a lot of them and they are well described in the literature, including, for example, Handbook of Proteolytic Enzymes (1998) AJ Barrett, ND Rawlings, and JF Woessner, eds., Academic Press. Proteolytic cleavage sites include, but are not limited to, the enterokinase cleavage site: (Asp) 4Lys (SEQ ID NO: 1307); Factor Xa cleavage site: Ile-Glu-Gly-Arg (SEQ ID NO: 1308); thrombin cleavage site, for example, Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO: 1309); a renin cleavage site, for example, His-Pro-Phe-His-Leu-Val-Ile-His (SEQ ID NO: 1310); collagenase cleavage site, for example, X-Gly-Pro (where X is any amino acid); trypsin cleavage site, for example, Arg-Lys; viral protease cleavage sites, such as 2A or 3C, protease cleavage sites, including but not limited to picornovirus protease cleavage site 2A (see, for example, Sommergruber et al. [0635] (1994) Virol. 198: 741-745), 3C hepatitis A virus cleavage site (see, for example, Schultheiss et al. [0636] (1995) J. Virol. 69: 1727-1733), protease cleavage site of human rhinovirus 2A (see, for example, Wang et al. (1997) Biochem. Biophys. Res. Comm. 235: 562-566), a protease 3 cleavage site of picornovirus (see, for example, Walker et al. (1994) Biotechnol. 12: 601-605).

Obtaining a hyperglycosylated, protease-resistant variant of the polypeptide

The claimed synthetic type I interferon receptor agonist polypeptide can be simply prepared using any known methods, including chemical synthesis methods, production of recombinant DNA using standard technologies, and combinations thereof. For example, the claimed synthetic type I interferon receptor agonist polypeptide can be synthesized using automated solid-phase tert-butyloxycarbonyl and benzyl protective strategies.

The claimed synthetic type I interferon receptor agonist polypeptide can be synthesized by native chemical ligation of, for example, fragments from about 15 to about 40 amino acids in length (for example, fragments from about 15 to about 20 amino acids long; from about 20 to about 25 amino acids in length; from about 25 to about 30 amino acids in length; from about 30 to about 35 amino acids in length; from, about 35 to about 40 amino acids) can be synthesized using standard chemical synthesis methods; and fragments subsidized using the process described by Dawson, et al. (1994) Science 266: 176-779. Purification of the synthesized peptides can be assessed by reverse phase high performance liquid chromatography (HPLC) and isoelectric focusing. Primary ligand structures can be verified using Edman sequence methods.

In many applications of the invention, the expression vector includes a nucleotide sequence that encodes the claimed synthetic type I interferon receptor agonist polypeptide, obtained using convenient methods, and is introduced into host cells, especially eukaryotic cells that are capable of glycosylating proteins. The expression vector provides for the production of the claimed synthetic type I interferon receptor agonist polypeptide in host cells. Therefore, the presented invention provides a method for producing a synthetic type I interferon receptor agonist polypeptide, comprising culturing eukaryotic host cells, for the implementation of which the host cells include the claimed recombinant expression vector providing favorable conditions for the production of a synthetic type I interferon receptor agonist polypeptide; and obtaining a synthetic interferon type I receptor agonist polypeptide from a culture. The claimed agonist polypeptide can be isolated and purified to more than 80%, to more than 90%, to more than 95%, to more than 98%, or to more than 99%.

Polypeptides can be expressed in prokaryotes or eukaryotes, in the usual way, the choice of which depends on the purpose of expression. As noted above, in many applications, the claimed synthetic type I interferon receptor agonist polypeptide was synthesized in eukaryotic cells. For large-scale protein production, as expression host cells, the following can be used: a unicellular organism such as S. cerevisiae; insect cells in combination with baculovirus vectors; cells of higher organisms, such as vertebrates, in particular mammals, for example, COS 7 cells, CHO cells, HEK293 cells and the like. In many applications of the invention, gene expression is required in a eukaryotic cell, where the protein must retain native folding and post-translational modification.

Protein or fragments thereof, obtained in large quantities by expression in host cells, can be isolated and purified conveniently. Lysates can be prepared from expression host cells and purified by HPLC, hydrophobic interaction chromatography (HIC), anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, ultrafiltration, gel electrophoresis, affinity chromatography, or other purification methods.

The claimed synthetic type I interferon receptor agonist polypeptide can also be isolated and purified conveniently from cell culture supernatant or from cell lysates. For example, a lysate can be prepared from expressing host cells and purified using HPLC, hydrophobic interaction chromatography (HIC), anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, ultrafiltration, gel electrophoresis, affinity chromatography, or other purification methods. In most cases, the mixtures that are used should include at least 20% by weight of the desired product; preferably they should include at least about 75% of the product by weight; most preferably, they should include about 95% of the product by weight; and for therapeutic purposes, usually at least about 99.5% of the product by weight, relative to contaminants associated with methods of obtaining the product and its purification. Typically, percentages are based on total protein content.

In many applications of the invention, the claimed synthetic type I interferon receptor agonist polypeptide is purified, for example, the claimed synthetic type I interferon receptor agonist polypeptide is free of other, not claimed proteins, and free of other macromolecules (e.g., carbon hydrates, lipids, etc. ) In many applications, the claimed synthetic type I interferon receptor agonist polypeptide is at least about 75% purified; at least approximately 80% purified; at least approximately 85% purified; at least approximately 90% purified; at least approximately 95% purified; at least approximately 98% purified; or at least about 99% purified, or more than 99%. Methods for determining whether a protein is truly free of other proteins and other macromolecules are known to those skilled in the art.

Hyperglycosylated, protease-resistant polypeptide variants can be obtained by recombinant DNA methods using convenient techniques known to those skilled in the art. Separate sequences and preparation methods can be selected depending on how convenient, economical, purity of the product is needed, etc.

Typically, oligonucleotides encoding the amino acid sequences of the desired polypeptide variant are prepared by chemical synthesis, for example, by using an oligonucleotide synthesizer. The necessary oligonucleotides are selected according to the amino acid sequence of the desired polypeptide and in many different embodiments, choosing those codons that are most preferred for the host cell in which the recombinant polypeptide will be synthesized. For example, individual small oligonucleotides encoding part of the function of the desired polypeptide can be synthesized and assembled by PCR, ligation or dot chain reaction (LCR). Individual oligonucleotides usually contain 5 'or 3' overlap for complementary assembly. Once the assembled nucleotide sequence encoding the polypeptide variant will be inserted into the recombinant vector and placed under the control of the sequence expressing the desired nucleic acid and subsequent production of the claimed polypeptide in the desired transformed cell.

In certain applications of the invention, the required nucleic acid is created in such a way that at least about 10%; at least about 20%; at least about 30%; at least about 40%; at least about 50%; at least about 60%; at least about 70%; at least about 80%; or at least about 90%; or more, the codons are codons, which are preferably human sequences. See, for example, below table. 8.

Table 8
Codons commonly found in humans ^* Amino acid Frequency of occurrence in human proteins (%) Codons and their occurrence in human proteins (%) Alanine 6.99 GCU (28.0) Gcc GCA (20.0) (41.6) Gcg (10.3) Arginine 5.28 CGU (8.9) Cgc CGA (5.4) (21.4) AGA (9.9) Cgg (10.4) AGG (11.1) Aspargin 3.92 AAU (42.3) Aac (57.7) Aspartic 5.07 GAU (42.8) Gac acid (57.2) Cysteine 2.44 UGU (40.6) Ugc (59.4) Glutamine 6.82 GAA (39.2) Gag acid (60.7) Glutamine 4.47 CAA (24.8) Cag (75.2) Glycine 7.10 GGU (15.8) Ggc GGA (24.1) (35.8) Ggg (24.3) Histidine 2,35 CAU (39.6) CAC (60.4) Isoleucine 4,50 AUU (33.1) Auc AUA (12.9) (54.0) Leucine 9.56 UUA (5.5) UUG CUU (11.1) (11.5) CUA (6.5) Cuc (20.8) Cug (44.5) Lysine 5.71 AAA (38.9) Aag (61.1) Methionine 2.23 AUG (100) Phenylalanine 3.84 UUU (41.1) Uuc (58.2) Proline 5.67 CCU (27.3) CCC SSA (25.7) (35.2) CCG (11.6) Series 7.25 UCU (18.3) UCC UCA (12.9) (23.7) AGU (13.2) UCG (5.9) AGC (25.9) Threonine 5.68 ACU (22.4) ACC ASA (25.4) (40.5) ACG (11.8) Tryptophan 1.38 UGG (100) Tyrosine 3.13 UAU (40.0) Uac (60.0) Valine 6.35 GUU (16.4) Guc GUA (9.3) (25.7) GUG (48.7) * Molecular Cloning: A Laboratory Manual. Sambrook J. and Russell D. W. Third Edition (c) 2001 by Cold Spring Harbor Press.

Polypeptide-coding nucleic acid molecules are typically propagated by insertion into a molecular vector. Viral and non-viral vectors, including plasmids, are used. The choice of plasmids depends on the type of cells in which they are expected to multiply, and the purpose of reproduction. Certain vectors are suitable for amplification and production of large quantities of the desired DNA sequences.

A recombinant expression vector is used to efficiently express polypeptides encoded by a nucleic acid molecule in a cell, for example, to produce a hyperglycosylated protease-resistant polypeptide variant. The selection of an appropriate vector is well known to those skilled in the art. Many such vectors are commercially available.

Expression vectors are suitable for expression in cell culture. These vectors typically include regulatory sequences (“control sequences” or “control regions”) that are necessary for efficient expression of the desired polynucleotide to which they are directly attached.

Expression vectors usually have convenient restriction sites located near the promoter sequence. They are designed to insert nucleic acid sequences encoding the desired protein or other proteins. In such a vector, selection markers that are efficiently expressed in the selected host can be represented. Expression vectors can be used to produce fusion proteins when exogenous fusion proteins provide additional functionality, for example, increased protein synthesis, stability, interaction with diagnostic antiserum, an enzymatic marker, for example, [beta] -galactosidase, luciferase, etc.

Expression cassettes are prepared in such a way as to include a transcription initiation region, a promoter region (eg, a promoter that functions in a eukaryotic cell), a necessary polynucleotide, and a transcription termination region. After the introduction of DNA, cells containing such constructs are selected using a selection marker, cells are propagated and used for expression.

Expression cassettes are introduced into various vectors suitable for expression in eukaryotic host cells, for example, a plasmid, HAC, YAC, vectors from animal viruses; for example, Moloni murine leukemia virus, SV40, vaccine viruses, baculoviruses, retroviruses; or plant viruses, for example, cauliflower mosaic virus, tobacco mosaic virus, and the like, where the vectors are typically capable of selecting cells including expression vectors. Vectors can be used for extrachromosomal existence, in particular, such as plasmids or viruses; or for integration into the host chromosome. When the extrachromosomal existence of a vector in the host is necessary, the origin sequence necessary for replication is taken from a plasmid, which may have a large or small number of copies. There is a wide variety of markers suitable for selection, especially those that protect against toxic substances, in particular, against antibiotics. An individual marker suitable for selection is selected according to the nature of the host; in some cases, complementation of the auxotrophic host can be used. Any convenient method can be used to introduce the DNA construct into the host cells, for example, calcium precipitation of DNA, electroporation, fusion, transfection, infection with viral vectors, bio-ballistics, etc.

The presented invention further provides for the use of production of a hyperglycosylated, protease-resistant polypeptide in genetically modified host cells, which can be isolated from host cells, including polynucleotides encoding the polypeptide variant; or, in certain applications of the invention, an expression vector capable of expressing such polynucleotides. Suitable eukaryotic cells include insect cells in combination with baculovirus vectors; yeast cells such as Saccharomyces cerevisiae; or cells of higher organisms such as vertebrates, including amphibian cells (e.g., Xenopus laevis oocytes) and mammals, in particular mammalian cells, e.g., COS cells, CHO cells, HEK293 cells, MA-IO cells, and the like, can be used as host cells for expression. In particular, host cells that are eukaryotic cells that are capable of glycosylating a protein.

A hyperglycosylated, protease-resistant polypeptide variant can be obtained from host cells and then isolated and purified by any convenient recombinant synthesis method. The lysate can be obtained from an expression host and purified by high resolution liquid chromatography, exclusion chromatography, gel electrophoresis, affinity chromatography and other purification technologies. In most cases, the compositions that are used include at least about 20% by weight of the desired product; most commonly, this value is about 75% by weight; preferably, it reaches approximately 95% by weight; and for therapeutic purposes, usually it is at least 99.5% by weight, in relation to contaminants associated with the method of isolation of products and methods of purification. Typically, the percentage is based on total protein.

PEGylated Type I Interferon Receptor Agonist Polypeptide

As noted above, in certain applications, the claimed synthetic type I interferon receptor agonist polypeptide is a modified one or more polyethylene glycol groups, i.e. Pegylated. The PEG molecule is attached to one or more amino acid chains of the claimed polypeptide agonist. In certain applications, the claimed PEGylated polypeptide agonist contains a PEG group on only one amino acid. In other embodiments of the invention, the claimed PEGylated polypeptide agonist contains a PEG group for two or more amino acids, for example, the claimed PEGylated polypeptide agonist contains a PEG group attached to two, three, four, five, six, seven, eight, nine or ten different amino acid residues.

The claimed polypeptide can be attached directly to the PEG (i.e., without a linker group) through an amino group, a sulfhydryl group, a hydroxyl group or a carboxyl group.

In certain applications, the PEGylated claimed polypeptide is PEGylated at or near the amino acid ends of the claimed polypeptide, for example, the PEG group is conjugated to the claimed polypeptide at one or more amino acid residues from amino acid 1, through amino acid 4; or from amino acid 5, through about 10. In other applications, the PEGylated claimed polypeptide is PEGylated at one or more amino acid residues from about 10 to about 28. In other applications, the PEGylated claimed polypeptide is PEGylated at the carboxyl ends (C-ends) or nearby, for example, in one or more residues from amino acids 156-166, or amino acids from 150 to 155. In other applications, the PEGylated claimed polypeptide is PEGylated in one or more amino acid residues in one or more amino acid residues 100-114.

Obtaining polyethylene glycol derivatives of amino acid residues in or near the receptor-binding site and / or the domain of active sites of the protein of interest may impair the function of these domains. In certain applications of the invention, amino acids are not pegylated at amino acid residues from amino acid 30 to amino acid 40, and from amino acid 113 to 149.

In individual applications, PEG was attached to the claimed polypeptide via a linker group.

A linker group is any biocompatible linker group where the “biocompatibility” indicates that the composition or group is non-toxic and can be disposed of in vitro or in vivo without harm, disease or death. PEG can be attached to the linker group, for example, through any group of ether, ester, thiol group or amide group. Suitable biocompatible linker groups include, but are not limited to, an ester group, an amide group, an imide group, a carbon hydrate group, a carboxyl group, a hydroxyl group, carbon hydrates, an succinic acid group (including, for example, succimidyl succinate (SS), succimidylpropanoate (SU), SBA), succimidyl carboxymethylate (SCM), succimidyl succinamide (SSA) or N-hydroxysuccinimide (NHS), epoxy group, hydroxycarbonylimidine group [including, for example, carbonyldiimidazole (CDI)], nitrophenyl group y [including, for example, nitrophenyl carbonate (NPC); or trichlorophenylcarbonate (TPC)] trisilatnuyu group, an aldehyde group, izotsianidnuyu group, a vinylsulfone group, a tyrosine group, a cysteine group, a histidine group or a primary amine.

Methods for preparing succinimidyl propyanate (SPA) and sucinimidyl butanoate (SBA) ether-activated PEGs are described in U.S. Pat. No. 5,672,662 (Harris, et al.) And WO 97/03106.

Methods of attaching PEG to polypeptides are known in the art, any known methods may be used for this. For example, see Park et al, Anticancer Res., 1: 373-376 (1981); Zaplipsky and Lee, Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, ed., Plenum Press, NY, Chapter 21 (1992); U.S. Patent No. 5,985,265; U.S. Pat. No. 5,672,662 (Harris, et al.) And WO 97/03106.

In many applications, PEG is a monomethoxyPEG molecule that has reacted with the primary amino groups of the claimed polypeptide. Methods for modifying monomethoxy-PEG polypeptides by reducing alkylation are known in the art. See, for example, Chamow et al. (1994) Bioconj. Chem. 5: 133-140.

Polyethylene glycol

A polyethylene glycol suitable for conjugation with the claimed polypeptide is soluble at room temperature and has the general formula R (O — CH ₂ —CH ₂ ) nO — R, where R is hydrogen or a protective group, such as an alkyl or alkanol group, and where n ranges from 1 to 1000. When R is a projective group, it usually has from 1 to 8 carbons.

In many applications, PEG has at least one hydroxyl group, for example, a terminal hydroxyl group, which is a hydroxyl group that is modified to form a functional group that interacts with an amino group, for example, epsilon, an amino group of a lysine residue, a free amino group of N-ends a polypeptide, or any other amino group, such as the amino group of aspargin, glutamine, arginine or histidine.

In other applications, PEG is a derivative designed to interact with the free carboxyl groups of the claimed polypeptide, for example, the free carboxyl group at the carboxyl ends of the claimed polypeptide. Suitable PEG derivatives that react with the free carboxyl group at the carboxyl ends of the claimed polypeptide include, but are not limited to, PEG amine and PEG hydrazine derivatives (e.g., PEG-NH-NH2).

In other applications, PEG is derivatized so that it includes a terminal thiocarboxylic acid group, —COSH, which selectively reacts with amino groups forming amide derivatives. The reaction nature of thioacid is selective to individual amino acids more to others. For example, -SH, under appropriate pH conditions, exhibits sufficient ability to react with the N-terminal [epsilon] amino groups of lysine residues protonated or non-nucleophilic. On the other hand, reactions under the influence of suitable pH conditions can give individual Lysine residues the ability to react selectively.

In other applications, PEG includes reactive esters, such as N-hydroxysuccinamidate at the end of the PEG chain. So the molecule of N-hydroxysuccinamidate-containing PEG reacts with individual amino acid groups under special pH conditions, such as neutral medium 6.5-7.5. For example, the N-terminal amino groups can be selectively modified under neutral pH conditions. However, if the reactivity of the reagent is excessive, the NHz group of lysine can also react.

PEG can be conjugated directly to the claimed polypeptide or via a linker. In certain applications, the linker is added to the claimed polypeptide, forming a link-modified polypeptide. Such linkers provide various functions, for example, reactive groups, such as sulfhydryl, amino or carboxyl, link the PEG with a linker-modified peptide.

In certain applications, the PEG conjugated to the claimed polypeptide is linear. In other applications, the PEG conjugated to the claimed polypeptide is branched. Branched derivatives of PEG, such as those described in U.S. Pat. No. 5,643,575, star PEGs and multi-reinforced PEGs, such as those described in the Shearwater Polymers, Inc. kA catalog. "Polyethylene Glycol Derivatives, 1997-1998." Star PEGs are known to those skilled in the art, including, for example, U.S. Patent No. 6,046,305.

PEG has a molecular weight ranging from about 2 kDa to about 100 kDa, where the commonly used term “about” in the context of PEG means that the molecular weight of the PEG is greater or less than the specified molecular weight. For example, the PEG most suitable for conjugation with the claimed polypeptide has a molecular weight ranging from about 2 kDa to about 5 kDa; from about 5 kDa to about 15 kDa to about 20 kDa; from about 20 kDa to about 25 kDa; from about 25 kDa to about 30 kDa; from about 30 kDa to about 40 kDa; from about 40 kDa to about 50 kDa; from approximately 50 kDa; up to approximately 60 kDa; from about 60 kDa to about 70 kDa; from about 70 kDa to about 80 kDa; from about 80 kDa to about 90 kDa; or from about 90 kDa to about 100 kDa.

The combination of the claimed synthetic polypeptides of the agonist receptor interferon type I

The invention itself provides a composition that includes a combination of synthetic type I interferon receptor agonist polypeptides, as described above. The claimed composition includes a combination of the claimed polypeptides, when the combination includes at least two different claimed synthetic polypeptides of an interferon type I receptor agonist (for example, agonist polypeptides that differ from each other in amino acid sequences by at least one amino acid).

Typically, the obtained claimed synthetic type I interferon receptor agonist polypeptide corresponds to about 0.5% to about 99.5% of the total synthetic polypeptides of type I interferon receptor agonists; for example, the resulting synthetic type I interferon receptor agonist polypeptide is from about 0.5% to about 1%; approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50 %, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 99%, or approximately 99.5% of the total population of polypeptide agonists type I interferon receptor.

Songs

The present invention describes compositions, including pharmaceutical compositions, comprising a synthetic type I interferon receptor polypeptide agonist of the present invention, a known hyperglycosylated polypeptide variant, a known protease-resistant polypeptide variant, or a known hyperglycosylated, protease-resistant polypeptide variant, for example, a polypeptide variant of the parent therapeutic protein, which comprises at least one mutant protease cleavage site in places the native protease cleavage site present in the parent therapeutic protein; and which includes (1) a carbohydrate moiety covalently linked to at least one non-native glycosylation site that is not present in the parent therapeutic protein, and / or (2) a carbohydrate moiety covalently linked to at least one native glycosylation site, which present, but not glycosylated in the parent therapeutic protein. The compositions may include a synthetic type I interferon receptor polypeptide agonist of the present invention, a known hyperglycosylated polypeptide variant, a known protease-resistant polypeptide variant, or a known hyperglycosylated, protease-resistant polypeptide variant; and one or more additional components that are selected depending on the polypeptide variant used. Suitable additional components include, but are not limited to, salts, buffers, solubilizers, stabilizers, detergents, protease inhibiting agents, and the like.

In some embodiments of the invention, the subject composition comprises a synthetic type I interferon receptor polypeptide agonist of the present invention, a known hyperglycosylated polypeptide variant, a known protease-resistant polypeptide variant, or a known hyperglycosylated, protease-resistant polypeptide variant and a pharmaceutically acceptable excipient. A wide variety of pharmaceutically acceptable excipients is known in the art and there is no need to discuss them in detail. Pharmaceutically acceptable excipients are described in various publications, e.g. A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, William, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., Eds., 7th ed., Lippincott, William, &Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3th ed. Amer. Pharmaceutical Assoc.

In some embodiments of the invention, in pharmaceutical dosage forms, a synthetic type I interferon receptor polypeptide agonist of the present invention, a known hyperglycosylated polypeptide variant, a known protease-resistant polypeptide variant, or a known hyperglycosylated, protease-resistant polypeptide variant are present in the form of pharmaceutically acceptable salts, used alone or in a suitable mixtures, as well as in combination with other pharmaceutically active compounds .

Formulations suitable for injection

In some embodiments of the invention, the synthetic type I interferon receptor polypeptide agonist of the present invention is formulated for injection (e.g., subcutaneous, intramuscular, intradermal, transdermal or other injection methods) by dissolving, suspending or emulsifying the agonist in an aqueous solvent (e.g., saline and the like) or in a non-aqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, complex Fira higher aliphatic acids or propylene glycol; and, if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

Compositions for delivery to the small intestine

In the manufacture of oral preparations, an object of the invention, an agent (for example, a synthetic type I interferon receptor polypeptide agonist) is used alone or in combination with additives suitable for the manufacture of tablets, powders, granules or capsules, for example, with conventional additives such as lactose, mannitol, corn or potato starch; with binders such as crystalline cellulose, cellulose derivatives, gum, corn starch or gelatin; with disintegrants such as corn starch, potato starch, sodium carboxymethyl cellulose; with lubricants such as talc or magnesium stearate; and, if desired, with diluents, buffering agents, moisturizing agents, preservatives and flavorings.

An object of the invention, an agonist may be included in the suppository by mixing with various bases, such as emulsifying bases or water-soluble bases. The subject of the invention is an agonist that can be administered rectally using a suppository. A suppository may include carriers, such as coconut oil, carbovax, and polyethylene glycol, which melt at human body temperature but solidify at room temperature.

Standard dosage forms for oral or rectal administration, such as syrups, elixirs and suspensions, can be formulated so that each dosage unit, for example, a full teaspoon, full tablespoon, tablet or suppository, contains a predetermined amount of a composition containing one or more active components. Similarly, unit dosage forms for injection or intravenous administration may include agonist (s) as a solution in sterile water, normal saline, or another pharmaceutically acceptable carrier.

In some embodiments of the invention, an object composition for enteral delivery includes an enteric coating material. Suitable enteric coating materials include succinate acetate hydroxypropyl methyl cellulose (HPMCAS), hydroxypropyl methyl cellulose phthalate (HPMC), cellulose acetate phthalate (CAP), polyvinyl phthal acetate (PVPA), Eudragit and alkali.

In one non-limiting example of a suitable oral formulation, a synthetic type I interferon receptor polypeptide agonist of the present invention can be formulated with one or more pharmaceutical excipients and coated with an enteric coating, as described in US Pat. No. 6,346,269. For example, a solution comprising a solvent, a synthetic polypeptide agonist the type I interferon receptor of the present invention and a stabilizer are applied to a core comprising pharmaceutically acceptable excipients to form a core coated with an active agent; a lower coating layer is applied to the core coated with the active agent, which is then coated with an enteric coating layer. The core mainly includes pharmaceutically inactive components, such as lactose, starch, mannitol, sodium carboxymethyl cellulose, sodium starch glycolate, sodium chloride, potassium chloride, pigments, salts of alginic acid, talc, titanium dioxide, stearic acid, stearate, microcrystalline glycol cellulicene , triethyl citrate, tributyl citrate, propanyl triacetate, dibasic calcium phosphate, tribasic sodium phosphate, calcium sulfate, cyclodextrin and castor oil. Suitable solvents for the active agent include aqueous solvents. Suitable stabilizers include alkali and alkaline earth metals, basic phosphates and salts of organic acids and organic amines. The lower coating layer includes one or more adhesives, a plasticizer, and an anti-caking agent. Suitable anti-caking agents include talc, stearic acid, stearate, sodium fumarate stearyl, glyceryl behenate, kaolin and aerosil. Suitable adhesives include polyvinylpyrrolidone (PVP), gelatin, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), vinyl acetate (VA), polyvinyl alcohol (PVA), methyl cellulose (hydroxymethyl cellulose (HCM), ethyl cellulose (hydroxymethyl), hydroxymethyl cellulose (hydroxymethyl cellulose (hydroxymethyl cellulose), hydroxymethyl cellulose (hydroxymethyl cellulose (hydroxymethyl cellulose), hydroxymethyl cellulose (hydroxymethyl cellulose (hydroxymethyl cellulose), hydroxyethyl cellulose (hydroxymethyl cellulose), ), cellulose acetate phthalate (CAP), xanthan gum, alginic acid, alginic acid salts, Eudragit ^™ , a copolymer of methyl acrylic acid and methyl methacrylate with phthalate polyvinyl acetate (PVAP). Suitable plasticizers include glycerin, polyethylene glycol, triethyl citrate, tributyl citrate, propanyl triacetate and castor oil. Suitable enteric materials include succinate acetate hydroxypropyl methyl cellulose (HPMCAS), hydroxypropyl methyl cellulose phthalate (HPMC), cellulose acetate phthalate (CAP), polyvinyl phthalate acetate (PVPA), Eudragit ^™ and alkali.

Suitable oral formulations also include a synthetic type I interferon receptor polypeptide agonist of the present invention, formed with any of the following: microbeads (see, for example, US Pat. No. 6,458,398); bi-degradable macromers (see, for example, US Pat. No. 6,703,037); biodegradable hydrogels (see, for example, Graham and McNeil (1989) Biomaterials 5: 27-36); individual biodegradable vectors (see, for example, US patent 5736371); biodegradable lactone-containing polymers (see, for example, US Pat. No. 5,631,015); slow release polymers (see, for example, US Pat. No. 6,699,504; Relias Technologes, Inc.); poly (lactide-glycolide) / polyethylene glycol block copolymer (see, for example, US Pat. No. 6,663,055); Atrix Laboratories, Inc.,); compositions comprising a biocompatible polymer and particles of a cation stabilized metal agent dispersed in the polymer (see, for example, US Pat. No. 6,397,701; Alkermers Controlled Therapeutics, Inc); or microspheres (see, for example, US Pat. No. 6,303,148); Octoplus, B.V.).

Suitable oral formulations also include a synthetic polypeptide agonist interferon type I receptor of the present invention formed from any of: a carrier such as ^{Emisphere ® (Emisphere Technologies, Inc.)} ; TIMERx hydrophilic combined matrix of xanthan gum and locust bean gum, which in the presence of dextrose forms a strong bonding gel in water (Penwest); Geminex ^TM (Penwest); Procise ^TM (GlaxoSmithKline); SAVIT ^TM (Mistral Pharma Inc.); RingCap ^TM (Alza Corp.); Smartrix ^® (Smatrix Technologies, Inc.); SQZgel ^TM (MacroMed, Inc.); ^{Geomatrix TM (Skye Pharma, Inc)} ; ^{Oros ® Tri-layer (Alza Corporation} ); etc.

Also suitable are those formulations described in US Pat. Nos. 6,298,642 (Alkermes Controlled Therapeutics, Inc.); 6187330 (Scios, Inc.); and the like.

Oral Delivery Formulations

The present invention also relates to pharmaceutical compositions comprising a synthetic type I interferon receptor polypeptide agonist of the present invention, a known hyperglycosylated polypeptide variant, a known proteazoresistant polypeptide variant, or a known hyperglycosylated, proteazoresistant polypeptide variant; and a pharmaceutical excipient suitable for oral delivery.

For oral preparations, the synthetic type I interferon receptor polypeptide agonist of the present invention, the known hyperglycosylated polypeptide variant, the known protease-resistant polypeptide variant, or the known hyperglycosylated, protease-resistant polypeptide variant are formed alone or in combination with suitable additives into tablets, powders, granules or capsules, for example, with conventional additives such as lactose, mannitol, corn or potato starch; with binders such as crystalline cellulose, cellulose derivatives, gum, corn starch or gelatin; with disintegrants such as corn starch, potato starch or sodium carboxymethyl cellulose; with lubricants such as talc or magnesium stearate; and, if desired, with diluents, buffering agents, moisturizing agents, preservatives and flavorings.

Standard dosage forms for oral administration, such as syrups, elixirs and suspensions, can be made in such a way that each dosage unit, for example, a full teaspoon, a full tablespoon, a tablet contains a predetermined amount of a composition containing one or more active ingredients.

In some embodiments of the invention, the subject composition for oral delivery comprises an enteric coating material. Suitable enteric coating materials include succinate acetate hydroxypropyl methyl cellulose (HPMCAS), hydroxypropyl methyl cellulose phthalate (HPMC), cellulose acetate phthalate (CAP), polyvinyl phthal acetate (PVPA), Eudragit and shellac.

In one non-limiting example of a suitable oral formulation, a synthetic type I interferon receptor polypeptide agonist of the present invention, a known hyperglycosylated polypeptide variant, a known protease-resistant polypeptide variant, or a known hyperglycosylated protease-resistant polypeptide variant can be formed together with one or more pharmaceutical excipients and enteric coating, as described in US patent 6346269. For example, a core including armatsevticheski acceptable excipients, coated with a solution comprising a solvent known hyperglycosylated protease-resistant polypeptide variant and a stabilizer to form a coated active agent core; a lower coating layer is applied to the core coated with the active agent, which is then coated with an enteric coating layer. The core mainly includes pharmaceutically inactive components such as lactose, starch, mannitol, sodium carboxy methyl cellulose, sodium starch glycolate, sodium chloride, potassium chloride, pigments, salts of alginic acid, talc, titanium dioxide, stearic acid, stearate, microcrystalline cellulose , polyethylene glycol, triethyl citrate, tributyl citrate, propanyl triacetate, dibasic calcium phosphate, tribasic sodium phosphate, calcium sulfate, cyclodextrin and castor oil. Suitable solvents for the active agent include aqueous solvents. Suitable stabilizers include alkali and alkaline earth metals, basic phosphates and salts of organic acids and organic amines. The lower coating layer includes one or more adhesives, a plasticizer, and an anti-caking agent. Suitable anti-caking agents include talc, stearic acid, stearate, sodium fumarate stearyl, glyceryl behenate, kaolin and aerosil. Suitable adhesives include polyvinylpyrrolidone (PVP), gelatin, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), vinyl acetate (VA), polyvinyl alcohol (PVA), methyl cellulose (hydroxymethyl cellulose (HCM), ethyl cellulose (hydroxymethyl), hydroxymethyl cellulose (hydroxymethyl cellulose (hydroxymethyl cellulose), hydroxymethyl cellulose (hydroxymethyl cellulose (hydroxymethyl cellulose), hydroxymethyl cellulose (hydroxymethyl cellulose (hydroxymethyl cellulose), hydroxyethyl cellulose (hydroxymethyl cellulose), ), cellulose acetate phthalate (CAP), xanthan gum, alginic acid, alginic acid salts, Eudragit ^™ , a copolymer of methyl acrylic acid and methyl methacrylate with phthalate polyvinyl acetate (PVAP). Suitable plasticizers include glycerin, polyethylene glycol, triethyl citrate, tributyl citrate, propanyl triacetate and castor oil. Suitable enteric materials include succinate acetate hydroxypropyl methyl cellulose (HPMC-AS), hydroxypropyl methyl cellulose phthalate (HPMC), cellulose acetate phthalate (CAP), polyvinyl phthal acetate (PVPA), Eudragit ^™ and shellac.

Suitable oral formulations also include the synthetic type I interferon receptor polypeptide agonist of the present invention, the known hyperglycosylated polypeptide variant, the known protease-resistant polypeptide variant, or the known hyperglycosylated protease-resistant polypeptide variant formed with any of: microbeads (see, for example, US Pat. ); bi-degradable macromers (see, for example, US Pat. No. 6,703,037); biodegradable hydrogels (see, for example, Graham and McNeil (1989) Biomaterials 5: 27-36); individual biodegradable vectors (see, for example, US patent 5736371); biodegradable lactone-containing polymers (see, for example, US Pat. No. 5,631,015); slow release polymers (see, for example, US Pat. No. 6,699,504; Relias Technologes, Inc.); poly (lactide-glycolide) / polyethylene glycol block copolymer (see, for example, US Pat. No. 6,663,055); Atrix Laboratories, Inc.,); a composition comprising a biocompatible polymer and dispersed in a polymer particles of a cation stabilized metal agent (see, for example, US patent 6379701; Alkermers Controlled Therapeutics, Inc); and microspheres (see, for example, US Pat. No. 6,303,148); Octoplus, B.V.).

Suitable oral formulations also include a synthetic polypeptide agonist interferon receptor I type of the present invention, known hyperglycosylated polypeptide variant, known protease-resistant polypeptide variant, or a known hyperglycosylated protease-resistant polypeptide variant formed with any of the following: a carrier such as Emisphere ^® (Emisphere Technologies , Inc.); TIMERx hydrophilic combined matrix of xanthan gum and locust bean gum, which in the presence of dextrose forms a strong bonding gel in water (Penwest); Geminex ^TM (Penwest); Precise ^TM (GlaxoSmithKline); SAVIT ^TM (Mistral Pharma Inc.); RingCap ^TM (Alza Sof.); Smartrix ^® (Smatrix Technologies, Inc.); SQZgel ^TM (MacroMed, Inc.); ^{Geomatrix TM (Skye Pharma, Inc)} ; ^{Oros ® Tri-layer (Alza Corporation} ); etc.

Also suitable are those formulations described in US Pat. Nos. 6,298,642 (Alkermes Controlled Therapeutics, Inc.); 6187330 (Scios.Inc.); and the like.

Formulations including an agent that enhances intestinal absorption are also suitable. Suitable intestinal absorption enhancers include, but are not limited to, calcium chelates (eg, citrate, ethylenediaminetetraacetic acid);

surfactants (e.g. sodium dodecyl sulfate, bile salts, palmitoylcarnitine and sodium fatty acid salts); toxins (e.g., small area blocking toxin); etc.

In one embodiment of the invention, the synthetic type I interferon receptor polypeptide agonist of the present invention, a known hyperglycosylated polypeptide variant, a known protease-resistant polypeptide variant, or a known hyperglycosylated protease-resistant polypeptide variant are present in a first oral dosage unit form. A known synthetic type I interferon receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant or a hyperglycosylated protease-resistant polypeptide variant is a parent therapeutic protein variant. In this embodiment of the invention, the first unit dosage form includes the first number of moles of a known synthetic type I interferon receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant, or a hyperglycosylated protease-resistant polypeptide variant. A parent therapeutic protein is one that is usually administered in a dose of a second number of moles of the parent therapeutic protein in a second unit dosage form, where the second unit dosage form is an immediate release form, for example, an immediate release formulation that is suitable for subcutaneous injection. Parent therapeutic protein is delivered via subcutaneous bolus injection at a selected dosing frequency. The proven therapeutic parental protein can be effective in treating disorders in a patient when administered to a patient in a second unit dosage form by subcutaneous bolus injection at a selected dosage frequency. The first number of moles in the first unit dosage form is greater than the second number of moles in the second unit dosage form. Moreover, when the first unit dosage form is orally administered to the patient, the time period for the release of the first number of moles of the known hyperglycosylated, protease-resistant polypeptide variant of the first unit dosage form is no more than the time interval between doses of the parent therapeutic protein at the selected dosage frequency.

In another aspect of the invention, the oral pharmaceutical composition of the present invention comprises a first dose of a known synthetic type I interferon receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant, or a hyperglycosylated protease-resistant polypeptide variant in a first unit dosage form. In these embodiments of the invention, the parent therapeutic protein is one which is usually administered in a second dose of the parent protein in the parenteral pharmaceutical composition, wherein the parenteral pharmaceutical composition is an immediate release composition, for example, an immediate release composition for a second dose bolus injection at a selected dosage frequency. It has been proven that the parent therapeutic protein is effective in treating the disease when it is administered to the patient by subcutaneous bolus injection in a parenteral pharmaceutical composition, where the patient receives a second dose of the parent therapeutic protein at a selected dosage frequency. When a patient is orally administered a first dose of a known synthetic type I interferon receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant or a hyperglycosylated protease-resistant polypeptide variant, the time required to release the entire amount of the known synthetic polypeptide interferon receptor type I polypeptide agonist, type I interferon receptor protease-resistant polypeptide variant Whether hyperglycosylated protease-resistant polypeptide variant in the first dose no greater than the time between doses in the selected dosing interval. The amount of a known synthetic type I interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease-resistant polypeptide variant or hyperglycosylated protease-resistant polypeptide variant in moles of a drug substance per kilogram of a patient’s body weight in the first dose is more than the amount of the parent therapeutic protein in moles of a drug substance kilogram of patient’s body weight in a second dose, where the first and second doses are calculated for the patient Independent user weight in the general population of patients suffering from the disease.

In some embodiments of the invention, the second dose is a dose dependent on the patient’s body weight, and the first dose in moles of the drug is greater than the product of the second dose in moles of the drug per kilogram of the patient’s body weight times the average patient’s body weight (for example, 75 kg) .

In other embodiments of the invention, the second dose is stratified depending on the patient’s body weight, that is, the second dose is selected from a set of two or more doses stratified according to the patient’s body weight (for example, 1000 mg of a drug substance for patients weighing ≤75 kg, and 1200 mg for patients weighing> 75 kg), and the first dose is more in moles of the drug than the highest dose of a set of stratified DOS depending on the patient’s body weight.

In still other embodiments of the invention, the second dose is a fixed dose and the first dose is greater than the second dose in moles of drug substance.

In one non-limiting example, the invention relates to any oral pharmaceutical compositions used for the oral administration of a known synthetic IFN-α receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant, or a hyperglycosylated protease-resistant polypeptide variant as described below in the section below "Treatment Methods Using IFN-α," a Treatment Method.

In another non-limiting example, the invention relates to any oral pharmaceutical compositions used for the oral administration of the known synthetic IFN-β receptor polypeptide agonist of the present invention, the known hyperglycosylated polypeptide variant, the known protease-resistant polypeptide variant, or the known hyperglycosylated protease-resistant polypeptide variant in described below in the section "Treatment Methods Using IFN-β", a method of treatment.

In another non-limiting example, the invention relates to any oral pharmaceutical compositions used for oral administration of a known synthetic IFN-γ receptor polypeptide agonist of the present invention, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant, or a hyperglycosylated protease-resistant polypeptide variant in the following, in the section "Treatment Methods Using IFN-γ", a method of treatment.

Peptide-borne oral formulations

Additional oral formulations suitable for the indicated use include the known inventive synthetic variant of the type 1 interferon receptor polypeptide, the known hyperglycolized polypeptide variant, or the known hyperglycolized, protease-resistant polypeptide variant mixed with an oral delivery vehicle as described in WO 03/066859. For example, a suitable oral form includes the desired synthetic Type 1 interferon receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant, or a hyperglycosylated protease-resistant polypeptide variant; and a penetrating peptide (also referred to as a "peptide carrier"). A penetrating peptide is any peptide that facilitates the translocation of a substance along a biological barrier, for example, the epithelial layer lining the gastrointestinal tract. Suitable peptide carriers include those derived from various proteins, including, but not limited to, integral membrane protein, bacterial toxin, non-pathogenic bacteria, viral protein, extracellular protein, and the like proteins. The amino acid sequence of the peptide carrier may be the same as the amino acid sequence of a peptide of natural origin, or may be a modified version of such a peptide (for example, include one or more amino acid substitutions in a peptide of natural origin)

Peptide carriers include from 10 to 30 amino acids in length, for example from 10 to 15 amino acids, from 15 to 20 amino acids, from 20 to 25 amino acids, or from 25 to 30 amino acids in length.

Suitable peptide carriers include, but are not limited to any single peptide 1-34, as shown in table 9 below (SEQ ID NOs: 1311-1326).

Table 9 Peptide / Organism Sequence Peptide 1: from ORF H10638 Haemophilus influenzae NYHDIVLALAGVCQSAKLVHQLA Peptide 2: from PM 1850 Pasteutrella multoclda NYYDITLALAGVCQAAKLVQQFA Peptide 3: from YCFC Escherichia coli NYYDITLALAGICQARLVQQLA Peptide 4: from VCI127 Vibrio cholerae AIYDRTIAFAGICQAVALVQQVA Peptide 5: from BU262 Buchnera aphidicola KIHLITLSLAGICQSAHLVQQLA Peptide 6: from PA2627 Pseudomonas acruginasa DPRQQLIALGAVFESAALVDKLA Peptide 7: from XP1439 Xylella fastidiasa LIDNRVLALAGVVQALQQVRQLA Peptide 8: from MLRO 187 Rhizoblum loti NLPPIVLAVIGICAAVFLLQQYV Peptide 9: from the NK-2 Human Receptor NYFIVNLALADLCMAAFNAAFNF Peptide 10: from CPN0710 / C Chlamydia pneumoniae TAFDFNKMLDGVCTYVKGVQQY
L Peptide 11: from MLR4119 RA1LIPLALAGLCQVARAGDISS Rhizoblum loti Peptide 12: from NprB bacillus subillis MRNLTKTSLLLAGLCTAAQMVFV TH Peptide 13: from Pilin Kingella dentrificans IELMIVIAIIGILAAIALPAYQEYV Peptide 14: from Pilin Eikenella corrodens reLMrVIAnOILAAIAU »AYQOYV Peptide 15: from zonula occludens toxin (ZOT) ASFGFCIGRLCVQDGF Peptide 29: from the human receptor NK-1 NYFLVNLAFAEASMAAFNTVVNF Peptide 30: from YCFC Escherichia coli MNYYDITLALAGICQSARLVQQL A Peptide 31: from YCFC Escherichia coli MYYDITLALAGICQSARLVQQLA Peptide 32: from YCFC Escherichia coli MYDITLALAGICQSARLVQQLA Peptide 33: from NprB Bacillus subtilis MRNLTRTSLLLAGLCTAAQMVFV Peptide 34: from ORF NI0638 Haemophilus influenzae NYHDIVLALAGVCQSARLVHQLA

Natural peptide carriers also include variants of any one peptide 1-34, as shown in table 9, for example, a variant that differs from one to one peptide 1-34 from one to five amino acids; and fragments of any one peptide 1-34. Variants of any one peptide 1-34 include those that have one to five conservative amino acid substitutions of any one peptide of 1-34. Fragments of any one peptide from 1-34 include fragments containing from 10 to 15 amino acids continuously following each other, fragments containing from 15 to 20 continuously following each other amino acids, fragments containing from about 20 to about 25 continuously following each other the other amino acids of any one peptide from 1-34.

The peptide carrier may be “associated with” (also referred to as “fused to,” “paired with,” “coupled to,” or “attached to”) the desired synthetic type 1 interferon receptor, hyperglycosylated, protease-resistant, or hyperglycosylated, protease- protein-resistant by any of the methods, including, for example, covalent interaction, ionic interaction, hydrophobic interaction, hydrogen bond or other type of association (for example, interaction through Vander-Waals forces; nonspecific asso citation due to the preferred solvent; and the like). Attachment of the peptide carrier to the desired protein is accomplished by any chemical, biochemical, enzymatic, or genetic mating method known in the art.

If the peptide carrier is paired with the desired synthetic type 1 interferon receptor, a hyperglycosylated, protease-resistant, or hyperglycosylated, protease-resistant protein, typically the N-terminus of the desired protein is paired with the carboxyl terminus of the peptide carrier. The desired synthetic type 1 interferon receptor, hyperglycosylated, protease-resistant or hyperglycosylated, protease-resistant protein can be paired with a peptide carrier directly or indirectly, for example, by covalent binding. For example, the covalent bond may be a peptide bond, or the covalent bond may be achieved by a homo- or hetero-functional binding reagent. The coupling reagent may be a succinimidyl- (N-maleimidomethyl) -cyclohexane-1-carboxylate (SMCC) type of carrier. Covalent binding can be achieved using a peptide linker.

In some embodiments of the invention, the desired synthetic type 1 interferon receptor, a hyperglycosylated, protease-resistant or hyperglycosylated, protease-resistant protein, is coupled to a peptide carrier via a linker peptide, which may be cleavable. The linker peptide may include any variety of amino acid sequences. Proteins can be linked by a spacer peptide of generally flexible structure, although other chemical bonds are not excluded. It is currently believed that the most useful linker sequences will be primarily peptides between about 6 to about 40 amino acids in length, or between about 6 to about 25 amino acids in length. These linkers are mainly obtained using synthetic, linker-coding oligonucleotides used for pairing with proteins. Peptide linkers with some degree of flexibility are preferred. Linker peptides can have virtually any amino acid sequence, bearing in mind that preferred linkers will have sequences that will usually lead to a flexible peptide. The use of small amino acids, such as glycine and alanine, is useful for creating a flexible peptide. The creation of such sequences is a routine work based on the prior art. A variety of various commercially available linkers for use in accordance with the present invention are contemplated.

It is known that amino acid sequences rich in alanine and proline residues are important for the flexibility of multi-domain protein structures. For example, such sequences bind domains of the so-called E2 components of 2-oxohydrogenase acid complexes, such as pyruvate dehydrogenase complex and 2-oxo glutarate dehydrogenase complex. Alanine proline enriched areas are also found in myosin light chains. Typical linkers used according to the invention have combinations of glycine, alanine, proline and methionine residues, such as AAAGGM (SEQ ID NO: 1332); AAAGGMPPAAAGGM (SEQ ID NO: 1333); AAAGGM (SEQ ID: 1334) and PPAAAGGM2 (SEQ ID NO: 1335). Other typical linker peptides include IEGR (SEQ ID NO: 1336); which can be cleaved by factor Xa) and GGKGGK (SEQ ID NO: 1337). However, any flexible linkers can generally be used with a length between about 6 to about 40 amino acids between amino acids. Linkers can have virtually any sequence that will result in the creation of a flexible peptide, including alanine-proline enriched sequences of the type of the sequences described above.

In some embodiments, the desired synthetic type 1 interferon receptor is a hyperglycosylated, protease-resistant or hyperglycosylated, protease-resistant protein coupled to a peptide carrier via a linker peptide that is cleaved by a peptide. In some embodiments, the enzyme is conveniently activated under special physiological conditions.

In other embodiments, the desired synthetic type 1 interferon receptor, a hyperglycosylated, protease-resistant or hyperglycosylated, protease-resistant protein is paired with a peptide carrier via a non-covalent bond, where the non-covalent bond is achieved by adhering the hydrophobic moiety to the peptide carrier so that the hydrophobic moiety allows be incorporated into the surface of a hydrophobic vesicle in which the desired synthetic type 1 interferon receptor is located, g perglikozilirovanny, protease-resistant, or a hyperglycosylated, protease-resistant polypeptide. In other embodiments, the non-covalent bond is a non-covalent, high affinity bond, such as a biotin-avidin or biotin-streptavidin bond.

Peptides can be synthesized chemically or enzymatically, can be produced by a recombinant method, can be isolated from a natural source, or by a combination of methods. Peptides can be isolated from natural sources using standard protein purification methods known in the art, including, but not limited to, high performance liquid chromatography, exclusive chromatography, gel electrophoresis, affinity chromatography, or other purification methods. A solid phase synthesis technique may be used where such a technique is also well known in the art. See Jones, The Chemical Synthesis of Peptides (Clarendon Press, Oxford) (1994). Basically, in such methods, the peptide is obtained by sequentially adding activated monomer units to the solid phase associated with the growth of the peptide chain. Well-designed recombinant DIC technologies can also be used to produce peptides.

A typical oral form includes enteric coated tablets and gelatin capsules, which include a peptide carrier; and the desired synthetic type 1 interferon receptor, hyperglycosylated, protease-resistant or hyperglycosylated, protease-resistant polypeptide; and one or more: a) a diluent, for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine; b) a protease inhibitor such as arotinin or trasylol; c) a lubricant, for example, silicon, talc, stearic acid, their magnesium and calcium salts, poloxamer or polyethylene glycol; d) a binder (e.g., for tablets), e.g. magnesium aluminosilicate, starch paste, gelatin, tragacanth gum, methyl cellulose, carboxymethyl cellulose sodium and / or polyvinyl pyrrolidone; e) an ionic surfactant such as bile acids; f) disintegrants, for example, starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and g) one or more adsorbents, a colorant, a flavor corrector, and a sweetener. In some embodiments, the oral form further includes one or more preservatives, a stabilizer, a humectant, an emulsifying agent, a dissolution promoter, a salt, and a buffer.

The oral form in some embodiments of the invention further includes one or more nonionic detergents, an ionic detergent, a protease inhibitor, and a reducing agent. The nonionic detergent may be a poloxomer, such as Pluronic F-68; the ionic detergent may be a bile salt, such as taurodeoxycholate; the protease inhibitor may be aprotinin or a soybean trypsin inhibitor; the restoring agent may be N-acetyl-L-cysteine.

Combined Composition

The present invention also describes a pharmaceutical composition comprising a type I interferon receptor synthetic polypeptide agonist of the present invention that is glycosylated; glycosylated IFN-γ; and a pharmaceutically acceptable excipient. In some embodiments of the invention, a glycosylated synthetic type I interferon receptor polypeptide agonist of the present invention and glycosylated IFN-γ are mixed together. In some embodiments of the invention, the glycosylated synthetic type I interferon receptor polypeptide agonist of the present invention and the glycosylated IFN-γ are mixed in a separate liquid composition, which is placed in a separate reservoir for use in a drug delivery device. In some embodiments of the invention, a glycosylated synthetic type I interferon receptor polypeptide agonist of the present invention and glycosylated IFN-γ are formulated for injection delivery. In other embodiments of the invention, a glycosylated synthetic type I interferon receptor polypeptide agonist of the present invention and glycosylated IFN-γ are included in a composition suitable for oral delivery. Compositions for oral delivery include the compositions described above.

The present invention also describes a pharmaceutical composition comprising a single dose of a glycosylated synthetic interferon type I receptor polypeptide agonist of the present invention and a single dose of glycosylated IFN-γ, which is sufficient for use in any method described here that uses the co-administration of a glycosylated synthetic type I interferon receptor polypeptide agonist of the present the invention and glycosylated IFN-γ in the treatment of a patient. In some aspects, the present invention describes a medicament reservoir or other container comprising a glycosylated synthetic polypeptide agonist of the type I interferon receptor of the present invention and glycosylated IFN-γ, co-formulated as a liquid, where, as a glycosylated synthetic polypeptide agonist of the type II interferon receptor of the present invention, both glycosylated IFN-γ are present in the composition in a quantity suitable for a single dose each. Quantitative doses are described herein. The tank can be made in any variety of forms, including, but not limited to, a cartridge, a syringe, a reservoir of a device for continuous delivery, etc.

In some embodiments of the invention, a pharmaceutical composition comprising a glycosylated synthetic interferon type I receptor polypeptide agonist of the present invention and a glycosylated IFN-γ polypeptide is prepared by mixing (a) a pharmaceutical composition comprising a glycosylated synthetic type I interferon receptor polypeptide agonist of the present invention in a sterile aqueous solution; and (b) a pharmaceutical composition comprising glycosylated IFN-γ in a sterile aqueous solution.

Polynucleotides, vectors and host cells

[00626] The claimed invention is a nucleic acid comprising a nucleotide sequence encoding the claimed synthetic type I interferon receptor agonist polypeptide; vectors comprising the claimed polynucleotide; and host cells comprising the claimed polynucleotide or vector. The claimed polynucleotide is used to create the claimed expression vector and genetically modified host cells, which are used to produce the claimed peptide agonist.

The claimed invention provides nucleic acid compositions encoding the claimed synthetic type I interferon receptor agonist polypeptide. As used here, the term “nucleic acid composition” encompasses a nucleic acid sequence including an open reading frame that encodes the claimed synthetic type I interferon receptor agonist polypeptide and is capable, under the influence of appropriate conditions, to begin its expression so that the synthetic interferon receptor agonist polypeptide Type I was produced in host cells, including nucleic acid. The term also refers to a nucleic acid that is homologous or substantially similar to a nucleic acid encoding a synthetic type I interferon receptor agonist polypeptide.

Type I interferon receptor agonist polypeptide. As used herein, the term “nucleic acid composition” refers to a composition comprising an open reading frame nucleic acid sequence that encodes a claimed synthetic type I interferon receptor agonist polypeptide and is capable of initiating expression under appropriate conditions such that the synthetic agonist polypeptide type I interferon receptor was produced in host cells containing nucleic acid. The term also implies that the nucleic acid is homologous or substantially similar or identical to the nucleic acid encoding the claimed synthetic type I interferon receptor agonist polypeptide.

Therefore, the claimed invention provides nucleic acids comprising nucleotide sequences encoding the claimed synthetic type I interferon receptor agonist polypeptide and nucleic acids having substantial identity with such a nucleic acid (i.e. homologs). In many embodiments, the claimed nucleic acid comprises a nucleotide sequence that encodes the claimed synthetic type I interferon receptor agonist polypeptide and which has at least about 75%; at least about 80%; at least about 85%; at least about 90%; at least about 95%; at least about 98%; at least about 99%; or more, a nucleotide sequence identical to the nucleotide sequence (especially the claimed polypeptide-coding region of the nucleotide sequence) encoding the claimed synthetic type I interferon receptor agonist polypeptide.

In certain embodiments, the claimed nucleic acid comprises nucleotide sequences encoding a synthetic type I interferon receptor agonist polypeptide comprising the amino acid sequence as set forth below in any one of SEQ ID NOs: 9-19. In separate embodiments, the claimed nucleic acid comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 24-34.

In certain embodiments, the claimed nucleic acid comprises nucleotide sequences encoding a synthetic type I interferon receptor agonist polypeptide comprising the amino acid sequence as set forth below in any of SEQ ID NOs: 48-52. In certain embodiments of the invention, the claimed nucleic acid comprises nucleotide sequences encoding a synthetic type I interferon receptor agonist polypeptide comprising the amino acid sequence as set forth below in any of SEQ ID NOs: 55-59.

A similar sequence is when it is calculated on the basis of a reference sequence, which can be many large sequences, such as a conservative motif, a coding region, a flanking region, etc. etc. The reference sequence is usually at least about 18 nucleotides long, usually at least 30 nucleotides long, and can be expanded to the complete sequence with which it will be compared. Algorithms for sequence analysis are already known, such as BLAST described by Altschul et al. (1990), J. Mol. Biol. 215: 403-10 (the use of replacement units, i.e., parameters w = 4 and T = I1).

Also includes nucleic acids that hybridize with the above nucleic acids under strictly controlled conditions. An example of stringent hybridization is hybridization at 50 ° C or higher, and at 0.1 × SSC (15 mM sodium chloride / 1.5 mM sodium citrate). Another example of stringent hybridization is incubation overnight at 42 ° C in the following solution: 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhard solution, 10% dextran sulfate, and 20 [mu] g / ml denatured, cut salmon sperm DNA, after washing the filters in 0.1 × SSC at a temperature of about 65 ° C. Stringent hybridization conditions are hybridization conditions that at least correspond to the above conditions. Other stringent hybridization conditions are known in the art and can also be used to identify nucleic acids in particular embodiments of the invention.

Nucleic acids encoding proteins and polypeptides of the claimed invention have many embodiments of DNA, including cDNA. The term “nucleic acid of a synthetic type I interferon receptor agonist polypeptide”, as used herein, corresponds to an open reading frame encoding the claimed specific polypeptides, as well as adjacent 5 ′ and 3 ′ non-coding nucleotide sequences involved in the regulation of expression, for example, from 100 bp to about 20 kb beyond the coding region, but advancement in any direction is possible. Nucleic acids can be inserted into an appropriate vector for extrachromosomal maintenance in the cell, or for integration into the host genome, as described in more detail below.

The nucleic acid composition of the claimed invention can encode all or part of the claimed synthetic type I interferon receptor agonist polypeptide. Double or single-stranded fragments can be obtained from DNA sequences through chemically synthesized oligonucleotides in accordance with selected convenient methods, by digestion with restriction endonucleases, by amplification by polymerase chain reaction, etc. ways.

In particular embodiments, the claimed nucleic acid may be prepared by chemical synthesis, i.e. using an oligonucleotide synthesizer, when oligonucleotides are selected based on the amino acid sequence of the desired polypeptide, and in many embodiments, selecting codons that are most suitable for the host cell in which the recombinant polypeptide will be synthesized.

For example, individual small oligonucleotides, for encoding regions of the desired polypeptides, can be synthesized and assembled by PCR, ligation, or chain ligation reaction (LCR). Individual oligonucleotides typically contain 5'- or 3'-ends for complementary assembly. After assembly, the nucleotide sequence encoding the claimed polypeptide is inserted into the recombinant vector under the control of the sequence required for expression of the claimed nucleic acids, and subsequent production of the claimed polypeptide in the desired transformed cells.

In separate embodiments of the invention, the claimed nucleic acid can be created in such a way that at least about 10%; at least about 20%; at least about 30%; at least about 40%; at least about 50%; at least about 70%; at least about 80%; or at least about 90% or more of the codons that are preferred in human DNA sequences. See, for example, Table 8 (below).

The claimed nucleic acid molecules are mainly propagated through incorporation into a molecular vector. Viral and non-viral vectors are used, including plasmid. The choice of plasmid will depend on the type of cell in which nucleic acid propagation is preferred and the purpose of its propagation. Individual vectors are used to amplify and prepare large quantities of the desired DNA sequence.

The present invention further relates to recombinant vectors (“constructs”) including the claimed polynucleotide. Recombinant vectors include vectors used to propagate the claimed polynucleotide, and expression vectors. Recombinant vectors are suitable for propagating the claimed polynucleotides (cloning vectors). The claimed recombinant expression vector is necessary for the efficient expression of the claimed polynucleotide in the cell, for example, for the production of the claimed synthetic type I interferon receptor agonist polypeptide. The selection of an appropriate vector is well represented in the prior art. Many such vectors are commercially available.

Expression vectors are suitable for expression in cell cultures. Such vectors typically include regulatory sequences ("control sequences" or "control regions") that are necessary for the efficient expression of the claimed polynucleotide with which they are operatively linked. In addition, other vectors can be used for transfer and expression in cells in the whole organism or patient.

Expression vectors usually have convenient restriction sites located close to promoter sequences that allow expression of nucleic acid sequences encoding heterologous proteins. Selection markers showing expression in the host may be provided. Expression vectors can be used for the production of fusion proteins, where the exogenous fusion peptide additionally provides the functional purpose of the claimed, i.e. increases protein synthesis by the cell, increases its stability, reaction with antiserum. As an enzyme marker, for example, [beta] -galactosidase, luciferase and the like can be used.

Expression cassettes can be prepared so that they include a transcription initiation region, a promoter region (eg, a promoter that functions in a eukaryotic cell), a claimed polynucleotide, and a transcription termination region. After the introduction of DNA, the cells containing the construct can be selectively selected using a selection marker, the cells are expanded and then used for expression.

Expression cassettes can be introduced into various vectors, for example, plasmids, [0758] BAC, HAC, YAC, bacteriophages such as lambda, P1, M1 3, etc. animal or plant viruses, and the like, where the vectors are typically characterized by their ability to select cells including the expression vector. Vectors can provide extrachromosomal gene existence, especially when plasmids or viruses are used as vectors; or intended to integrate into the host chromosome. When an extrachromosomal gene existence is desired, the claimed sequence is replicated using a plasmid, which may be low or high copy number. A wide variety of selection markers are available, in particular those that protect against toxic substances, and especially against antibiotics. A selectable marker that is selected and which for selection in accordance with the nature of the host; in some cases, complementation with auxotrophic hosts may be used. The introduction of the DNA construct into the host cell can be achieved using a convenient method, for example, conjugation, bacterial transformation, calcium precipitation of DNA, electroporation, fusion, transfection, infection by viral vectors, etc.

General aspects of the transformation of mammalian cell systems have been described by Axel in U.S. Pat. No. 4,399,216 published August 16, 1983. Transformation into yeast is usually carried out according to the standard method of Van Solingen et al., J. Bact., 130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA) 76: 3829 (1979). An optimized method for calcium phosphate transfection of eukaryotic host cells is described by Wumi and Jordan in U.S. Pat. Nos. 5,484,720 and 5,593,875. However, other methods of introducing DNA into cells can be used, such as introducing DNA by injection into the nucleus, electroporation or fusion of protoplasts.

The presented invention allows to genetically modify the host cells, which can be isolated from the host cells, introducing the claimed polynucleotide or, in separate embodiments, the claimed expression vector into them. Suitable host cells include prokaryotes, such as E. coli, B. subtilis; eukaryotes; insect cells in combination with baculovirus vectors; yeast cells such as Saccharomyces cerevisiae; or cells of higher organisms, such as vertebrates, including amphibians (e.g., Xenopus laevis oocytes); and mammals, for example, COS cells, CHO cells, HEK293 cells, MA-10 cells, and the like, can be used to express host cells. Host cells can be used for the purpose of propagating the claimed polynucleotide, for the production of a synthetic type I interferon receptor agonist polypeptide. In many embodiments of the invention, eukaryotic host cells are used as host cells. In particular, in many embodiments of the invention, eukaryotic host cells that are capable of glycosylating proteins are used.

Mammalian cells are used to synthesize the claimed synthetic type I interferon receptor agonist polypeptide, which can be cultured in many different environments. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium [(MEM), Sigma], RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium [(DMEM), Sigma)] are used to cultivate such cells. As additions, media described by Ham and Wallace, Meth can be used. Enz., 58: 44 (1979); Bames and Sato, Anal Biochem., 102: 255 (1980); U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; or U.S. Pat. No.4,560,655; WO 90/03430; WO 87/00195; U.S. Pat. Re. No. 30.985; or U.S. Pat. No. 5,122,469. All media described in these sources can be used as culture media for host cells. Any of these media can be enriched as needed with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphates), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic components, usually represented by final concentrations in the micromolar interval) and glucose or its equivalent energy source. Any other necessary additives may also be included in appropriate concentrations, in accordance with the knowledge of a qualified professional. Culturing conditions, such as temperature, pH, and the like, previously used for the expression of selected host cells and understood by a qualified person as obvious from the prior art.

Antibody Compositions

Antibodies are also provided that are specific for the claimed synthetic type I interferon receptor agonist polypeptide. Suitable antibodies were obtained by immunizing an animal host with peptides comprising the claimed protein in whole or in part. Suitable host animals include mice, sheep, rats, goats, hamsters, rabbits, etc. In many embodiments, the claimed antibodies have been isolated; and in many embodiments, the claimed antibodies have been purified.

An immunogen may include a whole protein or its derivatives and fragments. Typical immunogens include all or part of a protein if it undergoes a post-translational modification found in the native target protein. Immunogens are created in various ways known to a person skilled in the art, for example, by expressing cloned genes using suitable recombinant methods, chemical synthesis, chemical synthesis of type I interferon receptor agonist polypeptides, and the like.

To prepare polyclonal antibodies, the first step was to immunize the host animal with the target protein, when the target protein was in highly purified form, comprising less than 1% contaminating impurities. An immunogen may include the complete target protein, fragments and derivatives thereof. To increase the immune response of the host animal, the target protein may be in combination with adjuvants, including aluminum, dextran sulfate, large polymer anions, oil and water emulsions, for example, Freund's adjuvant, Freund's complete adjuvant and the like. The target protein may also be conjugated to a synthetic protein carrier or synthetic antigens. Various hosts may be immunized to form polyclonal antibodies. Such hosts include rabbits, guinea pigs, rodents, for example, mice, rats, goats, rams, and the like. The target protein is usually administered subcutaneously to the host, with an initial dose administered once or more, usually at least twice, with an additional booster dose. After immunization, blood is taken from the animal, then the serum is separated from the blood cells. The immunoglobulins (Ig) present in the resulting antiserum can be further fractionated using known methods such as ammonium sulfate fractionation, DEAE chromatography and the like.

Monoclonal antibodies are produced by known methods. Typically, the spleen and / or lymph nodes of the immunized host animal were the source of plasma cells. Plasma cells were immortalized by fusion with myeloma cells to form hybridoma cells. The culture supernatant of individual hybridomas was investigated using standard techniques to identify those that produce antibodies of the desired specificity. Suitable animals for producing monoclonal antibodies to a human protein are mice, rats, hamsters, and the like. To obtain antibodies to mouse protein, hamsters, guinea pigs, rabbits, etc. were used. Antibodies could be purified from the supernatant of hybridoma cells or ascitic fluid using standard techniques, for example, affinity chromatography, using a protein bound to an insoluble support, protein A-sepharose, and the like.

Antibodies can be produced as separate chains instead of normal multimeric structures. Single chain antibodies are described by Jost et al. (1994) J Biol. Chem. 269: 26267-73 and other authors. DIC sequences encoding the variable regions of the heavy chains and the variable regions of the light chains were ligated using a spacer encoded so that it includes at least four small neutral amino acids, glycine and / or series. The protein encoded by this fusion allows the collection of a functionally variable region that retains the specificity and affinity of the original antibody.

Of particular interest are individual embodiments of humanized antibodies. The humanization method of antibodies is known to those skilled in the art. Humanized antibodies may be the product of animals having genes with a constant region of human immunoglobulin (see International Patent Applications WO 90/10077 and WO 90/04036). Alternatively, such antibodies can be constructed by recombinant DNA technology by replacing CH1, CH2, CH3, hinge domains, and / or the skeleton domain with the corresponding sequences from the human genome (see WO 92/02190).

The use of Ig cDNA for the construction of chimeric immunoglobulin genes is already known [Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439 and (1987) J. Immunol. 139: 3521]. mRNA isolated from hybridomas or other cells producing antibodies was used to obtain cDNA. The cDNA of interest can be amplified by PCR using a specific primer (U.S. Patent nos. 4,683,195 and 4,683,202). Alternatively, a library can be obtained and an isolate with the sequence of interest selected. The resulting DNA sequence encoding the variable region of the antibody is then fused to the human constant sequence. The sequence of constant regions of human genes can be found in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, N.I.H. publication no. 91-3242. Preferably, C genes of the region of people from known clones are used. Isotype selection is based on criteria for the desired effector function, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity. Exemplary isotypes are IgG1, IgG3 and IgG4. Kappa and lambda regions from the human immunoglobulin light chain can also be used. Chimeric, humanized antibodies are then expressed by standard methods.

Antibody fragments, such as Fv, F (ab ') 2 and Fab, can be prepared by digesting intact antibodies, for example, a protease or a chemical agent. As an alternative, a truncated gene can be used. For example, a chimeric gene encoding part of the F (ab ') 2 fragment includes a DNA sequence encoding the CH1 domain and the region of the H chain loop following the translational stop codon, which allows a truncated molecule to be obtained.

The consensus sequences of the H and L J regions can be used to select oligonucleotides to be used as primers for introducing the desired restriction sites into the J region for subsequent binding of segments of the V region to segments of the human C region. Region cDNA can be modified by site-directed mutagenesis at a restriction site site in a similar position in humans.

Expression vectors include plasmids, retroviruses, YAC and EBV derivatives of Epis and the like. A suitable vector is one that encodes a functionally complete CH or CL human immunoglobulin sequence with corresponding restriction sites designed so that any VH or VL sequence can be easily inserted and expressed. In such vectors, splicing is usually performed between the splicing donor site inserted in the J region and the splicing acceptor site preceding the human C region; as well as in the splicing regions that are within the limits of human HF exons. Polyadenation and transcriptional termination occur in native chromosome sites below coding regions. The resulting chimeric antibodies can be combined with any strong promoter, including retroviral LTR; for example, with the early promoter SV-40 (Okayama et al. (1983) Mol. Cell. Bio. 3: 280), LTR of Routh sarcoma virus (Gorman et al. (1982) Proc. Natl. Acad. Sci. USA 79: 6777), LTR of Moloney mouse leukemia virus (Grosschedl et al. (1985) Cell 41: 885); native Ig promoters and the like.

Diagnostic use

The synthetic type I interferon receptor agonist polypeptides of this invention are unique research reagents that provide type I interferon activity matrices for use in screening in a chemical library, where the practitioner can use signal transduction in the test as an initial high-resolution test, intended for agents that inhibit in a wide range of activity of type I interferons of the claimed synthetic receptor agonist in terferon type I. In this direction, candidate agents are likely to inhibit a wide range of type I interferon activities (similar to the activity profile of the claimed synthetic type I interferon receptor agonist polypeptide), which could have been obtained earlier, in order to avoid excessive appreciation and organizationally impossible number of studies on the inhibition of viral growth or cell proliferation in large chemical libraries.

In one embodiment of the invention, a synthetic type I interferon receptor agonist polypeptide was used to screen chemical libraries in a kinase-receptor activation test (KIRA), as described in WO 95/14930 (published June 1, 1995). The KIRA test is suitable for use in this invention because the ligand attaches in situ to the type I interferon receptor complex on the surface of the host cell expressing the receptor and induces a rapid increase in tyrosine residue phosphorylation in the intracellular space of both components (IFNAR1 and IFN AR2 ) a receptor as set forth by Platanias and Colamonici, J. Biol. Chem., 269: 17761-17764 (1994). Tyrosine phosphorylation can be used to measure signal transduction. The effect of library components on the tyrosine phosphorylation level induced by the synthetic type I interferon receptor agonist polypeptide in the KIRA test is an indication of the complex inhibitory activity directed against a large array of type I interferons mimicked by the synthetic type I interferon receptor agonist polypeptide.

The KIRA test can be used to determine (a) host cells expressing the type I interferon receptor (both components of the receptor are IFNAR1 and IFN AR2), and (b) cells expressing the claimed synthetic type I interferon receptor agonist polypeptide whose inhibitory profile of interest. As cells that naturally express a type I interferon receptor, human cells such as Daudi or U-266 myeloma cells described by Colamonici and Domanski, J. Biol can be used. Chem. 268: 10895-10899 (1993). Additionally, cells that have been transfected with IFNAR1 and IFNAR2 components and containing the intracellular signaling protein necessary for signal transduction of type I interferon, such as L-929 mouse cells described by Domanski et al, J. Biol, can be used. Chem., 270: 21606-21611 (1995). For research in the KIRA test, the candidate antagonist was incubated with the claimed synthetic type I interferon receptor agonist polypeptide, and the incubation mixture contained cells expressing the type I interferon receptor. The treated cells were lysed and the IFNAR2 protein in the lysate was immobilized on a solid phase containing an anti-IFNAR2 antibody. Signal transduction was evaluated by measuring the amount of phosphorylated tyrosine in the intracellular domain captured by IFN AR2 and the amount of phosphorylated tyrosine that is present in the intracellular domain of any co-captured IFNAR1. Alternatively, cell lysis and immunoprecipitation can be performed under denaturing conditions to avoid co-capture of IFNAR1 and allow measurement of tyrosine phosphorylation of IFNAR2 separately, i.e. as described by Platanias et al., J. Biol. Chem., 271: 23630-23633 (1996). Tyrosine phosphorylation can be accurately measured using labeled antiphosphothyrosine antibodies that recognize phosphorylated tyrosine residues.

In another embodiment of the invention, host cells coexpress IFNAR1 and a chimeric construct containing IFNAR2 fused at its carboxyl end to an affinity polypeptide that is used in the KIRA test. The chimeric construct of IFNAR2 allows sorption of the construct from the cell lysate by using a solid phase sorbent agent (instead of anti-IFNAR2 antibodies) specific for the affinity polypeptide. In a preferred embodiment, the affinity linked peptide is derived from Herpes simplex virus glycoprotein D (gD); the binding agent is anti-gD monoclonal antibody, as described in examples 2 and 3 of WO 95/14930.

In this system, a synthetic type I interferon receptor agonist polypeptide that has a type I interferon activity profile is interesting for use as a standard in analyzing the ability to inhibit tyrosine phosphorylation activity in samples formed by members of screened chemical libraries. IFNAR2 ICD tyrosine phosphorylated sample generated by a synthetic type I interferon polypeptide receptor agonist as a standard was compared with tyrosine phosphorylated samples obtained using the standard in the presence of library compounds; and it was found that the samples indicating tyrosine phosphorylation inhibition are identical to candidate agents, which are likely to inhibit the range of type I interferon activities mimicked by the standard. Accordingly, the synthetic type I interferon receptor agonist polypeptide of the invention provides a means for quickly and efficiently screening compounds that are likely to inhibit the individual activity spectra of type I interferon represented by the claimed synthetic type I receptor agonist polypeptide.

In addition, the synthetic type I interferon receptor agonist polypeptide of the present invention is useful in diagnostic assays for the expression of type I interferon receptors in specific cells or tissues. In these assays, the claimed synthetic type I interferon receptor agonist polypeptide is labeled as described below and / or immobilized on an insoluble matrix that allows detection of the type I interferon receptor in the sample.

The claimed synthetic type I interferon receptor agonist polypeptide can be used to detect type I interferon receptor by any of the well-known diagnostic methods. For example, biological samples can be tested for type I interferon receptor activity by obtaining a sample from a selected source, mixing samples with the claimed synthetic type I interferon receptor agonist polypeptide, allowing the type I interferon receptor agonist synthetic polypeptide to form a type I interferon agonist / receptor complex with any type I interferon receptor presented in the mixture. Biological samples can be prepared for research using methods known from the prior art, which are suitable for special cases. Methods of mixing the samples with the claimed synthetic type I interferon receptor agonist polypeptide and detecting the type I interferon agonist / receptor complex can be selected according to the type of study used. Such assays include competitive and sandwich assays, and steric inhibition assays. Competitive and sandwich methods use the phase separation step as an integral part of the method, while steric inhibition analysis can only be carried out in a homogeneous reaction mixture.

All analytical methods for the type I interferon receptor use one or more of the following reagents: labeled type I interferon receptor analog, immobilized type I interferon receptor analog, labeled synthetic type I interferon receptor agonist polypeptide, immobilized synthetic type I interferon receptor agonist polypeptide, and steric conjugates. Tag agents are also known as tracers.

A label is used with any detectable (detector) function that does not interfere with binding to the type I interferon receptor and the claimed synthetic type I interferon receptor agonist polypeptide. A large number of labels are known for use in immunoassays, for example, including groups that can be detected directly, such as fluorochrome, chemiluminescent and radioactive labels, as well as fragments, such as enzymes, which must be reacted or derivatized to be detected. Examples of such labels include the radioisotopes ^<32> P, ^<14> C, ^<125> 1, ^<3> H, and ^<131> I; fluorophores such as rare earth chelates; or fluorescein and its derivatives; rhodamine and its derivatives; dansil; umbelliferon; luciferase, for example firefly luciferase and bacterial luciferase (US Pat. No. 4737456]; 72,3-Hihydropnthalazinediones, horseradish peroxidase, alkaline phosphatase; beta-galactosidase; glucoamylase, lysozyme, saccharide oxidase, for example, glucose oxidase, 6-phosphate dehydrogenase; heterocyclic oxidases, such as uricase and xanthine oxidase, linked to an enzyme that uses hydrogen peroxide to oxidize a dye precursor, such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals and the like.

Convenient methods suitable for covalently linking tags to proteins or polypeptides. For example, to label antibodies with the fluorescent, chemiluminescent, and enzymes described above, coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, benzidine bis-diazotides, and the like can be used. See U.S. for an example. Pat. Nos. 3,940,475 (fluorimetry) and 3,645,090 (enzymes); Hunter et al., Nature, 144: 945 (1962); David et al., Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol. Methods 40: 219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407-412 (1982). Preferred labels are enzymes such as horseradish peroxidase and alkaline phosphatase.

Conjugation of such a label involves the attachment of an enzyme to an antibody, and is a standard method for immunological testing of samples. For an example, see O'Sullivan et al. "Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay", in Methods in Enzymology, ed. J. J. Langone and N. Van Vunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), p. 147-166.

For the implementation of individual methods of analysis requires the immobilization of reagents. The immobilization act separates the synthetic polypeptide of the type I interferon receptor agonist from any type of interferon receptor that remains in solution in the free state. What is usually done either by bringing the synthetic polypeptide of the type I interferon receptor agonist into an insoluble state, or the interferon receptor analog is adsorbed with a water-insoluble matrix or surface before testing (Bennich et al., US Pat. No. 3,720,760), through covalent binding (for example, using cross-linking with glutaraldehyde), or by rendering the synthetic polypeptide of an interferon type I receptor agonist synthetic polypeptide, for example, by immunoprecipitation.

Other analytical methods, known as competing or sandwich methods, are well developed and widely used in the commercial diagnostic industry.

Competing methods based on the ability of a labeled type I interferon receptor analog to compete for a limited number of sites with a type I interferon receptor contained in a test sample and binding to a synthetic type I interferon receptor agonist polypeptide. The synthetic type I interferon receptor agonist polypeptide is usually insoluble either before or after competition, and then the labeled dried type I interferon receptor polypeptide is coupled to the synthetic type I interferon receptor agonist polypeptide, separated from the unbound label and type I interferon receptor. This separation is completed by decantation (the binding agent was previously soluble) or by centrifugation (the binding agent was precipitated after completion of the competition reaction). The amount of test sample of type I interferon receptor is inversely proportional to the amount of bound label, measured by the amount of labeling substance. The dose-response curve for the amount of type I interferon receptor is known; and compared with the results of a quantitative determination of the content of the type I interferon receptor in the test sample. In cases where enzymes are used as detection markers, these studies are performed using ELISA systems.

There is another kind of competitive analysis, called "homogeneous" analysis and does not require phase separation (phase separation). To perform such an analysis, an enzyme conjugate with a type I interferon receptor is prepared and used so that when a synthetic type I interferon receptor agonist polypeptide binds to a type I interferon receptor, the presence of a synthetic type I interferon receptor agonist polypeptide modifies the enzymatic activity. In this case, the type I interferon receptor or immunologically active fragments thereof were conjugated by bifunctional organic bridges to an enzyme such as peroxidase. Conjugates are selected for use with a synthetic type I interferon receptor agonist polypeptide so that binding of the synthetic type I interferon receptor agonist polypeptide inhibits or potentiates the enzymatic activity of the label. This method per se is already widely practiced under the name EMIT.

Steric conjugates are used in steric hindrance methods for homogeneous analysis. These conjugates are synthesized by covalently linking a low molecular weight hapten to a small fragment of the type I interferon receptor, so that the anti-hapten antibody is able to firmly bind the conjugate at the same time as the synthetic type I interferon receptor agonist polypeptide. Under the conditions of this study, the type I interferon receptor presented in the test sample binds the synthetic polypeptide of the type I interferon receptor agonist, thereby allowing the antigapten to bind the conjugate, leading to a change in the properties of the conjugated hapten, for example, a change in fluorescence when the hapten is a fluorophore.

Sandwich studies are usually performed to determine if a type I interferon receptor is present in a sample. In the sandwich assay sequence, the immobilized synthetic type I interferon receptor agonist polypeptide is used to adsorb the type I interferon receptor in the test sample, the test sample is removed by washing, the coupled type I interferon receptor is used to adsorb labeled antibodies to the type I interferon receptor, and the resulting material is then Separated from the marks. The amount of bound label is directly proportional to the amount of type I interferon receptor in the test sample. In a “simultaneous” sandwich test, the test sample is not separated before the addition of pre-labeled antibodies to the type I interferon receptor.

The above are only examples of typical diagnostic studies for the type I interferon receptor. Other methods, today and in the future, developed to use the synthetic type I interferon receptor agonist polypeptide for determining type I interferon receptors are included within these boundaries, including the biological assays described above.

Therapeutic methods

The presented invention provides a method for the treatment of fibrotic diseases. The claimed methods involve mainly the introduction of effective combinations of a synthetic polypeptide of an interferon type I receptor agonist, a hyperglycosylated version of the peptide, a protease-resistant variant of the peptide and a type II interferon receptor agonist. In certain embodiments of the invention, the claimed method of treatment comprises administering to the patient at least one additional antifibrotic agent.

The presented invention in a more distant future can be used for the treatment of cancer. The claimed methods usually include administering to the patient individual effective amounts of the claimed synthetic interferon type I receptor agonist polypeptide, hyperglycosylated variant of the peptide, protease-resistant variant of the peptide or hyperglycosylated, protease-resistant variant of the polypeptide. In certain embodiments, the claimed method involves the future inclusion of at least one additional anti-cancer agent.

The presented invention additionally provides the implementation of methods of treating a viral infection. The claimed methods typically include administering to the patient, if necessary, effective amounts of the claimed synthetic polypeptide of an interferon type I receptor agonist, a hyperglycosylated variant of the peptide, a protease-resistant variant of the peptide, or a hyperglycosylated, protease-resistant variant of the polypeptide. In separate embodiments, the claimed method in the future involves the inclusion in the treatment regimen of at least one additional antiviral agent.

In separate embodiments, the claimed treatment method in the future involves the inclusion in the scheme of an agent aimed at eliminating the side effects of treatment for the treatment of side effects caused by a therapeutic agent.

Fibrotic diseases

The presented invention provides the implementation of methods of treating fibrotic diseases in an individual having such a disease. The method typically includes administering effective combinations of the claimed synthetic type I interferon receptor agonist polypeptide, a hyperglycosylated variant of the peptide, a protease-resistant variant of the peptide, or a hyperglycosylated, protease-resistant variant of the polypeptide and type II interferon receptor agonist. The methods allow the treatment of fibrotic diseases, including those affecting the lungs, such as idiopathic pulmonary fibrosis, pulmonary fibrosis of known etiology, liver fibrosis or cirrhosis, cardiac fibrosis and renal fibrosis. The etiology can be the result of any acute or chronic lesions, including toxic, metabolic, genetic disorders or infectious agents.

Fibrosis is usually characterized by a pathological or excessive accumulation of collagenized connective tissue. Fibrotic diseases include, but are not limited to collagen disease, interstitial lung disease, human fibrotic lung disease (e.g. obliterative bronchiolitis, idiopathic pulmonary fibrosis, pulmonary fibrosis of known etiology, lung stroma cancer, systemic sclerosis with lung damage, Hermansky-Pudlak syndrome, pneumonia coal mines, asbestosis, silicosis, chronic pulmonary hypertension, AIDS-associated pulmonary hypertension, sarcoidosis and the like), fibrotic vascular disease, arterial sclerosis, atherogenic sclerosis, atherosclerosis, varicose veins, coronary infarction, cerebral infarction, myocardial fibrosis, musculoskeletal fibrosis, post-surgical adhesion, human kidney disease (eg, nephritic syndrome; Alpert syndrome; HIV-associated nephropathy; polycystic disease; polycystic disease; polycystic disease; diabetic nephropathy; chronic glomerulonephritis; nephritis associated with systemic lupus erythematosus and similar diseases), keloid scarring of wounds, progressive systemic sclerosis (PSS), primary sclerosis hepatitis cholangitis (PSC), liver fibrosis, cirrhosis of the liver, renal fibrosis, pulmonary fibrosis, chronic post-grafting disease, scleroderma (local and systemic), Grave ophthalmopathy, diabetic retinopathy, glaucoma, Perron’s disease, penile fibrosis, urestrostenosis, internal cystoscopy after surgery, multiple scarring, myelofibrosis, idiopathic retroperitoneal fibrosis, peritoneal fibrosis of known etiology, drug-induced ergotism, fibrosis after the development of cancer, fibrotic manifestations of a microbial infection (e.g., viral, bacterial, parasitic, fungal, etc.), Alzheimer's disease, fibrotic degeneration of the intestine, developing against the background of inflammatory diseases (including the formation of stricture against Crohn’s disease and microscopic colitis), fibrosis caused by chemical or external injuries (for example, cancer caused by chemotherapy, pesticides, radiation and similar influences; for example, cancer radiotherapy; and the like), and the like.

In certain embodiments, effective amounts of a synthetic type I interferon receptor agonist polypeptide and a type II interferon receptor agonist can be used in any combination of doses that, when administered to a patient with fibrotic disease, will effectively reduce the development of fibrosis, or reduce the rate of its progression, according to at least about 19%; at least about 15%; at least about 20%; at least about 25%; at least about 30%; at least about 35%; at least about 40%; at least about 45%; or at least about 50%; or more, comparing with the degree of development of fibrosis in individuals not previously treated, or comparing with the rate of progression of fibrosis, which was observed in patients in the absence of treatment.

In certain embodiments, effective amounts of the claimed synthetic polypeptide of an interferon type I receptor agonist, a hyperglycosylated version of the peptide, a protease resistant variant of the peptide or hyperglycosylated, protease resistant variant of the polypeptide, and an interferon type II receptor agonist can be used in combined doses in patients with fibrotic disease, they will effectively reduce the development of fibrosis, or reduce the rate of its progression of at least one organ function , Subject to fibrotic changes (e.g., lung, liver, kidney, etc.) at least about 15%; at least about 20%; at least about 25%; at least about 30%; at least about 35%; at least about 40%; at least about 45%; or at least about 50%; or more, comparing with the baseline level of organ function in individuals before treatment, or comparing with the rate of deterioration of organ function studied in an individual in the absence of treatment.

Methods for measuring the incidence of individual organ fibrosis and methods for measuring organ function are known in the art.

Idiopathic pulmonary fibrosis

The presented invention provides the implementation of methods for the treatment of idiopathic pulmonary fibrosis (IPF).

The method basically involves administering to IPF sufferers effective amounts of the claimed synthetic polypeptide of an interferon type I receptor agonist, a hyperglycosylated variant of the peptide, a protease-resistant variant of the peptide or a hyperglycosylated, protease-resistant variant of the polypeptide, and an interferon type II receptor agonist.

In certain embodiments, the diagnosis of IPF is made by detecting common interstitial pneumonia (1LR) on histopathological lung tissue samples obtained by surgical biopsy. Diagnosis criteria are well known: Ryu et al. (1998) Mayo Clin. Proc. 73: 1085-1101.

In other embodiments, the diagnosis of IPF is established or suspected using high-resolution computed tomography (HRCT). When making a diagnosis using HRCT, the following known characteristics are taken into account: (1) the presence of a reticular anomaly and / or signs of bronchiectasis with a basal or peripheral predominance; (2) the presence of voids with basal or peripheral predominance; and (3) the absence of atypical features such as micronodules, perivascular nodes, seals, isolated cysts (not cellular) and mediastinal adenopathy (mild, but sufficient to be detected by chest x-ray).

The diagnosis is established as the corresponding IPF if the following characteristics are identified: (1), (2), and (3) occur together. A presumptive diagnosis of IPF is made if the characteristics given in (1) and (3) were found together

In other embodiments of the invention, “effective amounts” of the claimed synthetic type I interferon receptor agonist polypeptide, a hyperglycosylated peptide variant, a proteasoresistant peptide variant or a hyperglycosylated, proteasoresistant polypeptide variant, and a type II interferon receptor agonist are used in combination at a dose that is effective to slow down disease progression of at least about 10%; at least about 15%; at least about 20%; at least about 25%; at least about 30%; at least about 35%; at least about 40%; at least about 45%; at least about 50%; at least about 55%; at least about 60%; at least about 65%; at least about 70%; or more, compared to placebo control or control without treatment.

Disease progression is assessed by one or more of the following criteria: (1) a 10% or more decrease in predicted FVC; (2) or more in a single breath DL _co . Any of the two criteria for disease progression can be determined by measuring one or more of these parameters over a period of two 4 to 14 weeks and comparing the obtained values with the baseline.

Thus, for example, when an untreated patient or a patient receiving a placebo shows a decrease in FVC of 50% or more in a specified period of time, then the patient who was administered effective combinations of a synthetic interferon type I receptor agonist polypeptide and a type II interferon receptor agonist FVC by 45%, about 42%, about 40%, about 37%, about 35%, about 32%, about 30%, or less, over the same period or longer.

In certain embodiments, “effective amounts” of the claimed synthetic type I interferon receptor agonist polypeptide, hyperglycosylated variant peptide, protease resistant peptide variant or hyperglycosylated, protease resistant variant polypeptide and type II interferon receptor agonist, in any combination and dose, which are effective for increasing survival time without progression of the disease, for example, time from the baseline (for example, at a point in time from 1 to 28 days before cure), the number of deaths or cases of disease progression increase by at least about 10%; at least about 20%; at least about 25%; at least about 30%; at least about 40%; at least about 50%; at least about 60%; at least about 70%; at least about 80%; at least about 90%; at least about 2 times; at least about 3 times; at least about 4 times; at least about 5 times; or more, compared with a placebo control or a control in which patients did not receive treatment. Thus, for example, in certain embodiments, effective amounts of the claimed synthetic polypeptide of an interferon type I receptor agonist, a hyperglycosylated version of the peptide, a protease-resistant variant of the peptide or a hyperglycosylated, protease-resistant variant of the polypeptide, and an interferon type II receptor agonist, in any dose combination that effectively increases the survival time patients at least about 1 week; at least about 2 weeks; at least about 3 weeks; at least about 4 weeks; at least about 2 months; at least about 3 months; at least about 4 months; at least about 5 months; at least about 6 months; at least about 8 months; at least about 10 months; at least about 12 months; at least about 18 months; at least about 2 years; at least about 3 years; and more, in comparison with patients who received a placebo, or who did not receive treatment at all.

In certain embodiments of the invention, effective amounts of the claimed synthetic polypeptide of an interferon type I receptor agonist, a hyperglycosylated version of a peptide, a protease-resistant variant of a peptide or a hyperglycosylated, protease-resistant variant of a polypeptide, and an interferon type II receptor agonist are used with any combination of doses that are effective to improve, according to at least one lung function parameter, for example, a combination of a dose that increases at least one a parameter of at least about 10%; at least about 20%; at least about 25%; at least about 30%; at least about 40%; at least about 50%; at least about 60%; at least about 20%; at least about 80%; at least about 90%; at least twice; at least three times; at least four times; at least five times; or more, compared to untreated people or taking a placebo; in individual of these embodiments, the actual parameters of pulmonary function, which are increased, were determined by comparing the base value with the value at any time point after the start of treatment, for example, after 48 weeks of treatment, or between two time points, for example, intervals from 4 to 14 weeks after start treatment.

In certain embodiments of the invention, effective amounts of the claimed synthetic type I interferon receptor agonist polypeptide, hyperglycosylated peptide variant, protease resistant peptide variant or hyperglycosylated, protease resistant polypeptide variant and type II interferon receptor agonist are used with any combination of dose that is effective to increase FVC at least about 10%, at least about 20%, at least about 25%, around at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, or more compared to the original measurements in the range from 4 to 14 weeks.

In certain embodiments of the invention, effective amounts of the claimed synthetic polypeptide of an interferon type I receptor agonist, a hyperglycosylated variant of the peptide, a protease-resistant variant of the peptide or a hyperglycosylated, protease-resistant variant of the polypeptide and type II interferon receptor agonist are used in any combination of doses, which leads to a decrease in the alveoli: arterial ( Aa) a gradient of at least about 5 mm Hg; no less than about 7 mm Hg; at least about 10 mm Hg; at least about 12 mm Hg; at least about 15 mm Hg; or more than the original.

In certain embodiments of the invention, effective amounts of the claimed synthetic type I interferon receptor agonist polypeptide, hyperglycosylated peptide variant, protease resistant peptide variant or hyperglycosylated, protease resistant polypeptide and type II interferon receptor agonist are used in any dose combination that increases DLCO inhalation, at least by 15%; at least 20%; at least 30%; at least 40%; at least 50%; at least 60%; at least 70%; at least 80%; at least 90%;

at least 2 times; at least 3 times; at least 4 times; at least 5 times; or more, and compared with the original value. CL _C0 is a measure of lung carbon dioxide capacity and is expressed as ml CO / mm Hg / second.

Pulmonary function parameters include, but are not limited to, forced vital capacity (FVC); forced expiratory volume (FEV1); total pulmonary capacity; partial pressure of arterial oxygen at rest; partial arterial oxygen pressure at maximum effort.

Pulmonary function can be determined using any known method, including but not limited to spirometry.

Hepatic fibrosis

The presented invention includes a method for the treatment of hepatic fibrosis, including reducing the clinic of fibrosis, reducing the likelihood of developing hepatic fibrosis and reducing the parameters associated with hepatic fibrosis itself. The methods include administering a combination of effective amounts of the claimed synthetic type I interferon receptor agonist polypeptide, a hyperglycosylated peptide variant, a proteasoresistant peptide variant or a hyperglycosylated, proteasoresistant polypeptide variant, and type II interferon receptor agonist in individually needed amounts. Of particular interest for the embodiment of the invention is the treatment of humans.

Hepatic fibrosis is a precursor to a number of diseases. It is difficult associated with cirrhosis of the liver, portal hypertension, progressive liver failure, hepatocellular carcinoma. A decrease in hepatic fibrosis reduces the risk of such complications. Accordingly, the present invention further suggests the use of methods to reduce the likelihood that the patient will develop cirrhosis associated with fibrosis.

The presented methods mainly include the introduction of therapeutically effective amounts of the claimed synthetic polypeptide of an interferon type I receptor agonist, a hyperglycosylated version of the peptide, a proteasoresistant variant of the peptide or a hyperglycosylated, proteasoresistant variant of the polypeptide, and an interferon type II receptor agonist. As used herein, the term “effective amounts” of the claimed synthetic type I interferon receptor agonist polypeptide, hyperglycosylated peptide variant, protease resistant peptide variant or hyperglycosylated, protease resistant polypeptide and type II interferon receptor agonist means a dose combination that effectively reduces hepatic fibrosis or reduces liver progression ; and / or which effectively reduces the parameters associated with hepatic fibrosis; and / or which effectively reduce liver cirrhosis disorders.

The invention also allows a method for the treatment of hepatic fibrosis in individuals receiving individual amounts of the claimed synthetic polypeptide of an interferon type I receptor agonist, a hyperglycosylated variant of the peptide, a protease-resistant variant of the peptide or hyperglycosylated, protease-resistant variant of the polypeptide, and an interferon type II receptor agonist, which in combination are effective for prevention or therapy for hepatic fibrosis, for example, increasing the likelihood of survival of patients, decreasing I risk of death, making it easier to "burden of disease" or slowing its progression in patients.

Is treatment with combinations of the claimed synthetic polypeptide of an interferon type I receptor agonist, a hyperglycosylated variant of a peptide, a proteasoresistant variant of a peptide or a hyperglycosylated, proteasoresistant variant of a polypeptide and an interferon type II receptor agonist is effective to reduce hepatic fibrosis by any well-developed methods for the diagnosis of hepatic fibrosis impaired liver function. Whether hepatic fibrosis actually decreases under the influence of treatment is determined by analysis of liver samples obtained by biopsy. Liver biopsy analyzes include the study of two main components: necrotic inflammation, identified by "ranking", by assessing the severity and activity of the disease in the current time period; and lesions of fibrosis and parenchymal or vascular changes, as defined by the "stage", reflecting the progression of the disease over a long period of time. See, for example, Brunt (2000) Hepatol. 31: 241-246; and METAVIR (1994) Hepatology 20: 15-20. An analysis of hepatic biopsy is based on generally accepted assessment criteria. There are various quantitative standardized assessment systems (scoring system or scoring system) that allow quantitative assessment of the degree and severity of fibrosis. These include systems such as METAVIR, Knodell, Scheuer, Ludwig, and Ishak.

The METAVIR scoring system is based on an analysis of various features of a liver biopsy, including fibrosis (portal fibrosis, centrolobular fibrosis and cirrhosis); necrosis (partial and lobular necrosis, acidophilic reaction and ballooning degeneration); inflammation (inflammation of the portal tract, portal lymphoid aggregates and common portal inflammation); changes in the bile ducts; and Knodell index (multiple periportal necrosis, lobular necrosis, portal inflammation, fibrosis, and general disease activity). Determination of each stage by means of the METAVIR system by scoring according to established criteria. Score in points: 0, without fibrosis; score in points: 1, stellate extension of the portal tract, but without the formation of partitions; score in points: 2, expansion of the portal tract with a rare formation of partitions; score in points: 3, numerous partitions, but without cirrhosis; and a score in points: 4, cirrhosis.

The Knodell score system, also called the Hepatitis Activity Index, classifies samples based on scores in four categories of histological features: I, periportal and / or overlapping necrosis; II, intralobular degeneration and focal necrosis; III, portal inflammation; and IV, fibrosis. In the Knodel staging system, scores are calculated as follows: 0, without fibrosis; 1, moderate fibrosis (portal fibrous expansion); 2, medium fibrosis; 3, severe fibrosis (overlapping fibrosis); 4, cirrhosis. Higher score, more serious liver damage. Knodell (1981) Hepatol. 1: 431.

In the Sheur point system, points are defined as follows: 0, no fibrosis; 1, enlarged, fibrotic changes in the portal tracts; 2, periportal, portal-portal septa detected, but intact architecture; 3, fibrosis with architectural distortion, but there is no obvious cirrhosis; 4, suspected or apparent cirrhosis. The system is described by Sheur in J. Hepatol. (199I) 13: 372.

The scoring system of Ishak is described by him in J. Hepatol. (1995) 22: 696-699. Stage 0, [0825] no fibrosis; stage 1, fibrosis spread to separate portal areas, with short septa or without short septa (septa); stage 2, fibrosis spread to most portal areas, with short fibrous septa or without short fibrous septa; stage 3, fibrotic expansion of most portal areas with random portal-portal (PP) overlaps; stage 4, fibrotic expansion of portal areas with explicit portal-portal overlap (RR), as well as portal-central (R-C); stage 5, marked overlap (PP and / or RC) with random nodes (incomplete cirrhosis); stage 6, cirrhosis, probable or accurate. The benefits of anti-fibrotic therapy can be assessed using the Child-Pug-digital system, which includes a multi-component point system based on an assessment of serum bilirubin level, prothrombin time, the presence and risk of ascites, the presence and risk of encephalopathy. Based on the presence and severity of these deviations, the patient can be placed in one of three categories according to the severity of the disease: A, B or C.

In certain embodiments of the invention, therapeutically active combinations of the claimed synthetic type I interferon receptor agonist polypeptide, hyperglycosylated variant peptide, protease-resistant peptide variant or hyperglycosylated, protease-resistant variant polypeptide and type II interferon receptor agonist are used in any dose combination effective to change one units or more in the stage of fibrosis, assessed by the results of biopsies done before and after treatment. In certain embodiments of the invention, therapeutically effective dose combinations reduce liver fibrosis by at least one unit according to the METAVIR, Knodell, Scheuer, Ludwig or Ishak scoring system.

Auxiliary or indirect, to evaluate the effectiveness of treatment with the claimed synthetic polypeptide agonists of the type I interferon receptor, hyperglycosylated variant of the peptide, protease-resistant variant of the peptide or hyperglycosylated, protease-resistant variant of the polypeptide and agonist of the type II interferon receptor, use their ability to induce liver function.

To quantify the degree of liver fibrosis, a morphometric computerized semi-automatic system based on specific staining of collagen and / or serum markers of liver fibrosis is used. These indicators can also be measured as evidence of the effectiveness of the claimed method of treatment.

Secondarily induced liver function includes, but does not limit, serum transaminase levels, prothrombin time, bilirubin, platelet count, portal pressure, albumin level and determination of the number of Child-Pugh points.

In another embodiment of the invention, an effective combination of the claimed synthetic type I interferon receptor agonist polypeptide, hyperglycosylated peptide variant, protease resistant peptide variant or hyperglycosylated, protease resistant polypeptide variant, and type II interferon receptor agonist are used in any dose combination that is effective to increase the hepatic index functions by approximately 10%; approximately 20%; approximately 25%; approximately 30%; approximately 35%; approximately 40%; approximately 45%; approximately 50%; approximately 55%; approximately 60%; approximately 65%; approximately 70%; approximately 75%; or at least about 80%; or more, compared with the liver function index of untreated people or those taking a placebo. A qualified person can easily measure such liver function indices using standard diagnostic methods, many of which are commercially available and used in routine clinical trials.

Serum markers of liver fibrosis can be measured as indicators of the effectiveness of the claimed method of treatment. Serum markers of liver fibrosis include, but are not limited to, hyaluronate, an N-terminal procollagen III peptide, a 7S domain of collagen type IV, a C-terminal procollagen I peptide, and laminin. Additional biochemical markers of liver fibrosis include alpha-2-macroglobulin, haptoglobulin, gammaglobulin, apolipoprotein A and gammaglutamyltransferase.

In another embodiment of the invention, a therapeutically effective combination of the claimed synthetic interferon type I receptor agonist polypeptide, hyperglycosylated variant peptide, protease-resistant peptide variant or hyperglycosylated, protease-resistant polypeptide variant, and type II interferon receptor agonist is used in any dose combination that is effective to reduce serum hepatic fibrosis markers by at least 10%; at least 20%; at least 25%; at least 30%; at least 35%; at least 40%; at least 45%; at least 50%; at least 55%; at least 60%; at least 65%; at least 70%; at least 75%; or at least 80%; or more, compared with the level of hepatic markers of untreated people, or who took a placebo. A qualified person can easily identify these markers of liver fibrosis using standard diagnostic methods, many of which are commercially available and used in routine clinical trials.

Methods for measuring serum markers include immunological methods, for example, an immunosorbent binding assay test, for example, an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay and the like, using antibodies specific for the serum marker to be studied.

To measure the effectiveness of treatment with a synthetic polypeptide of an interferon type I receptor agonist, a hyperglycosylated variant of the peptide, a protease-resistant variant of the peptide or a hyperglycosylated, protease-resistant variant of the polypeptide, and an interferon type II receptor agonist, reserve quantitative tests of the liver functional state are used. These include: Indocyanine Green Clearance (ICG), Galactose Elimination Capacity (GEC), Exhaled Aminopurine Test (ABT), Antipyrine Clearance, Monoethyl Glycine Clearance (MEG-X), and Caffeine Clearance.

As used herein, a “complication associated with cirrhosis of the liver” refers to a disorder that results from decompensation of liver disease, i.e. or occurs in consequence and as a result of developed liver fibrosis, and includes, but is not limited to, the detection of ascites; varicose bleeding; portal hypertension; jaundice; progressive liver failure; encephalopathy; hepatocellular carcinoma; liver failure requiring liver transplantation; and liver related mortality.

In another embodiment of the invention, the therapeutically effective combination of the claimed synthetic interferon type I receptor agonist polypeptide, hyperglycosylated variant peptide, protease-resistant peptide variant or hyperglycosylated, protease-resistant polypeptide variant, and type II interferon receptor agonist can be at any dose of components that is effective in reducing the complications (for example, those that develop due to individual tolerance) associated with cirrhosis m liver, at least about 10%; at least about 20%; at least about 30%; at least about 35%; at least about 40%; at least about 45%; at least about 50%; at least about 55%; at least about 60%; at least about 65%; at least about 70%; at least about 75%, or at least about 80%, or more, compared to untreated patients or placebo patients.

Whether combination therapy with the claimed synthetic type I interferon receptor agonist polypeptide, a hyperglycosylated peptide variant, a proteazoresistant peptide variant or a hyperglycosylated, proteasoresistant polypeptide variant and a type II interferon receptor agonist can be easily determined to reduce disorders associated with cirrhosis of the liver prior art.

[00715] A decrease in liver fibrosis is manifested by an improvement in its function. Therefore, the invention provides methods for improving liver function, typically comprising administering a therapeutically effective dose combination of the claimed synthetic type I interferon receptor agonist polypeptide, hyperglycosylated variant peptide, protease resistant variant peptide or hyperglycosylated, protease resistant polypeptide variant, and type II interferon receptor agonist. Liver functions include, but are not limited to, the following: 1) synthesis of proteins, such as serum proteins (eg, albumin; blood coagulation factors; alkaline phosphatase; aminotransferases, for example, alanine transaminase; aspartate trans-transaminase; 5'-nucleosidase, [gamma ] -glutaminyl transpeptidase and the like); 2) synthesis of bilirubin; 3) cholesterol synthesis; 4) and the synthesis of bile acids. The metabolic function of the liver includes, but is not limited to: carbon hydrate metabolism, amino acid and ammonium metabolism, hormone metabolism and lipid metabolism; detoxification of exogenous drugs; maintaining hemodynamic function, including internal and portal hemodynamics; and the like.

Whether liver function is really improving is easy to ascertain from the prior art using known liver function tests. Therefore, the synthesis of liver function markers, such as albumin, alkaline phosphatase, alanine transaminase, aspartate transaminase, bilirubin and the like, can be determined by measuring the level of these serum markers using standard immunological and enzymatic methods. Internal circulation and portal hemodynamics can be measured by determining pressure and / or resistance in the portal vein using standard methods. Metabolic functions can be measured by measuring serum ammonia levels.

Whether whey proteins are actually synthesized in the liver in the normal range can be determined by measuring the levels of such proteins in serum using standard immunological and enzymatic methods. Normal ranges for such whey proteins are known in the art. Partially they are given below. The normal range for alanine transaminase ranges from about 7 to about 56 units per liter of serum. The normal range for aspartate transaminase ranges from about 5 to about 40 units per liter of serum. Bilirubin is measured in a standard manner. Normally, bilirubin levels are usually less than about 1.2 mg / dL. The level of serum bilirubin is determined in a standard way. Normally, it is in the range of from about 35 to about 55 g / L. The duration of prothrombin time is determined in a standard way. Normally, the duration of the prothrombin time is less than or more than the control within 4 seconds.

In another embodiment of the invention, [0841] a therapeutically effective combination of a synthetic type I interferon receptor agonist polypeptide, a hyperglycosylated variant peptide, a protease resistant peptide variant or a hyperglycosylated, protease resistant polypeptide variant, and a type II interferon receptor agonist are combined in a dose that is effective to improve liver function by at least about 10%; at least about 20%; at least about 30%; at least about 40%; at least about 50%; at least about 60%; at least about 70%; at least about 80%; or more. For example, a therapeutically effective combination of the claimed synthetic polypeptide of an interferon type I receptor agonist, a hyperglycosylated variant of a peptide, a protease-resistant variant of a peptide or a hyperglycosylated, protease-resistant variant of a polypeptide, and an interferon type II receptor agonist, includes any dose combination that is effective to reduce elevated serum function markers liver, at least about 10%; at least about 20%; at least about 30%; at least about 40%; at least about 50%; at least about 60%; at least about 70%; at least about 80%; or more; or a decrease in serum markers to normal intervals. A therapeutically effective combination of the claimed synthetic polypeptide of an interferon type I receptor agonist, a hyperglycosylated variant of a peptide, a protease-resistant variant of a peptide or a hyperglycosylated, protease-resistant variant of a polypeptide and an interferon type II receptor agonist also includes any dose combination that increases the level of liver function markers lower than normal, at least about 10%; at least about 20%; at least about 30%; at least about 40%; at least about 50%; at least about 60%; at least about 70%; at least about 80%; or more; or increases the level of markers of liver function to normal intervals.

Renal fibrosis

The presented invention allows the implementation of methods for the treatment of renal fibrosis. The methods mainly consist in administering to a patient with renal fibrosis effective amounts of the claimed synthetic polypeptide of an interferon type I receptor agonist, a hyperglycosylated version of the peptide, a protease resistant variant of the peptide or a hyperglycosylated, protease resistant variant of the polypeptide and type II interferon receptor agonist. As used herein, the term “effective amounts” of the claimed synthetic type I interferon receptor agonist polypeptide, hyperglycosylated variant peptide, protease resistant variant peptide or hyperglycosylated, protease resistant variant polypeptide and type II interferon receptor agonist means any dose combination that effectively reduces renal fibrosis; and / or which is effective in reducing the likelihood that the patient will develop renal fibrosis; and / or, in other words, effective in reducing the parameters associated with renal fibrosis; and / or effective in reducing the complications associated with kidney fibrosis.

In one embodiment of the invention, effective amounts of the claimed synthetic type I interferon receptor agonist polypeptide, hyperglycosylated peptide variant, protease resistant peptide variant or hyperglycosylated, protease resistant polypeptide and type II interferon receptor agonist are used in any dose combination that is sufficient to reduce renal fibrosis, or reduces the rate of progression of renal fibrosis by at least about 10%; at least about 15%; at least about 20%; at least about 25%; at least about 30%; at least about 35%; at least about 40%; at least about 45%; at least about 50%; in comparison with the degree of renal fibrosis in individuals before treatment, or in comparison with the rate of progression of renal fibrosis, which may occur in patients without treatment.

To what extent there is a decrease in kidney fibrosis under the influence of treatment is determined using known methods. For example, by performing a histochemical analysis of a kidney biopsy sample to determine ECM deposition and / or fibrosis. Other methods are known in the art. See, for example, Masseroli et al. (1998) Lab. Invest. 78: 511-522; U.S. Patent No. 6,214,542.

In certain embodiments, effective amounts of the claimed synthetic type I interferon receptor agonist polypeptide, hyperglycosylated peptide variant, protease resistant peptide variant or hyperglycosylated, protease resistant polypeptide and type II interferon receptor agonist are used in any combination of doses that are effective for enhancing renal function, at least at least about 10%; at least about 15%; at least about 20%; at least about 25%; at least about 30%; at least about 35%; at least about 40%; at least about 45%; at least about 50%; in comparison with the baseline level of kidney function in an individual before treatment.

In certain embodiments, effective amounts of the claimed synthetic type I interferon receptor agonist polypeptide, hyperglycosylated peptide variant, protease resistant peptide variant or hyperglycosylated, protease resistant polypeptide and type II interferon receptor agonist are used in any dose combination that effectively slows down the decrease in renal function, at least approximately 10%; at least about 15%; at least about 20%; at least about 25%; at least about 30%; at least about 35%; at least about 40%; at least about 45%; at least about 50%; compared with the deterioration of kidney function, which will occur if untreated.

Renal function can be measured using known tests, including but not limited to plasma creatinine levels (when normal levels are in the range of about 0.6 to about 1.2 mg / dL); creatinine clearance (when the normal creatinine clearance is in the range of from about 97 to about 137 mL / minute in men and from about 88 to 128 mL / minute in women); glomerular filtration rate (either calculated or determined by inulin clearance or other methods), blood urea nitrogen (normal range is from 7 to about 20 mg / dL); and protein content in urine.

Additional antifibrotic agents

Any of the above combinations of therapy can be adapted to treat fibrotic disorder, including co-administration of one or more additional antifibrotic agents. Accordingly, the present invention allows a method of treating fibrotic disorder, mainly including the administration of the claimed synthetic polypeptide, an interferon type 1 receptor agonist, a hyperglycosylated version of the peptide, a protease resistant variant of the peptide or hyperglycosylated, protease resistant variant of the polypeptide, and an interferon type II receptor agonist, in combination therapy, which at least one additional antifibrotic agent. Suitable antifibrotic adjuvants include, but are not limited to, SAPK inhibitors (eg, pirfenidone or its analogs), TNF antagonists, TOR [beta] antagonists, endothelin receptor antagonists, and the like.

Non-limiting examples, any of the above methods of treatment, especially therapy using a combined dosage of the claimed synthetic type 1 interferon receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant or a hyperglycosylated, protease resistant polypeptide variant and an effective interferon type II receptor agonist, disorders in patients can be modified for the desired continuation treatment duration by including, together with them, amounts of an SAPK inhibitor (for example, pirfenidone or its analogue) used to treat patients, effective to increase the antifibrotic effect of combination therapy with a synthetic type 1 interferon receptor polypeptide agonist and type II interferon receptor agonist.

[00727] As non-limiting examples, any of the above methods of treatment, especially therapy using a combined dosage of the claimed synthetic type I interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant agonist and interferon receptor type II agonist for the treatment of fibrotic disorders in a patient, can be modified for the desired continuation treatment duration by including as an additional therapeutic agent a TNF antagonist (e.g., etanercept, influximab, or adalimumab), effective to increase the antifibrotic effect of combination therapy with a synthetic type 1 interferon receptor polypeptide agonist and type II interferon receptor agonist.

[00728] As non-limiting examples, any of the above methods of treatment, especially therapy using a combined dosage of the claimed synthetic type I interferon receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant and an II type receptor agonist, for the treatment of fibrotic disorders in a patient, can be modified to continue The treatment should include the TOR [beta] antagonist (for example, GLEEVEC) as an additional therapeutic agent in amounts effective to increase the antifibrotic effect of combination therapy with a synthetic type 1 interferon receptor polypeptide agonist and type II interferon receptor agonist.

[00729] As non-limiting examples, any of the above methods of treatment, especially therapy using a combined dosage of the claimed synthetic type I interferon receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant or a hyperglycosylated, protease resistant polypeptide variant agonist, and an interferon receptor type II agonist for the treatment of fibrotic disorders in a patient, can be modified to continue The treatment should include amounts of an endothelin receptor antagonist (e.g., TRACLEER), which are effective for enhancing the antifibrotic effect of combination therapy with a synthetic type 1 interferon receptor polypeptide agonist and type II interferon receptor agonist.

[00730] As non-limiting examples, any of the above methods of treatment, especially therapy using a combined dosage of the claimed synthetic type I interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant agonist and interferon receptor type II agonist for the treatment of fibrotic disorders in a patient, can be modified to continue the treatment by including as an additional therapeutic agent in patients through a combined dose of an SAPK inhibitor (e.g., pirfenidone or its analogue) and a TNF antagonist (e.g., etanercept, influximab or adalimumab), effective to enhance the antifibrous effect of combination therapy with a synthetic interferon receptor polypeptide agonist Type 1 and type II interferon receptor agonist.

[00731] As non-limiting examples, any of the above methods of treatment, especially therapy using a combined dosage of the claimed synthetic type I interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant, or hyperglycosylated, protease resistant polypeptide variant agonist and interferon receptor type II agonist for the treatment of fibrotic disorders in a patient, may be modified to include as an additional therapeutic agent in patients with a combined dose of an SAPK inhibitor (for example, pirfenidone or its analogue) and a TOP-beta beta antagonist (for example, GLEEVEC), effective to enhance the antifibrous effect of combination therapy with a synthetic type 1 interferon receptor polypeptide agonist and interferon II receptor agonist type, for the desired duration of treatment.

[00732] As non-limiting examples, any of the above methods of treatment, especially therapy using a combined dosage of the claimed synthetic type I interferon receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant or a hyperglycosylated, protease resistant polypeptide variant and an interferon receptor type II agonist for the treatment of fibrotic disorders in a patient, can be modified to continue The treatment should include the inclusion of a combined dose of an SAPK inhibitor (e.g., pirfenidone or its analogue) and an endothelin receptor antagonist (e.g. TRACLEER) as an additional therapeutic agent, which are effective for enhancing the antifibrous effect of combination therapy with a synthetic type 1 interferon receptor polypeptide agonist and receptor agonist interferon type II,

[00733] As non-limiting examples, any of the above methods of treatment, especially therapy using a combined dosage of the claimed synthetic type I interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant agonist and interferon receptor type II agonist for the treatment of fibrotic disorders in a patient, can be modified to continue The treatment should include the inclusion of a combined dose of a TNF antagonist (for example, etanercept, influximab or adalimumab) and TOR [beta] antagonist (for example, GLEEVEC) as an additional therapeutic agent, which are effective for enhancing the antifibrous effect of combination therapy with a synthetic interferon receptor polypeptide agonist 1 type and type II interferon receptor agonist.

[00734] As non-limiting examples, any of the above methods of treatment, especially therapy using a combined dosage of the claimed synthetic type I interferon receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant or a hyperglycosylated, protease resistant polypeptide variant and an interferon receptor type II agonist for the treatment of fibrotic disorders in a patient, can be modified to continue The treatment should include the inclusion of a combined dose of a TNF antagonist (e.g., etanercept, influximab or adalimumab) and an endothelin receptor antagonist (e.g. TRACLEER) as an additional therapeutic agent, which are effective in enhancing the antifibrous effect of combination therapy with a synthetic type I agonist interferon receptor polypeptide agonist type I agonist type II interferon receptor.

[00735] As non-limiting examples, any of the above methods of treatment, especially therapy using a combined dosage of the claimed synthetic type I interferon receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant or a hyperglycosylated, protease resistant polypeptide variant and an interferon receptor type II agonist for the treatment of fibrotic disorders in a patient, can be modified to continue The treatment should include the inclusion of a combined dose of a TOP-beta [beta] antagonist (eg, GLEEVEC) and an endothelin receptor antagonist (eg, TRACLEER) as an additional therapeutic agent, effective to enhance the antifibrotic effect of combination therapy with a synthetic type 1 interferon receptor polypeptide agonist and receptor agonist interferon type II ,.

[00736] As non-limiting examples, any of the above methods of treatment, especially therapy using a combined dosage of the claimed synthetic type I interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant and interferon receptor type II agonist for the treatment of fibrotic disorders in a patient, can be modified to continue The treatment should include the inclusion of a combined dose of an SAPK inhibitor (for example, pirfenidone or its analogue), TNF antagonist (for example, etanercept, influximab or adalimumab) and TOR- [beta] antagonist (for example GLEEVEC) as an additional therapeutic agent in patients to enhance the antifibrotic effect of combination therapy with a synthetic type 1 interferon receptor polypeptide agonist and a type II interferon receptor agonist.

[00737] As non-limiting examples, any of the above methods of treatment, especially therapy using a combined dosage of the claimed synthetic type I interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant agonist and interferon receptor type II agonist for the treatment of fibrotic disorders in a patient, can be modified to continue The treatment should include the inclusion of a combined dose of an SAPK inhibitor (e.g., pirfenidone or its analogue), a TNF antagonist (e.g., etanercept, influximab, or adalimumab) and an endothelin receptor antagonist (e.g. TRACLEER), which are effective in enhancing antifibrous effect in combination therapy with a synthetic polypeptide agonist of an interferon type 1 receptor and an agonist of an interferon type II receptor.

[00738] As non-limiting examples, any of the above methods of treatment, especially therapy using a combined dosage of the claimed synthetic type I interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant agonist, and interferon receptor type II agonist for the treatment of fibrotic disorders in a patient, can be modified to continue The treatment should include the inclusion of a combined dose of a TNF antagonist (for example, etanercept, influximab or adalimumab), TOR [beta] antagonist (for example, GLEEVEC) and an endothelin receptor antagonist (for example, TRACLEER), which are effective in enhancing antifibrous the effect of combination therapy with a synthetic type 1 interferon receptor polypeptide agonist and a type II interferon receptor agonist.

[00739] As non-limiting examples, any of the above methods of treatment, especially therapy using a combined dosage of the claimed synthetic type I interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant agonist and interferon receptor type II agonist for the treatment of fibrotic disorders in a patient, can be modified to continue The treatment should include the inclusion of a combined dose of an SAPK inhibitor (for example, pirfenidone or its analogue), a TOP-beta antagonist (for example GLEEVEC) and an endothelin receptor antagonist (for example TRACLEER) as an additional therapeutic agent, which are effective for enhancing the antifibrotic effect combination therapy with a synthetic type 1 interferon receptor polypeptide agonist and type II interferon receptor agonist, for the desired duration of treatment.

[00740] As non-limiting examples, any of the above methods of treatment, especially therapy using a combined dosage of the claimed synthetic type I interferon receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant or a hyperglycosylated, protease resistant polypeptide variant and an interferon receptor type II agonist for the treatment of fibrotic disorders in a patient, can be modified to continue The treatment should include the inclusion of a combined dose of an SAPK inhibitor (for example, pirfenidone or its analogue), TNF antagonist (for example, etanercept, influximab or adalimumab), TOR- [beta] antagonist (for example, GLE-EVEC) as an additional therapeutic agent in patients and an endothelin receptor antagonist (e.g., TRACLEER), effective to enhance the antifibrotic effect of combination therapy with a synthetic type 1 interferon receptor polypeptide agonist and a type II interferon receptor agonist.

[00741] As non-limiting examples, any of the above methods of treatment, especially therapy using a combined dosage of the claimed synthetic type I interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant and interferon receptor type II agonist for the treatment of fibrotic disorders in a patient, with or without co-administration of one or b more additional antifibrotic (antifibrotic) agent (s) may be modified in the future for the desired duration of treatment by the joint administration of amounts of N-acetylcysteine (NAC) effective to increase the antifibrotic effect of combination therapy.

CANCER

[00742] The present invention provides a method of treating proliferative disorders (eg, cancer), the method typically comprising administering to an individual effective amounts of a synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant

[00743] The methods effectively reduce tumor growth rate by approximately at least 5%; approximately at least 10%; approximately at least 20%; approximately at least 25%; approximately at least 50%; at least 75%; at least 85%; or approximately at least 90%; up to complete inhibition of its growth in comparison with a suitable control.

Thus, in this embodiment of the invention, an “effective amount” of a synthetic type 1 interferon receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant, or a hyperglycosylated, protease-resistant polypeptide variant are that amount sufficient to reduce the tumor size growth by approximately at least at least , by 5%; approximately at least 10%; approximately at least 20%; approximately at least 25%; approximately at least 50%; at least 75%; at least 85%; or approximately at least 90%; up to complete inhibition of its growth in comparison with a suitable control. In experimental animal systems, genetically identical animals not treated with a synthetic type 1 interferon receptor polypeptide agonist may be a suitable control. In non-experimental systems, a suitable control may be a tumor that developed prior to treatment with a synthetic type 1 interferon receptor polypeptide agonist. Another suitable control may be a placebo control.

[00744] Whether tumor growth is actually inhibited can be determined using known methods, including but not limited to evaluating proliferation, as described in the example; ³ H-thymidine uptake test and the like.

[00745] The methods can be used to treat various cancers, including carcinomas, sarcomas, leukemias and lymphomas.

[00746] Carcinomas that can be treated using the claimed method include, but are not limited to, esophagial carcinoma, hepatocellular carcinoma, basal cell carcinoma (in the form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including intermediate cell carcinoma (malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma lung cancer, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat carcinoma, sebaceous carcinoma, papillary carcinoma, papillary carcinoma, cystic adenocarcinoma, cystoadenocarcinoma, ducts in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilma tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, oste fire carcinoma, epithelial carcinoma, nasopharyngeal carcinoma and similar diseases.

[00747] Sarcomas that can be treated using the claimed method include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, sanguinomelioma, synangelomeosomyeloma, sanguinomesoma, , leiomyosarcoma, rhabdomyosarcoma and other soft tissue sarcomas.

[00748] Other solid tumors that can be treated using the claimed method include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngoma, ependioma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retin.

[00749] Leukemias that can be treated using the claimed method include, but are not limited to, the following: a) chronic myeloproliferative syndromes (neoplastic disorders of multipotential hematopoietic stem cells); b) acute myelogenous leukemias (neoplastic transformation of multipotential hematopoietic stem cells or hematopoietic cells with limited potential; c) chronic lymphatic leukemias (CLL; clonal proliferation of immunologically immature and functionally incompetent small lymphocytes), including B-cell polymorphism CLL, T-cell leukemia CLL, and hairy cell leukemia; and d) acute lymphoblastic leukemias (characterized by the accumulation of lymphoblasts). Lymphomas that can be treated using the claimed method include, but are not limited to, B-cell lymphomas (eg, Burkett's lymphoma); Hodgkins lymphoma; and the like.

Combination therapy

[00750] In certain applications, the present invention allows for combination therapy of cancer. Accordingly, the present invention provides for the implementation of a method for treating cancer, mainly comprising administering the claimed synthetic type 1 interferon receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant in combination therapy with at least adjuvant therapeutic agents.

[00751] In other embodiments, the present invention provides methods for treating cancer, which include administering synergistic combinations of the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant, or hyperglycosylated, protease resistant polypeptide variant and adjuvant therapeutic agent. As used here, a “synergistic combination” of the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease-resistant polypeptide variant or hyperglycosylated, protease-resistant polypeptide variant and adjuvant therapeutic agent is a combination of doses that are more effective in therapeutic than therapeutic step-by-step improvements in treatment, the result of which can be predicted whether forward of only simple combinations of (i) therapeutic or prophylactic benefits of the claimed synthetic polypeptide agonist receptor type 1 interferon hyperglycosylated polypeptide variant, the polypeptide variant or proteazorezistentnogo hyperglycosylated, proteazorezistentnogo polypeptide variant when they are administered in a dosage adopted for monotherapy; and (ii) the therapeutic and prophylactic benefits of the adjuvant therapeutic agent when it is administered at a dose taken for monotherapy.

[00752] In certain applications, the claimed type 1 interferon receptor synthetic polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant are used as adjunctive therapy for standard cancer therapy. Standard cancer treatments include surgical treatment (e.g., surgical removal of cancerous tissue), radiation therapy, bone marrow transplantation, chemotherapy treatment, treatment with biological response modifiers, and the use of individual combinations of the above methods.

[00753] Radiation therapy includes, but is not limited to, x-rays or gamma radiation, which are generated either by special devices or through the implantation of small radioactive sources.

[00754] Chemotherapeutic agents are non-peptide (ie, non-protein) compounds that reduce the proliferation of cancer cells, cytotoxic and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolics, antitumor antibiotics, plant (periwinkle) alkaloids, and steroid hormones.

[00755] Cell proliferation reducing agents are known and widely used in the art. Such agents include alkylating agents such as nitrogen mustards, nitrosoureas, ethyleneimine derivatives, alkyl sulfonates and triazenes, including (but not limited to) mechlorethamine, cyclophosphamide (Cytoxan (TM)), melphan (L-sarcolysin), carmustine (BCU), BCM (CCNU), semistin (methyl-CCNU), streptozocin, chlorozotocin, uraciliprite, chloromethine and phosphamide, chlorambicyl, pipobromane, triethylene melamine, triethylene thiophosphoromine, bisulfan, dicarbazine and temozolomide.

[00756] Antimetabolic agents include folic acid analogs, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors, including but not limited to cytarabine (CYTO-SAR-LJ), cytosine arabinose, fluorouracil (5-FU), phloxuridine (Fud) (Fud) -thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazofolate (PDDF, CB3717), 5,8-dideazotetraxydrofolate acid (DDATHF) leucovorin, fludarobin phosphate, pentostatin and gemcitabine.

[00757] Suitable natural products and their derivatives (eg, vinca alkaloids, antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxin) include (but are not limited to) Ara-C, paclitaxel (Taxol (R)), docetaxel (Taxotere (R)) , deoxycoformicin, mitomycin-C, L-asparginase, azathioprine; brequinar; alkaloids, for example, vincristine, vinblastine, vinorelbine, vindesine and the like; podophyllotoxins, e.g. etoposide, teniposide, and the like; antibiotics, for example, anthracycline, dianorbicin hydrochloride (dianomycin, rubidomycin, cerubidin), idarubicin, doxorubicin, epirubicin and morvoline derivatives, etc .; phenoxysone biscyclopeptides, for example, diastomycin; basic glycopeptides, for example, bleomycin; antraquin glycosides, for example plicamycin (mitramycin); anthracendiones, for example, mitoxantrone; azirinopyrroloindoledione, for example mitomycin; macrocyclic immunosuppressants, for example, cyclosporin, FK-506 (tacrolimus, prograf), rapamycin, etc. etc.

[00758] Other antiproliferative cytoxic agents include Navelben, CPT-11, Anastrozole, Letrazole, Capecitabine, Reloxafin, CIC-Lophosphamide, Ifosamide and Drolocafine.

[00759] Microtubule acting agents are also suitable for use and include, but are not limited to, allocolhamicin (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (eg, NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol (R)), derivatives of Taxol (R), docetaxel (Taxotere (R)), thiocolchicine (NSC 361-792), trityl cysterine, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones, including (but not limited to) eoptilon A, eoptilon B, discordemolide; estramustine, nocodazole and similar drugs.

[00760] Hormonal modulators and steroids (including synthetic analogs) that are suitable for use include, but are not limited to, hadronocorticosteroids, for example, prednisone, dexamethasone, and the like; estrogens and progestins, for example, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, chlomiphene, tamoxifen; etc.; and adrenocortical suppressants, for example, aminoglutemide; 17 [alpha] ethynyl estradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromestanolona propionate, testolactone, methylprednisolone, triamcinolone, hlorotrianizin, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil), Torimefen (Fareston), and Zoladex (R). Estrogens stimulate proliferation and differentiation, therefore, compounds that bind estrogen receptors can be used to block their activity. Corticosteroids can inhibit T cell proliferation.

[00761] Other chemotherapeutic agents include metal complexes, for example, cisplatin (cis-DDP), carboplatin, and the like; urea, for example, hydroxyurea; and hydrazines, for example, N-methylhydrazine; epidophylotoxin; polymerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur, etc. Other antiproliferative agents of interest include immunosuppressants, for example, mycophenolic acid, thalidomide, deoxyspergualin, azasporin, leflunomide, misoribine, azaspiran (SKF 105685); Iressa (R) (ZD 1839, 4- (3-chloro-4-fluorophenyl) -7-methoxy-6- (3- (4-orpholinyl) propoxy) quinazoline); etc.

[00762] "Taxanes" include paclitaxel, as well as any active derivative of taxanes or a prodrug. Paclitaxel (which is contemplated herein includes analogues, formulations and derivatives, for example, docetaxel, TAXOL (TM), TAXOTERE (TM) (a derivative of doxetaxel), 10-desacetyl analogues of paclitaxel and 3'N-desbenzoyl-3'Nt-bitoxycarboxylic paclitaxel analogs) can be easily prepared using techniques known in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; US Pat. Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or obtained from various commercial sources, including, for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; or T-1912 from Taxus yannanensis).

[00763] Paclitaxel corresponds not only to the general chemically suitable form of paclitaxel, but to analogs and derivatives (for example, Tachotere (TM) docetaxel, and paclitaxel conjugates (for example, paclitaxel-PEG, paclitaxeladextran, or paclitaxelxylose).

[00764] Various known derivatives, including hydrophilic and hydrophobic derivatives, are also included within the term "taxane". Derivatives of taxane include, but are not limited to, the galactose and mannose derivatives described in the appendix to international patent No. WO 99/18113; piperazine and other derivatives described in WO 99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, and U.S. Patent No. 5,869,680; 6-thio derivatives described in W098 / 28288; sulfenamide derivatives described in U.S. Patent No. 5,821,263; and taxol derivatives described in U.S. Patent No.5,415,869. In the future, paclitaxel prodrugs will be included, including but not limited to those described in WO 98/58927; WO 98/13059; and U.S. Patent No. 5,824,701.

[00765] Biological response modifiers are included suitable for use in conjunction with the methods of the invention, but they do not limit them with respect to (1) tyrosine kinase activity inhibitors (RTK); (2) serine / threonine kinase activity inhibitors; (3) tumor-associated antigenic antagonists, such as antibodies specifically binding to a tumor antigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6) 1PM- [alpha]; (7) IFN- [gamma] (8) colony stimulating factors; and (9) angiogenesis inhibitors.

[00766] In one aspect, the invention contemplates a combination of the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant, or hyperglycosylated, protease resistant polypeptide variant, as an adjuvant in any therapy in which an emergency patient is treated for at least one additional antineoplastic drug, where the adjuvant is and tyrosine kinase inhibitor. In particular applications, the tyrosine kinase inhibitor is tyrosine kinase receptor (RTK) inhibitors such as type I tyrosine kinase receptor inhibitors (e.g., epidermal growth factor receptor inhibitors), type II tyrosine kinase receptor inhibitors (e.g., insulin receptor type II tyrosine kinase inhibitors) (e.g., platelet growth factor receptor inhibitors) and type IV tyrosine kinase receptor inhibitors (e.g., fibroblast growth receptor). In other embodiments of the invention, tyrosine kinase inhibitors are inhibitors not related to tyrosine kinase receptors, such as inhibitors of DHS kinases or j anus kinases.

[00767] In another aspect, the invention contemplates a combination of the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant, or hyperglycosylated, protease resistant polypeptide variant, as adjunctive to any therapy in which an extreme cancer patient is treated with at least one additional antineoplastic agent, where, as an additional drug, changing receptor tyrosine kinase inhibitor involved in the signal (signal), the path (path) growth factor. In certain applications, the inhibitor is Genistein. In other applications, the inhibitor is an EGFR tyrosine kinase-specific antagonist such as IRESSA (TM) gefitinib (ZD1 8398; Novartis), TARCEVA (TM) erolotinib (OSI-774; Roche; Genentech; OSI Pharmaceuticals), or tyrphostin AG1 478 4- (3-chloroanilino) -6,7-demi-toxicoquinazoline In the remaining other applications, the inhibitor is any indolinone antagonist of FIk-1 / KDR (VEGF-R2) tyrosine kinase activity described in US Pat. No. 2002/0183364 A1, such as the indolinone antagonists of Flk-1 / KDR (VEGF-R2) tyrosine kinase activity disclosed in table 1 on pages 4-5 of this document. of the invention, the inhibitor may be any of the substituted 3 - [(4,5,6,7-tetrahydro-1H-indole-2-y1) methylene] -1,3-dihydroin-dol-2-one Flk-1 / KDR antagonists (VEGF-R2), FGF-R1, or PDGF-R tyrosine kinase activity disclosed in Sun, L., et al., J. Med. Chem .. 43 (14: 2655-2663 (2000). In further embodiments, the inhibitor can be any substituted is any substituted 3 - [(3- or 4-carboxyethyl-pyrrol-2-y1) methylidenyl] indoline-2-one antagonist FIt-I (VEGF-R1), Flk-1 / KDR (VEGF- R2), FGF-R1 or PDGF-R tyrosine kinase activity disclosed by Sun, L., et al., J. Med. Chem., 42 (25): 5120-5130 (1999).

[00768] In another aspect, the invention contemplates a combination of the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease-resistant polypeptide variant, or hyperglycosylated, protease-resistant polypeptide variant, as adjunctive to any therapy in which a patient with a cancer patient receives treatment with at least one additional antineoplastic agent, where, as an additional drug, called an inhibitor of non-receptor tyrosine kinase involved in the signal (s) pathway (s) of growth factor. In certain embodiments, the inhibitor is a JAK2 antagonist of tyrosine kinase activity, such as tyrphostin AG490 (2-cyano-3- (3,4-dehydroxyphenyl) -N- (benzyl) -2-propenamide). In other embodiments, the inhibitor is a bcr-ab1 antagonist of tyrosine kinase activity, such as GLEEVEC (TM) imatinib mesylate (STI-571; Novartis).

[00769] In another aspect, the invention contemplates a combination of the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant, or hyperglycosylated, protease resistant polypeptide variant, as adjunctive to any therapy in which an extreme cancer patient is treated with at least one additional antineoplastic agent, where, as an additional drug, changing an inhibitor of one or more of the kinases involved in cell cycle regulation. In certain embodiments, the inhibitor is a CDK2 activation antagonist, such as tryptostin AG490 (2-cyano-3- (3,4-dihydroxyphenyl) -N- (benzyl) -2-propenamide). In other embodiments, the inhibitor is a CDK1 / cyclin B activity antagonist, such as alsterpaillon. In the remaining other embodiments, the inhibitor is a CDK2 antagonist of kinase activity, such as indirubin-3'-monoxime. In further embodiments, the inhibitor is an ATP pool antagonist such as lometrexol (described in U.S. No. 2002/0156023 A1).

[00770] In another aspect, the invention contemplates a combination of the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant, or hyperglycosylated, protease resistant polypeptide variant, as adjunctive to any therapy in which an extreme cancer patient is treated with at least one additional antineoplastic agent, where, as an additional drug, changing antagonist tumor-associated antigen, such as an antibody antagonist. In certain embodiments, including the treatment of HEK2-expressing tumors, the tumor associated antigenic antagonist is an anti-HEK2 monoclonal antibody such as HERCEPTIN ( ^TM ) trastuzumab. In certain embodiments, including the treatment of CD20-expressing tumors, such as B-cell lymphomas, a tumor antagonist of an associated antigen, such as an anti-CD20 monoclonal antibody, such as RITUXAN ( ^TM ) rituximab, is used.

[00771] In another aspect, the invention contemplates a combination of the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant, or hyperglycosylated, protease resistant polypeptide variant, as adjunctive to any therapy in which an extreme cancer patient is treated with at least one additional antineoplastic agent, where, as an additional drug, Olza tumor growth factor antagonist. In certain embodiments, the tumor growth factor antagonist is an epidermal growth factor EGF antagonist, such as an anti-EGF monoclonal antibody. In certain embodiments, the tumor growth factor antagonist is an epidermal growth factor receptor antagonist erbB1 (EGFR), such as an anti-EGFR monoclonal body, an EGFR activation or signal transduction inhibitor, for example, ERBITUX ( ^TM ) cetuximab.

[00772] In another aspect, the invention contemplates a combination of the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant, or hyperglycosylated, protease resistant polypeptide variant, as adjunctive to any therapy in which an extreme cancer patient is treated with at least one additional antineoplastic agent, where, as an additional drug, changing agonist Apo-2 ligand. In certain embodiments, an Apo-2 ligand agonist is any Apo-2 ligand peptides described in WO 97/25428.

[00773] In another aspect, the invention contemplates a combination of the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant, or hyperglycosylated, protease resistant polypeptide variant, as adjunctive to any therapy in which an extreme cancer patient is treated with at least one additional antineoplastic agent, where, as an additional drug, changing the anti-angiogenic agent. In certain embodiments, the anti-angiogenic agent is an antagonist of vascular endothelial cell growth factor (VEGF), such as an anti-VEGF monoclonal antibody, for example, AVASTIN ( ^TM ) bevacizumab (Genentech). In other embodiments, the anti-angiogenic agent is a VEGF-R1 antagonist, such as an anti-VEGF-R1 monoclonal antibody. In other embodiments, the anti-angiogenic agent is a VEGF-R2 antagonist, such as an anti-VEGF-R2 monoclonal antibody. In other embodiments, the anti-angiogenic agent is an antagonist of basic fibroblast growth factor (bFGF), such as an anti-bFGF monoclonal antibody. In other embodiments, the anti-angiogenic agent is a bFGF receptor antagonist, such as a monoclonal antibody anti-bFGF receptor. In other embodiments, the anti-angiogenic agent is a TOR- [beta] antagonist, such as an anti-TOR- [beta] monoclonal antibody. In other embodiments, the anti-angiogenic agent is an antagonist of the TOR [beta] receptor, such as an anti-TOR [beta] receptor monoclonal antibody. In other embodiments, the anti-angiogenic agent is a retinoic acid receptor (RXR) ligand, such as any RXR ligand described in US Pat. 2001/0036955 A1 or in any of the following: US Pat. Nos. 5,824,685; 5,780,676; 5,399,586; 5,466,861; 4,810,804; 5,770,378; 5,770,383; or 5,770,382. In the remaining other embodiments, the anti-angiogenic agent is the gamma ligand of the peroxomo proliferating activating receptor (PPAR), such as any PPAR gamma ligand described in the description of the patent US No. 2001/0036955 A1.

[00774] As non-limiting examples, any of the above methods of treatment, especially therapy with effective amounts of the claimed synthetic polypeptide agonist of the type 1 interferon receptor, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant, for treating cancer in patients, can be for the desired duration of treatment, including co-treatment of the patient IFN-γ [gamma], taken in quantities effective in increasing the anti-cancer effect of therapy with a synthetic polypeptide agonist of type I interferon receptor.

[00775] As non-limiting examples, any of the above methods of treatment, especially therapy with effective amounts of the claimed synthetic polypeptide agonist of the type 1 interferon receptor, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant, for treating cancer in patients, can be for the desired duration of treatment, including co-treatment of the patient with an SAPK inhibitor (e.g. pirfenidone or its analogue), taken in amounts effective to achieve the anti-cancer effect of therapy with a type 1 interferon receptor polypeptide synthetic agonist.

[00776] As non-limiting examples, any of the above methods of treatment, especially therapy with effective amounts of the claimed synthetic polypeptide agonist of the type 1 interferon receptor, hyperglycosylated polypeptide variant, protease-resistant polypeptide variant or hyperglycosylated, protease-resistant polypeptide variant, for treating cancer in patients, can be for the desired duration of treatment, including co-administration of IFN- [gamma] and SAPK-inhibitory to patients a (e.g., pirfenidone, or analogs thereof) taken in amounts sufficient to exhibit an anticancer effect of a synthetic polypeptide agonist therapy interferon receptor type 1.

[00777] As non-limiting examples, any of the above methods of treatment, especially therapy with effective amounts of the claimed synthetic polypeptide agonist of the type 1 interferon receptor, hyperglycosylated polypeptide variant, protease resistant polypeptide variant, or hyperglycosylated, protease resistant polypeptide variant, for treating cancer in patients, can be for the desired duration of treatment by including coadministration of additional antirust An agent other than IFN-γ [gamma] effective for treating cancer in a patient can be modified by including co-administering IFN-γ [gamma] to the patient in amounts effective for the anticancer effect of combination therapy with a synthetic type I interferon receptor polypeptide agonist and an additional anti-cancer agent.

[00778] As non-limiting examples, any of the above methods of treatment, especially therapy with effective amounts of the claimed synthetic polypeptide agonist of the type 1 interferon receptor, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant, for treating cancer in patients, can be the inclusion of co-administration to patients of an additional anti-cancer agent other than a SAPK inhibitor (n an example, pirfenidone or its analogue), effective for treating cancer in patients, can be modified for the desired duration of treatment by co-administering to patients a SAPK inhibitor (for example, pirfenidone or its analogues) in amounts effective for the anticancer effect of combination therapy with a synthetic polypeptide receptor agonist interferon type 1 and an additional anti-cancer agent.

[00779] As non-limiting examples, any of the above methods of treatment, especially therapy with effective amounts of the claimed synthetic type I interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant, for treating cancer in patients, can be for the desired duration of treatment by including coadministration of additional antirust an agent other than an IFN- [gamma] or SAPK inhibitor (eg, pirfenidone or its analogs) for the effective treatment of cancer in patients can be modified for the desired duration of treatment by co-administering to patients amounts of IFN- [gamma] and amounts of SAPK -inhibitor (for example, pirfenidone and its analogues), which effectively increases the anti-cancer effect of combination therapy with a synthetic polypeptide agonist of the type 1 interferon receptor and an additional anti-cancer agent.

VIRAL INFECTIONS

[00780] The present invention provides a method of treating a viral infection and a method of reducing the viral load or reducing the time of the body's release of viruses, or reducing morbidity or mortality in the clinical outcomes of a disease in patients suffering from a viral infection. The presented invention further provides methods for reducing the risk of complications arising in individuals as a consequence of a developed viral infection.

The methods mainly include administering therapeutic effective amounts of the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant, for treating a viral infection.

[00781] In certain embodiments, the claimed treatment method is prophylactic. In those cases where the claimed method is used as a prophylactic, it reduces the risk of developing a pathological viral infection in individuals. Effective amounts of the claimed synthetic type I interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant are amounts that reduce the risk or reduce the likelihood of an individual developing a viral pathological infection. For example, an effective amount reduces the risk of developing a pathological infection in an individual by approximately at least 10%; approximately at least 20%; approximately at least 30%; approximately at least 35%; approximately at least 40%; approximately at least 50%; at least 60%; approximately at least 70%; approximately at least 80%; or approximately at least 90%; or more, in comparison with the risk of developing a pathological viral infection in the absence of treatment with the claimed agent.

[00782] In certain embodiments, effective amounts of a claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant are amounts that reduce the viral load by approximately at least 10%; approximately at least 20%; approximately at least 30%; approximately at least 35%; approximately at least 40%; approximately at least 50%; at least 60%; approximately at least 70%; approximately at least 80%; or approximately at least 90%; or more, in comparison with the viral load in the absence of treatment with the claimed agent.

[00783] In certain embodiments, effective amounts of a claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant, or hyperglycosylated, protease resistant polypeptide variant are amounts that reduce the virus release time by at least about 10% ; approximately at least 20%; approximately at least 30%; approximately at least 35%; approximately at least 40%; approximately at least 50%; at least 60%; approximately at least 70%; approximately at least 80%; or approximately at least 90%; or more, compared with the duration of the release of the body from the virus in the absence of treatment.

[00784] In certain embodiments, effective amounts of the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant are amounts that reduce the incidence or mortality due to viral infection by at least about 10 %; approximately at least 20%; approximately at least 30%; approximately at least 35%; approximately at least 40%; approximately at least 50%; at least 60%; approximately at least 70%; approximately at least 80%; or approximately at least 90%; or more, compared with morbidity or mortality in the absence of treatment.

[00785] Whether or not the claimed method of treatment is effective in reducing the risk of a pathological viral infection, reducing the viral load and reducing the time of the body to free the virus, or to reduce the morbidity or mortality from the viral infection, is readily apparent from the prior art. Viral load can easily be determined by the titer or amount of virus in the serum. The amount of virus in the serum can be determined using any known tests, including, for example, a quantitative polymerase chain reaction using oligonucleotide primers specific for the test virus. Whether the incidence actually decreases can be determined by identifying any symptoms associated with a viral infection, including, for example, fever, respiratory symptoms (eg, coughing, weakness or difficulty breathing, and the like).

[00786] In certain embodiments, the present invention provides a method of reducing viral load and / or reducing the duration of an organism's release from a virus, and / or reduces morbidity or mortality in individuals who have been exposed to the virus (for example, persons who have had contact with persons infected virus), the method comprises administering an effective amount of a claimed synthetic type 1 interferon receptor polypeptide agonist. In such an embodiment of the invention, treatment is started from about 1 hour to about 14 days following exposure to the virus, for example, from about 1 hour to about 24 hours; from about 24 hours to about 48 hours; from about 48 hours to about 3 days; from about 48 hours to about 3 days; from about 3 days to about 4 days; from about 4 days to about 7 days; from about 7 days to about 10 days; or from about 10 days to about 14 days after exposure to the virus.

[00787] In certain embodiments, the present invention provides a method of reducing the risk that an individual exposed to a virus (for example, an individual who has been in contact with an individual infected with a virus) with a detected pathological viral infection with clinical manifestations, the method comprises administering effective amounts of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide var ianta or hyperglycosylated, protease-resistant polypeptide variant. In such an embodiment of the invention, treatment is started from about 1 hour to about 36 days following exposure to the virus, for example, from about 1 hour to about 24 hours; from about 24 hours to about 48 hours; from about 48 hours to about 3 days; from about 48 hours to about 3 days; from about 3 days to about 4 days; from about 4 days to about 7 days; from about 7 days to about 10 days; from about 10 days to about 14 days; from about 14 days to about 21 days; or from about 21 days to about 35 days after exposure to the virus.

[00788] In certain embodiments, the present invention provides a method of reducing viral load and / or shortening the duration of an organism's release from a virus, and / or reducing morbidity or mortality in individuals who may have been infected with the virus and who have had no signs of infection. In certain of these embodiments, the methods include administering effective amounts of the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant within 24 hours of exposure to the virus.

[00789] In certain embodiments, the present invention provides a method of reducing viral load and / or reducing the duration of an organism's release from a virus, and / or reduces morbidity or mortality in individuals who have not been infected with the virus and who have been exposed to the virus. In particular embodiments, the methods comprise administering effective amounts of an SAPK inhibitor (e.g., pirfenidone or an analog thereof) and a type 1 interferon receptor agonist within 48 hours after exposure to the virus.

[00790] In certain embodiments, the present invention provides a method of reducing viral load and / or reducing the duration of an organism's release from a virus, and / or reduces morbidity or mortality in individuals who have not been infected with the virus and who have been exposed to the virus. The methods include administering the claimed agent more than 48 hours after exposure to the virus, for example, from about 72 hours to about 35 days; for example, within 72 hours, 4 days, 5 days, 6 days or 7 days after exposure; or from about 7 days to about 10 days; from about 10 days to about 14 days; from about 14 days to about 17 days; from about 17 days to about 21 days; from about 21 days to about 25 days; from about 25 days to about 30 days; or from about 30 days to about 35 days after exposure to the virus.

In certain of these embodiments, the methods comprise administering effective amounts of the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant more than 48 hours after exposure to the virus.

[00791] In certain embodiments, the present invention provides a method of reducing the risk an individual exposed to a virus with a developed pathological viral infection with clinical manifestations is exposed. In certain of these embodiments, the methods include administering effective amounts of the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant within 24 hours of exposure to the virus.

[00792] In certain embodiments, the present invention provides a method of reducing the risk to an individual exposed to a virus (eg, an individual who has been in contact with an individual infected with a virus) with a developed pathological viral infection with clinical manifestations. In certain of these embodiments, the methods comprise administering effective amounts of the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant within 48 hours after exposure to the virus.

Hepatitis B infection

[00793] The present invention provides methods for treating hepatitis virus infection. In certain embodiments, the present invention provides methods for treating hepatitis C virus (HCV) infection; methods for reducing the occurrence of complications associated with HCV and cirrhosis; and methods for decreasing the viral load, or decreasing the duration of the organism's release from the virus, or decreasing morbidity or mortality as a result of the clinical course of the disease in patients suffering from HCV infection. The methods generally include administering effective amounts of a claimed synthetic type 1 interferon receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant, or a hyperglycosylated, protease-resistant polypeptide variant.

[00794] In many embodiments, the claimed method of treatment is effective to reduce the viral load in an individual and achieve a sustained viral response. Additionally, the claimed method in the future will provide the introduction of individuals effective amounts of nucleoside analogues, such as ribavirin, levorin and viramidine. Of particular interest in many applications is the treatment of people.

[00795] Whether the claimed method is really effective in the treatment of HCV infection can be determined by determining the viral load or by measuring the parameters of the infection associated with HCV, including (but not limited to) liver fibrosis, increasing serum transaminase levels of non-inflammatory activity in the liver. Indicators of liver fibrosis will be given in detail below.

[00796] Viral load can be determined by measuring the titer or level of the virus in the serum. These methods include, but are not limited to, quantitative polymerase chain reaction (PCR) and Branchial DNA (bDNA) test. Quantitative tests have been developed to measure the viral load (titer) of HCV RNA. Many commercially available assays include quantitative reverse transcription-PCR (RT-PCR) (Amplicor HCV Monitor ( ^TM ), Roche Molecular Systems, New Jersey); and branched DNA signal amplification test (deoxyribonucleic acid) (Quantiplex ( ^TM ) HCV RNA Assay (bDNA), Chiron Corp., Emeryville, California). See, for example, Gretch et al. (1995) Ann. Intern. Med. 123: 321-329. Also of interest is the nucleic acid test (NAT) developed by Gen-Probe Inc. (San Diego) and Chiron Corporation sold by Chiron Corporation under the trade name Procleix (R), in which NAT is simultaneously a test for the presence of HIV-I and HCV. See, for example, Vargo et al. (2002) Transfusion 42: 876-885.

[00797] Basically, effective amounts of the claimed agent (for example, the claimed synthetic type 1 interferon receptor polypeptide agonist, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant) are used in amounts that effectively reduce the viral load to not detect for example, less than about 5000, less than about 1000, less than about 500, or less than about 200 genomic copies / ml of serum tki. In certain embodiments, effective amounts of the claimed agent are amounts that reduce the viral load to less than 100 genomic copies / ml of serum. In many embodiments, the methods of the invention achieve a sustained virus response, for example, the viral load decreases to an undetectable level for a period of at least about one month; for a period of at least up to approximately two months; for a period of at least up to approximately three months; for a period of at least up to about four months; for a period of at least up to about five months; or for a period of at least up to approximately six months after discontinuation of treatment.

[00798] Whether the claimed method is really effective in the treatment of HCV infection can be established by measuring the parameters associated with HCV infection, such as liver fibrosis. Methods for determining the severity of liver fibrosis are discussed in detail below. In certain embodiments, the level of serum markers of hepatic fibrosis indicates the degree of hepatic fibrosis.

[00799] As a separate, non-limiting example, serum alanine aminotransferase (ALT) levels can be measured using a standard assay. Typically, an ALT level of less than approximately 45 international units (U / ml) is considered normal. In certain embodiments, effective amounts of a therapeutic agent that, when administered as part of the claimed combination therapy, are considered to be sufficiently effective if they lower ALT levels to less than 45 U / ml serum.

Combination therapy

[00800] In certain embodiments, the present invention provides combination therapy for treating viral infections. Therefore, the present invention provides a method for treating a viral infection, typically comprising administering a claimed synthetic type 1 interferon receptor polypeptide agonist, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant in combination therapy, with at least a second therapeutic agent. A suitable additional therapeutic agent includes, but is not limited to, nucleoside analogs such as ribavirin or viramidine; L-nucleosides, such as levorin; type II interferon receptor agonists; (e.g. IFM- [gamma]); TNF antagonists; thymosin- [alpha]; SAPK inhibitors (e.g., pirfenidone or its analogues); amantadine and the like. In combination with combination therapy for treating HCV infection, suitable therapeutic agents include, but are not limited to, nucleoside analogs such as ribavirin, levorin, or viramidine; type II interferon receptor agonists; (for example, IFN- [gamma]); TNF antagonists; NS3 inhibitors; NS5B inhibitors; alpha glycosidase inhibitors; thymosin- [alpha]; SAPK inhibitors (e.g., pirfenidone or its analogues); amantadine and the like.

[00801] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant for treating a viral infection, for example, HC -infection in patients can be modified for the desired duration of treatment by co-administration of n to patients with amounts of IFN-γ [gamma] effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

[00802] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of a viral infection, for example HC infection in patients can be modified for the desired duration of treatment by co-administration of patients with amounts of an SAPK inhibitor (for example, pirfenidone or its analogue) effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

[00803] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of a viral infection, for example HC infection in patients can be modified for the desired duration of treatment by co-administration of to patients with amounts of a nucleoside analogue effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

[00804] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of a viral infection, for example HC infection in patients can be modified for the desired duration of treatment by co-administration of to patients with amounts of ribaverin effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

[00805] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of a viral infection, for example HC -infection in patients can be modified for the desired duration of treatment by co-administration of n patients with amounts of L-nucleoside (e.g., levorin) effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

[00806] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant for treating a viral infection, for example, HC infection in patients can be modified for the desired duration of treatment by co-administration of patients with amounts of pyramidine effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

[00807] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant, or a hyperglycosylated, protease-resistant polypeptide variant, designed to treat a viral infection, for example, HC infection in patients can be modified for the desired duration of treatment by co-administration of patients with amounts of TNF antagonist (eg, etanercept, influximab or adalimumab) effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

[00808] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant for treating a viral infection, for example, HC infection in patients can be modified for the desired duration of treatment by co-administration of patients with amounts of thymosin- [alpha] effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

[00809] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of a viral infection, for example HC infection in patients can be modified for the desired duration of treatment by co-administration of to patients with amounts of an NS5B inhibitor effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

[00811] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant, or a hyperglycosylated, protease-resistant polypeptide variant, designed to treat a viral infection, for example, HC -infection in patients can be modified for the desired duration of treatment by co-administration of n to patients with amounts of an alpha glucosidase inhibitor effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

[00812] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of the type 1 interferon receptor, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant intended for the treatment of viral infection (for example, HC infection) in patients, may be modified for the desired duration of treatment by co-administration atsientam amounts of IFN- [gamma] and amounts SAPK inhibitor (e.g., pirfenidone, or an analogue thereof) effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00813] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant, or a hyperglycosylated, protease-resistant polypeptide variant intended for the treatment of a viral infection (for example, HC infection) in patients can be modified for the desired duration of treatment by co-administration atsientam amounts of IFN- [gamma] and amounts of the nucleoside analog effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00814] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of a claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of a viral infection (for example, HC infection) in patients, may be modified for the desired duration of treatment by co-administration atsientam amounts of IFN- [gamma] and ribaverina amounts effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00815] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of a viral infection (for example, HC infection) in patients can be modified for the desired duration of treatment by co-administration atsientam amounts of IFN- [gamma] and amounts of L-nucleoside (e.g., levorin) effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00816] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant, or a hyperglycosylated, protease-resistant polypeptide variant, designed to treat a viral infection (for example, HC infection) in patients, may be modified for the desired duration of treatment by co-administration atsientam amounts of IFN- [gamma] and viramidine amounts effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00817] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of a viral infection (for example, HC infection) in patients can be modified for the desired duration of treatment by co-administration atsientam amounts of IFN- [gamma] and amounts of TNF antagonist (e.g., etanercept, or adalimumab inflyuksimab) effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00818] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of a viral infection (for example, HC infection) in patients can be modified for the desired duration of treatment by co-administration atsientam amounts of IFN- [gamma] and amounts timozina- [alpha], effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00819] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of a claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of a viral infection (for example, HC infection) in patients can be modified for the desired duration of treatment by co-administration atsientam amounts of IFN- [gamma] and amounts of NS 3 inhibitor effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00820] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of a viral infection (for example, HC infection) in patients, may be modified for the desired duration of treatment by co-administration atsientam amounts of IFN- [gamma] and amounts NS5B inhibitor effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00821] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of a viral infection (for example, HC infection) in patients can be modified for the desired duration of treatment by co-administration atsientam amounts of IFN- [gamma] and amounts of alpha-glycosidase inhibitor effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00822] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of a viral infection (for example, HC infection) in patients, may be modified for the desired duration of treatment by co-administration atsientam amounts of nucleoside analogue (e.g. ribavirin or viramidine L-nucleoside, such as levorin) and the amounts of IFN- [gamma] effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00823] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of the type 1 interferon receptor, hyperglycosylated polypeptide variant, protease resistant polypeptide variant, or hyperglycosylated, protease resistant polypeptide variant intended for the treatment of viral infection (for example, HC infection) in patients can be modified for the desired duration of treatment by co-administration atsientam amounts of nucleoside analogue (e.g. ribavirin or viramidine L-nucleoside, such as levorin) and the amounts of TNF antagonist (e.g., entanertsept, inflyuksimab or adalimunab) effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00824] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of the type 1 interferon receptor, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant intended for the treatment of viral infection (for example, HC infection) in patients can be modified for the desired duration of treatment by co-administration atsientam amounts of nucleoside analogue (e.g. ribavirin or viramidine L-nucleoside, such as levorin) and timozin- amounts [alpha], effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00825] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of a viral infection (for example, HC infection) in patients, may be modified for the desired duration of treatment by co-administration atsientam amounts of nucleoside analogue (e.g. ribavirin or viramidine L-nucleoside, such as levorin) and quantities SAPK inhibitor (e.g., pirfenidone, or an analogue thereof) effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00826] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of a claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by co-administering to the patient the amounts of nucleosis Nogo annaloga (e.g., ribavirin, viramidine or L-nucleoside, such as levorin) and quantities of NS3 inhibitor effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00827] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by co-administering to patients a number of nucleosides analogue of the (e.g., ribavirin, viramidine or L-nucleoside, such as levorin) and NS5B inhibitor amounts effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00828] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of the type 1 interferon receptor, hyperglycosylated polypeptide variant, protease resistant polypeptide variant, or hyperglycosylated, protease resistant polypeptide variant, intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by co-administering to the patient the amounts of nucleosis analogue of the (e.g., ribavirin, viramidine or L-nucleoside, such as levorin) and the amounts of TNF antagonist (e.g., etanercept, or inflyuksimab adalimunab) effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00829] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by co-administering to patients amounts of NS3 inhibitor ora and amounts of TNF antagonist (e.g., etanercept, or inflyuksimab adalimunab) effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00830] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of the type 1 interferon receptor, hyperglycosylated polypeptide variant, protease resistant polypeptide variant, or hyperglycosylated, protease resistant polypeptide variant, intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by coadministration of amounts of NS5B inhibitor a torus and amounts of a TNF antagonist (e.g., etanercept, influximab or adalimumab) effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

[00831] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant, or a hyperglycosylated, protease-resistant polypeptide variant, intended for treating HCV infection in patients can be modified for the desired duration of treatment by co-administering to the patient amounts of inhibitory and alpha-glycosidase and amounts of TNF antagonist (e.g., etanercept, or inflyuksimab adalimunab) effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00832] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant, or a hyperglycosylated, protease-resistant polypeptide variant, intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by co-administration of amounts of SAPK inhibitors to patients a torus (e.g., pirfenidone or an analog thereof) and amounts of a TNF antagonist (e.g., etanercept, influximab or adalimumab) effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

[00833] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant or a hyperglycosylated, protease resistant polypeptide variant, intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by co-administration of amounts of thymosin- [ lfa] and amounts of TNF antagonist (e.g., etanercept, or inflyuksimab adalimunab) effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00834] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant or a hyperglycosylated, protease resistant polypeptide variant, intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by co-administering to patients a number of nucleosides Nogo annaloga (e.g., ribavirin, viramidine or L-nucleoside, such as levorin) and timozin- amounts [alpha], effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00835] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by co-administration of amounts of SAPK inhibitors to patients a torus (for example, pirfenidone or its analogue) and amounts of thymosin- [alpha] effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

[00836] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of a claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant for treating HCV infection in patients can be modified for the desired duration of treatment by co-administering to patients amounts of NS3 inhibitor ora timozin- and amounts [alpha], effective to increase the antiviral effect of therapy synthetic polypeptide agonist type 1 interferon receptor.

[00837] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by co-administering to patients amounts of NS5B inhibitor a torus and amounts of thymosin- [alpha], effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

[00838] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant, or a hyperglycosylated, protease-resistant polypeptide variant, intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by co-administering to the patient amounts of inhibitory and alpha-glucosidase and timozin- amounts of [alpha], effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00839] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant, or a hyperglycosylated, protease-resistant polypeptide variant, intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by co-administering to the patient amounts of inhibitory a nucleoside analogue (e.g. ribavirin or viramidine L-nucleoside, such as levorin) and quantities SAPK inhibitor (e.g., pirfenidone, or an analogue thereof) effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00840] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by co-administering to patients amounts of NS3 inhibitor ora and amounts SAPK inhibitor (e.g., pirfenidone, or an analogue thereof) effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00841] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by co-administering to patients amounts of NS5B inhibitor a torus and amounts of an SAPK inhibitor (e.g., pirfenidone or its analogue) effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

[00842] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease-resistant polypeptide variant, or a hyperglycosylated, protease-resistant polypeptide variant, intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by co-administering to the patient amounts of inhibitory and alpha-glucosidase and amounts SAPK inhibitor (e.g., pirfenidone, or an analogue thereof) effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00843] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by co-administering to patients a number of nucleosides Nogo annaloga (e.g., ribavirin, viramidine or L-nucleoside, such as levorin) and amounts of alpha-glucosidase inhibitor effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00844] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of the type 1 interferon receptor, hyperglycosylated polypeptide variant, protease resistant polypeptide variant or hyperglycosylated, protease resistant polypeptide variant, intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by coadministration of amounts of NS5B inhibitor a torus and amounts of an NS3 inhibitor effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

[00845] As non-limiting examples, any of the above methods of treatment, especially with an effective amount of the claimed synthetic polypeptide agonist of an interferon type 1 receptor, a hyperglycosylated polypeptide variant, a protease resistant polypeptide variant, or a hyperglycosylated, protease resistant polypeptide variant intended for the treatment of HCV infection in patients can be modified for the desired duration of treatment by co-administering to the patient amounts of inhibitory and alpha-glucosidase inhibitors and NS3 amounts effective to increase the antiviral effect of therapy synthetic polypeptide receptor agonist type 1 interferon.

[00846] As non-limiting examples, any of the above treatment methods, especially with an effective amount of the claimed synthetic type 1 interferon receptor polypeptide agonist, for treating HCV infection in patients, can be modified for the desired duration of treatment by co-administering to the patient amounts of an NS5B inhibitor and amounts of an alpha glucosidase inhibitor effective to increase the antiviral effect of therapy with a synthetic type 1 interferon receptor polypeptide agonist.

Patient identification

[00847] In certain embodiments, the specific drug regimen used in the treatment of HCV patients is selected according to individual parameters of the disease manifested by the patient, such as the initial viral load, the genotype of the HCV infection in the patient, and / or the stage of hepatic fibrosis in the patient.

[00848] Therefore, in certain embodiments, the present invention allows any of the above methods for treating HCV infection, in which the claimed method is modified, to be used to treat severe patients for 48 weeks.

[00849] In other embodiments, the invention provides any of the above [0974] HCV treatments, wherein the claimed method is modified to treat non-responding patients when a patient has received a 48-week course of therapy.

[00850] In other embodiments, the invention provides any of the methods for treating HCV infection as described above, wherein the claimed method is modified to treat recurrent patients when a patient has received a 48-week course of treatment.

[00851] In other embodiments, the invention provides any of the above methods for treating HCV infection, in which the claimed method has been modified to treat a primary patient infected with HCV genotype 1 when the patient has taken a 48-week course of therapy.

[00852] In other embodiments, the invention provides any of the methods for treating HCV infection as described above, wherein the claimed method has been modified to treat a primary patient infected with HCV genotype 4 when the patient has taken a 48-week course of therapy.

[00853] In other embodiments, the invention provides any of the above methods for treating HCV infection, in which the claimed method has been modified to treat a primary patient infected with HCV genotype 1, when the patient had a high viral load (HVL), where "HVL" reflects that the HCV viral load exceeds 2 × 10 ⁶ HCV genomic copies per ml of serum when the patient took a 48-week course of therapy.

[00854] In one embodiment, the invention provides any of the methods for treating HCV infection as described above, wherein the claimed method has been modified to include the steps of: (1) identifying patients with advanced disease or a single serious stage of hepatic fibrosis using the Knodell score system corresponding to 3 or 4; and then (2) administering drug therapy to the patients according to the claimed method for a period of time from about 24 weeks to about 60 weeks; or about 30 weeks, up to about one year; or about 36 weeks, up to about 50 weeks; or about 40 weeks, up to about 48 weeks; or at least about 24 weeks; or at least about 30 weeks; or at least about 36 weeks; or at least about 40 weeks; or at least about 48 weeks; or at least about 60 weeks.

[00855] In another embodiment, the invention provides any of the methods for treating HCV infection as described above, wherein the claimed method has been modified to include the steps of: (1) identifying patients with advanced disease or a single serious stage of hepatic fibrosis using the Knodell score system corresponding to 3 or 4; (2) administering to the patient medicament therapy of the claimed method for a period of about 40 weeks to about 50 weeks, or about 48 weeks.

[00856] In another embodiment, the invention provides any of the methods for treating HCV infection as described above, wherein the claimed method has been modified to include the steps of: (1) identifying patients infected with HCV genotype 1 and with an initial viral load of more than 2 million viral genomic copies per ml patient serum; and then (2) administering drug therapy to the patients according to the claimed method for a period of about 24 weeks to about 60 weeks; or about 30 weeks, up to about one year; or about 36 weeks, up to about 50 weeks; or about 40 weeks, up to about 48 weeks; or at least 24 weeks; or at least 30 weeks; or at least 36 weeks; or at least 40 weeks; or at least 48 weeks; or at least 60 weeks.

[00857] In another embodiment, the invention provides any of the methods for treating HCV infection as described above, wherein the claimed method has been modified to include the steps of: (1) identifying patients infected with HCV genotype 1 and with an initial viral load of more than 2 million viral genomic copies per ml of patient serum; and then (2) administering the drug therapy to the patients according to the claimed method for a period of from about 40 weeks to about 50 weeks, or about 48 weeks.

[00858] In another embodiment, the invention provides any of the methods for treating HCV infection as described above, wherein the claimed method has been modified to include the steps of: (1) identifying patients infected with HCV genotype 1 and with an initial viral load of more than 2 million viral genomic copies per ml patient serum; and either did not have or had an early stage of fibrosis, estimated by Knodell as 0, 1, or 2; and then (2) administering drug therapy to the patients according to the claimed method for a period from 24 weeks to about 60 weeks; or about 30 weeks, up to about one year; or about 36 weeks, up to about 50 weeks; or about 40 weeks, up to about 48 weeks; or at least about 24 weeks; or at least about 30 weeks; or at least about 36 weeks; or at least about 40 weeks; or at least about 48 weeks; or at least about 60 weeks.

[00859] In another embodiment, the invention provides any of the methods for treating HCV infection as described above, wherein the claimed method has been modified to include the steps of: (1) identifying patients infected with HCV genotype 1 and with an initial viral load of more than 2 million viral genomic copies per ml patient serum; and either did not have or had an early stage of fibrosis, estimated by Knodell as 0, 1, or 2; and then (2) administering drug therapy to the patients according to the claimed method for a period from 40 weeks to about 50 weeks, or about 48 weeks.

[00860] In another embodiment, the invention provides any method of treating HCV infection as described above, wherein the claimed method has been modified to include the steps of: (1) identifying patients infected with HCV genotype 1 and with an initial viral load of less than or equivalent to 2 million viral genomic copies per ml of patient serum; (2) administering drug therapy to patients according to the claimed method for a period of from 20 weeks to about 50 weeks; or about 24 weeks, up to about 48 weeks; or about 30 weeks, up to about 40 weeks; or about 20 weeks, up to about 24 weeks; or about 24 weeks, up to about 48 weeks; or within 20 weeks; or within 24 weeks; or within 30 weeks; or within 36 weeks; or within 48 weeks.

[00861] In another embodiment, the invention provides any of the methods for treating HCV infection as described above, wherein the claimed method has been modified to include the steps of: (1) identifying patients infected with HCV genotype 1 and with an initial viral load of less than or equivalent to 2 million viral genomic copies per ml of patient serum; (2) administering drug therapy to patients according to the claimed method for a period of from 20 weeks to about 24 weeks.

[00862] In another embodiment, the invention provides any of the methods for treating HCV infection as described above, wherein the claimed method has been modified to include the steps of: (1) identifying patients infected with HCV genotype 1 and with an initial viral load of less than or equivalent to 2 million viral genomic copies per ml of patient serum; (2) administering drug therapy to patients according to the claimed method for a period of 24 weeks to about 48 weeks.

[00863] In another embodiment, the invention provides any of the methods for treating HCV infection as described above, wherein the claimed method has been modified to include the steps of: (1) identifying patients infected with HCV genotypes 2 or 3; and then (2) administering drug therapy to the patients in the claimed manner for a period of 24 weeks to about 60 weeks; or about 30 weeks, up to about one year; or about 36 weeks, up to about 50 weeks; or about 40 weeks, up to about 48 weeks; or at least about 24 weeks; or at least about 30 weeks, or at least about 36 weeks, or at least about 40 weeks, or at least about 48 weeks; or at least about 60 weeks.

[00864] In another embodiment, the invention provides any of the methods for treating HCV infection as described above, wherein the claimed method has been modified to include the steps of: (1) identifying patients infected with HCV genotypes 2 or 3; and then (2) administering drug therapy to the patients according to the claimed method for a period of 24 weeks to about 50 weeks; or about 24 weeks, up to about 48 weeks; or about 30 weeks, up to about 40 weeks; or, about 20 weeks, up to about 24 weeks; or within 30 weeks; or within 36 weeks; or within 48 weeks.

[00865] In another embodiment, the invention provides any of the methods for treating HCV infection as described above, wherein the claimed method has been modified to include the steps of: (1) identifying patients infected with HCV genotypes 2 or 3; and then (2) administering drug therapy to the patients in the claimed manner for a period of from 20 weeks to about 24 weeks.

[00866] In another embodiment, the invention provides any method of treating HCV infection as described above, wherein the claimed method has been modified to include the steps of: (1) identifying patients infected with HCV genotypes 2 or 3; and then (2) administering drug therapy to the patients according to the claimed method for a period of at least about 24 weeks.

[00867] In another embodiment, the invention provides any of the methods for treating HCV infection as described above, wherein the claimed method has been modified to include the steps of: (1) identifying patients infected with HCV genotypes 1 or 4; and then (2) administering drug therapy to the patients according to the claimed method over a period of at least about 24 weeks to about 60 weeks; or about 30 weeks, up to about one year; or about 36 weeks, until about 50 weeks; or about 40 weeks, up to about 48 weeks; or at least about 24 weeks; or at least about 30 weeks; or at least about 36 weeks; or at least about 40 weeks; or at least about 48 weeks; or at least about 60 weeks.

[00868] In another embodiment, the invention provides any of the above methods for treating HCV infection, in which the claimed method has been modified to include the steps of: (1) identifying patients infected with HCV by any of genotypes 5, 6, 7, 8 and 9; and then (2) administering drug therapy to the patients according to the claimed method for a period of at least about 20 weeks to about 50 weeks.

[00869] In another embodiment, the invention provides any of the methods for treating HCV infection as described above, wherein the claimed method has been modified to include the steps of: (1) identifying patients infected with HCV by any of genotypes 5, 6, 7, 8, and 9; and then (2) administering drug therapy to the patients according to the claimed method for a period of at least about 24 weeks to about 48 weeks.

Type II interferon receptor agonists

[00870] As used herein, the term "type II interferon receptor agonist" includes any natural or non-natural type II human interferon receptor ligands that bind to or induce signal transduction through this receptor. Interferon type II receptor agonists include interferons, including natural interferons, modified interferons, synthetic interferons, pegylated interferons, fusion proteins containing interferon and a heterologous protein, interferons obtained by shuffling; antibodies specific for the interferon receptor; non-peptide chemical agonists; and the like.

[00871] A particular example of an interferon type II receptor agonist is IFN-gamma and variants thereof. Although the present invention cites the use of the IFN-gamma polypeptide as an example, it will be readily apparent that any type II interferon receptor agonist can be used in the present method.

SAPK Inhibitors

[00872] SAPK inhibitors suitable for use in the subject treatment method particularly include pirfenidone and pirfenidone analogs; and in particular, include any compound of Formula I indicated in US Patent Publication No. 20030149041.

[00873] Additional SAPK inhibitors suitable for use in this invention include agents that inhibit the enzymatic activity of SAPK by at least about 10%, at least about 20%, at least about 25%, at least at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more, compared with enzymatic acti in particular, SAPK in the absence of an SAPK inhibitor.

[00874] Signal pathways involving mitogen-activated protein kinases (MAPKs) play an important role in a variety of cell responses, including growth, stress-activated gene expression, and compensatory functions for environmental changes. MAPKs include an SAPK group including c-Jun N-terminal kinase (JNK) and p38 kinases. The p38 group of MAPKinases includes at least four members designated p38 or p38α, p38β, p38γ, and p38δ. The amino acid sequences of p38α, p38β, and p38γ of various species are known. For example, the amino acid sequences of human p38α, p38β p38γ are under the following GenBank registration numbers: I) Q16539, NP_620583 and NP_001306 provide the amino acid sequences of human p38a polypeptides; 2) NP_620478, NP_002742 and Q15759 provide the amino acid sequences of human p38β polypeptides; and 3) NP_002960, P53778, and JC5252 provide the amino acid sequences of human p38γ polypeptides.

[00875] In some embodiments, a suitable SAPK inhibitor is an agent that inhibits the enzymatic activity of p38α, p38β and p38γ. In other embodiments, a suitable SAPK inhibitor is an agent that preferably inhibits the enzymatic activity of p38α and p38β, i.e. this agent is a more potent inhibitor of the enzymatic activity of p38α and p38β than the activity of p38γ, for example, the IC50 of this agent for p38α and p38β is at least about two times lower, or about five times lower, or about ten times lower, or even lower than the IC50 of this agent in relation to p38γ.

[00876] In other embodiments, a suitable SAPK inhibitor is an agent that preferably inhibits p38γ, i.e. this agent is a more potent inhibitor of p38γ enzymatic activity than p38α and p38β activity, for example, the IC50 of this agent for p38y is at least about two times lower, or about five times lower, or about ten times lower, or even lower than the IC50 of this agent in relation to p38α and p38β.

[00877] In some embodiments, the SAPK inhibitor is a competitive SAPK inhibitor, for example, p38α, a p38β, or a p38γ. In some of these wonders, a SAPK inhibitor is one that competes for adenosine triphosphate (ATP) for binding to the ATP binding site of p38α, p38β, or p38γ.

[00878] In addition, stress-activated protein kinase (SAPK) inhibitors that are suitable for use in the combination therapy under consideration include any 2-alkylimidazole described in US Pat. No. 6,548,520; any of the 1,4,5-substituted imidazole compounds described in US Pat. No. 6,489,325; 1,4,5-substituted imidazole compounds described in US Pat. No. 6,569,871; heteroarylaminophenylketone compounds described in US Published Patent Application No. 2003/0073832; pyridylimidazole compounds described in US Pat. No. 6,288,089; and heteroarylaminobenzophenones described in US patent No. 6432962. Also suitable for use are the compounds described in US patent No. 6214854. Heterocyclic compounds, which are discussed in international publication WO 99/61426, are also suitable for use.

Specially included pirfenidone and pirfenidone analogues are described in detail below. As already discussed above, compounds of formula I from US Patent Publication 20030149041 are particularly included. Formula I is as follows:

where R ^{1 is} selected from —H, hydrocarbon C ₁ -C ₂₀ , aminocarbonylalkyl, alkoxyalkyl, substituted arylalkyl, heteroaryl, heteroarylalkyl, heterocyclylalkyl and substituted heterocyclylalkyl;

R ^{2 is} selected from halogen, C ₁ -C ₂₀ hydrocarbon, hydroxy, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl;

R ^{5 is} selected from —H, substituted alkyl alkyl;

R ^{6 is} selected from a direct bond, alkyl, aryl, substituted aryl and heteroaryl;

R ^{7 is} selected from —H, acyl, alkyl, substituted alkyl, alkoxycarbonyl, amidine, aryl, aralkyl, heterocyclyl, heteroaryl, substituted heteroaryl 1, substituted alkoxy, heteroarylsulfonamido, dialkylsulfonamido,

-C (O) NR ⁸ R ⁹ , -C (NH) NR ⁸ R ⁹ and -NR ⁸ R ⁹ ;

R ^{8 is} selected from —H and alkyl;

R ^{9 is} selected from —H, alkyl, substituted alkyl, aryl, heteroaryl, alkylcarbonyl and arylcarbonyl;

R ³ choose from a direct connection,

where the left bond is the point of attachment to the ring and the right bond is the point of attachment to R ⁴ ;

R ^{4 is} selected from —H, halogen, alkyl, heterocyclyl, alkylamino, aminocarbonyl,

и

and

where R ^{10 is} selected from —H, —OH, alkyl, cycloalkyl and substituted cycloalkyl;

R ¹¹ choose —H, —OH, —COOH, aryl, substituted aryl, heteroaryl, substituted heteroaryl, aryl substituted alkyl, cycloalkyl, substituted cycloalkyl, alkoxy, aminocarbonyl, aminocarbonylalkyl,

R ^{12 is} selected from alkyl and aryl; R13 is selected from "H and aryl;

R ^{14 is} selected from aryl, substituted aryl, heteroaryl, substituted alkyl, aryl substituted alkyl, and alkoxy substituted alkyl;

R ^{15 is} selected from alkyl, aryl, substituted aryl, and substituted alkyl;

R ^{16 is} selected from aryl, substituted aryl, heteroaryl, carboxyl, alkoxy, substituted alkyl, cycloalkyl, substituted cycloalkyl, aminocarbonyl, substituted aminocarbonyl, heterocyclyl, and

R ^{17 is} selected from alkyl and dialkylamine; R ^{18 is} selected from a C ₁ -C ₂₀ hydrocarbon, a substituted C ₁ -C ₂₀ hydrocarbon, and heteroaryl; Y is selected from —H and lower alkyl, or Y and R ¹ taken together with attached N can be selected from heterocyclyl, substituted heterocyclyl, heteroaryl and substituted heteroaryl; where at least two X, X ¹ and X represent —N =, and the other is selected from —C (H) = and —N =.

Of particular interest in certain embodiments of the invention is the use of any of the following compounds of SAPK inhibitors, or their pharmaceutically acceptable salts, derivatives, esters or analogs thereof:

which has a name according to IUPAC (4-benzyl-piperidin-1-yl) - (1H-indol-5-yl) -methanone. Also suitable for use are any of the following compounds: (4-benzyl-piperidin-1-yl) - (6-chloro-1H-indol-5-yl) methanone; (4-chloro-H-indol-5-yl- [4- (4-fluoro-benzyl) -piperidin-1-yl] -methanone; (4-benzyl-piperidin-1-yl) - (4-methoxy- 1H-indol-5-yl) -methanone; (4-benzyl-piperidin-1-yl) - {1- [3- (cyclohexylmethylamino) -2-hydroxypropyl] -1H-indol-5-yl} -methanone; ( 4-benzyl-piperidin-1-yl) - {1- [2-hydroxy-3- (4-methylpiperazin-1-yl) propyl] -1H-indol-5-yl} methanone; [1- (3 -benzyl-mino-2-hydroxypropyl) -1H-indol-5-yl] - (4-benzyl-piperidin-1-yl) -methanone; (4-benzyl-piperidin-1-yl) - {1- [2 -hydroxy-3- (4-methoxy-benzylamino-but) -propyl] -1H-indol-5-yl} -methanone; (4-benzyl-piperidin-1-yl) - [1- (2-hydroxy-3 propylamino propyl) -1H-indol-5-yl] methanone; (4-benzyl- Iperidin-1-yl) - [1- (pyridin-4-carbonyl) -1H-indol-5-yl] methanone; 1- [5- (4-benzyl-piperidin-1-carbonyl) -indole-1- yl] ethanone; 2- [5- (4-benzyl-piperidin-1-carbonyl) -indol-1-yl] -N- (4-methoxy-benzyl) -acetamide; (2-methoxy-ethyl) -amide (2-Methoxy-ethyl) -5- (4-benzyl-piperidin-1-carbonyl) -1H-indole-3-carboxylic acid; (2-methylamino ethyl) -amide 5- (4benzylpiperidin-1-carbonyl) -1H -indole-3-carboxylic acid; (2-amino-ethyl) -amide 5- (4-benzyl-piperidin-1-carbonyl) -1H-indole-3-carboxylic acid; [3- (4-benzyl-piperidin-1-carbonyl) -1H-indol-5-yl] - (4-benzyl-piperidin-1-yl) methanone; [3- (4-benzyl-piperidin-1-carbonyl) -1H-indol-6-yl] - (4-benzyl-piperidin-1-yl) methanone; 5- (4-benzyl-piperidin-1-carbonyl) -1H-in-dol-3-carboxylic acid 4-fluoro-benzylamide; 5- (4-benzyl-piperidin-1-carbonyl) -1H-indole-3-carboxylic acid [2- (3,5-dimethoxy-phenyl) -ethyl] -amide; (4-benzyl-piperidin-1-yl) - (6-methoxy-1H-indol-5-yl) methanone; 1- [5- (4-benzyl-piperidin-1-carbonyl) -1H-indol-3-yl] -2,2,2-trifluoro-ethanone; (2-Dimethylamino-ethyl) -amide 5- (4-benzyl-piperidin-1-carbonyl) -6-methoxy-1H-indole-3-carboxylic acid; 5- (4-benzyl-piperidin-1-carbonyl) -1H-indole-3-carboxylic acid; 5- (4-benzyl-piperidin-1-carbonyl) -1H-indole-3-carboxylic acid (2-dimethylamino-ethyl) -amide; (1H-benzoimidazol-5-yl) - (4-benzyl-piperidin-1-yl) methanone; (1H-Benzoimidazol-5-yl) - [4- (4-fluoro-benzyl) piperidin-1-yl] methanone; (4-benzyl-piperidin-1-yl) - (3-morpholin-4-ylmethyl-1H-indol-5-yl) methanone; 1- [6- (4-benzyl-piperidin-1-carbonyl) -1H-indol-3-yl] -2,2,2-trifluoro-ethanone; (4-benzyl-piperidin-1-yl) - [1- (pyridin-4-carbonyl) -1H-indol-6-yl] methanone; (3-benzyl-8-aza-bicyclo [3.2.1] oct-8-yl) - (6-methoxy-1H-indol-5-yl) methanone; (3H-benzoimidazol-5-yl) - (3-benzyl-8-aza-bicyclo [3.2. 1] oct-8-yl) -methanone; [3- (4-fluoro-benzyl) pyrrolidin-1-yl] - (1H-indol-6-yl) methanone; (1H-benzimidazol-5-yl) - [4- (2,6-difluoro-benzyl) piperazin-1-yl] methanone; (1H-Benzoimidazol-5-yl) - [4- (4-methylsulfanyl-benzyl) piperazin-1-yl] methanone; (1H-benzoimidazol-5-yl) - [4- (2,3-difluoro-benzyl) piperazin-1-yl] methanone; (1H-benzoimidazol-5-yl) - [4- (3,5-difluoro-benzyl) piperazin-1-yl] methanone; (1H-Benzoimidazol-5-yl) - [4- (3-chloro-benzyl) piperazin-1-yl] methanone; 4- [4- (1H-benzo-imidazole-5-carbonyl) piperazin-1-yl-methyl] -benzoic acid methyl ester; (1H-Benzoimidazol-5-yl) - [4- (4-methoxy-benzyl) piperazin-1-y1] methanone; (1H-Benzoimidazol-5-yl) - [4- (4-trifluoromethoxy-benzyl) piperazin-1-yl] methanone; (1H-Benzoimidazol-5-yl) - [4- (4-methyl-benzyl) piperazin-1-yl] methanone; (1H-benzoimidazol-5-yl) - [4- (2,4-dichloro-benzoyl) piperazin-1-yl] methanone; (1H-benzoimidazol-5-yl) - [4- (3,4-dichloro-benzoyl) piperazin-1-yl] methanone; trans-1- [4- (1H-benzoimidazole-5-carbonyl) piperazin-1-yl] -3- (3-trifluoromethyl-phenyl) -propenone; (1H-Benzoimidazol-5-yl) - [4- (4-chloro-benzoyl) piperazin-1-yl] methanone; (1H-benzoimidazol-5-yl) - (4-benzoyl-piperazin-1-yl) methanone; (1H-Benzoimidazol-5-yl) - [4- (2-trifluoromethyl-benzoyl) piperazin-1-yl] methanone; (1H-Benzoimidazol-5-yl) - [4- (4-methoxy-benzoyl) piperazin-1-yl] methanone; (1H-benzoimidazol-5-yl) - [4- (3,4-dichloro-phenyl) piperazin-1-yl] methanone; (1H-benzoimidazol-5-yl) - {4 - [(4-chlorophenyl) phenylmethyl] piperazin-1-yl} methanone; trans- (1H-benzoimidazol-5-yl) - [4- (3-phenyl-allyl) piperazin-1-yl] methanone; (1H-benzoimidazol-5-yl) - {4- [bis- (4-fluoro-phenyl) methyl] piperazin-1-yl} methanone; (1H-benzoimidazol-5-yl) - [4- (4-chloro-benzyl) piperazin-1-yl] methanone; (1H-Benzoimidazol-5-yl) - [4- (2-chloro-benzyl) piperazin-1-yl] methanone; (1H-Benzoimidazol-5-yl) - [4- (3,4,5-trifluoromethoxy-benzyl) piperazin-1-yl] methanone; (1H-benzoimidazol-5-yl) - [4- (4-diethylaminro-benzyl) piperazin-1-yl] methanone; (1H-benzoimide-zol-5-yl) - (4-biphenyl-4-ylmethyl-piperazin-1-yl) methanone; (1H-Benzoimidazol-5-yl) - [4- (4-phenoxy-benzyl) piperazin-1-yl] methanone; (4-benzylpiperidin-1-yl) - (6-methoxy-1H-benzoimidazol-5-yl) methanone; (4-benzyl-piperidin-1-yl) - (1-isopropyl-1H-benzoimidazol-5-yl) methanone; (4-benzyl-piperidin-1-yl) - (3-isopropyl-3H-benzoimidazol-5-yl) methanone; (4-benzyl-piperidin-1-yl) - (1-isopropyl-1 H-indol-5-yl) methanone; [4- (4-chloro-benzyl) piperazin-1-yl] - (1-isopropyl-1H-indol-5-yl) methanone; (1H-benzotriazol-5-yl) - (4-benzyl-piperidin-1-yl) methanone; (4-benzyl-piperidin-1-yl) - (1-isopropyl-1H-benzotriazol-5-yl) methanone; [4- (4-chloro-benzyl) -piperidin-1-yl] - (1H-indol-5-yl) methanone; [4- (3-chloro-benzyl) -piperidin-1-yl] - (1H-indol-5-yl) methanone; [4- (2-chloro-benzyl) -piperidin-1-yl] - (1H-indol-5-yl) methanone; (4-benzyl-2-methyl-piperidin-1-yl) - (1H-indol-5-yl) methanone; (4-benzyl-piperidin-1-yl) - (4-chloro-1H-indol-5-yl) methanone; (4-benzyl-piperidin-1-yl) - [7-chloro-1- (pyridin-3-carbonyl) -1H-indol-6-yl] methanone; (4-benzyl-piperidin-1-yl) - (5-chloro-1H-indol-6-yl) methanone; (4-benzyl-piperidin-1-yl) - (7-chloro-1H-indol-6-yl) methanone; (2-Dimethylaminoethyl) -amide 6- (4-benzyl-piperidin-1-carbonyl) -7-chloro-1- (pyridin-3-carbonyl) -1H-indole-3-carboxylic acid; (4-benzyl-piperidin-1-yl) - (1-pyridin-4-ylmethyl-1H-indol-5-yl) methanone; (4-benzyl-piperidin-1-yl) - [6-methoxy-1- (pyridin-3-carbonyl) -1H-indol-5-yl] methanone; [5- (4-benzyl-piperidin-1-carbonyl) -indol-1-yl] -acetic acid methyl ester; 1- [5- (4-benzyl-piperidin-1-carbonyl) -indol-1-yl] -3-isopropylamino-propan-1-one; 1- [5- (4-benzyl-piperidin-1-carbonyl) -indol-1-yl] -3-piperazin-1-yl-propan-1-one; 3-benzylamino-1- [5- (4-benzyl-piperidin-1-carbonyl) indol-1-yl] propan-1-one; 1- [5- (4-benzyl-piperidin-1-carbonyl) -indol-1-yl] -3-morpholin-4-yl-propan-1-one; 2- [5- (4-benzyl-piperidin-1-carbonyl) indol-1-yl] -N-propyl-acetamide; (4-benzyl-piperidin-1-yl) - [1- (2-diethylaminoethyl) -6-methoxy-1H-indol-5-yl] methanone; (4-benzyl-piperidin-1-yl) - [1- (3-diethylaminopropyl) -1H-indol-5-yl] methanone; (4-benzyl-piperidin-1-yl) - [1- (2-diethylamino-ethyl) -1H-indol-5-yl] methanone; (4-benzyl-piperidin-1-yl) - [6-chloro-1- (3-diethylaminopropyl) -1H-indol-5-yl] methanone; [1- (2-diethylaminoethyl) -1H-indol-5-yl] - [4- (4-fluoro-benzyl) piperidin-1-yl] methanone; (4-benzyl-piperidin-1-yl) - [1- (3-diethylaminopropyl) -6-methoxy-1H-indol-5-yl] methanone; 5- (4-benzyl-piperidin-1-carbonyl) -1H-indole-3-carboxylic acid (2-aminoethyl) methyl amide; 5- (4-benzyl-piperidin-1-carbonyl) -1H-indole-3-carboxylic acid [2- (3,4-dimethoxy-phenyl) -ethyl] -amide; (4-benzyl-piperidin-1-yl) - (3-diethylaminomethyl-1H-indol-5-yl) methanone; [4- (4-fluoro-benzyl) -piperidin-1-yl] - (6-methoxy-1H-indol-5-yl) methanone; (4-benzyl-piperidin-1-yl) - (1-pyridin-4-yl-1H-indol-5-yl) methanone; and 4- (4-benzyl-piperidin-1-yl) - (4-chloro-2-methyl-1H-indol-5-yl) methanone; or pharmaceutically acceptable salts, or derivatives, or esters, or analogs of any of the preceding compounds.

Of particular interest in the embodiment of the invention is the use of any of the following compounds of SAPK inhibitors or their pharmaceutically acceptable salts, or derivatives, or esters, or analogues thereof:

which is named after IUPAC '[2- (2-chloro-phenyl) -quinazolin-4-yl] -pyridin-4-yl amine. Also suitable for use are any of the following compounds: [2- (2,6-dichlorophenyl) -quinazolin-4-yl] -pyridin-4-yl-amine; pyridin-4-yl- (2-o-tolyl-quinazolin-4-yl) -amine; [2- (2-Bromophenyl) -quinazolin-4-yl] pyridin-4-yl-amine; [2- (2-fluorophenyl) -quinazolin-4-yl] pyridin-4-yl-amine; [2- (2,6-difluorophenyl) -quinazolin-4-yl] pyridin-4-yl-amine; (2-phenyl-quinazolin-4-yl) -pyridin-4-yl-amine; [2- (4-fluorophenyl) -quinazolin-4-yl] -pridin-4-yl-amine; [2- (4-methoxyphenyl) -quinazolin-4-yl] pyridin-4-yl-amine; [2- (3-fluorophenyl) -quinazolin-4-yl] pyridin-4-yl-amine; isopropyl- (2-phenyl-quinazolin-4-yl) -pyridin-4-yl-amine; (4-methoxy-benzyl) - (2-phenyl-quinazolin-4-yl) -pyridin-4-yl-amine; (2-phenyl-quinazolin-4-yl) -pyridin-4-ylmethyl-amine; [2- (4-chloro-phenyl) -quinazolin-4-yl] -pyridin-4-ylmethyl-amine; (2-phenyl-quinazolin-4-yl) -pyridin-3-yl-amine; (2-phenyl-quinazolin-4-yl) -pyridin-2-ylmethyl-amine; (2-phenyl-quinazolin-4-yl) -pyridin-3-ylmethyl-amine; (2-phenyl-quinazolin-4-yl) - (2-pyridin-2-yl-ethyl) -amine; (2-phenyl-quinazolin-4-yl) -pyrimidin-4-yl-amine; (2-phenyl-chi-nazolin-4-yl) -pyrimidin-2-yl-amine; phenyl- (2-phenyl-quinazolin-4-yl) -amine; benzyl- [2- (3-chlorophenyl) -quinazolin-4-yl] -amine; 3- (2-phenyl-quinazolin-4-ylamino) -phenol; 2- (2-phenyl-quinazolin-4-ylamino) -phenol; 4- (2-phenyl-quinazolin-4-ylamino) -phenol; (1H-indol-4-yl) - (2-phenyl-quinazolin-4-yl) -amine; (1H-indol-5-yl) - (2-phenyl-quinazolin-4-yl) -amine; (4-methoxy-phenyl) - (2-phenyl-quinazolin-4-yl) -amine; (3-methoxy-phenyl) - (2-phenyl-quinazolin-4-yl) -amine; (2-methoxyphenyl) - (2-phenyl-quinazolin-4-yl) -amine; 2- [4- (2-phenyl-quinazolin-4-ylamino) phenyl] ethanol; 3- (2-phenyl-quinazolin-4-yl-amino) benzonitrile; (2,5-difluoro-benzyl) - (2-phenyl-quinazolin-4-yl) -amine; [4- (2-butyl) phenyl] - (2-phenyl-quinazolin-4-yl) -amine; N, N-dimethyl-N ' - (2-phenyl-quinazolin-4-yl) -benzene-1,4-diamine; [2- (2-chloro-phenyl) -6,7-dimethoxy-quinazolin-4-yl] pyridin-4-yl-amine; [2- (2-fluoro-phenyl) -6-nitro-quinazolin-4-yl] pyridin-4-yl-amine; 2- (2-fluoro-phenyl) -N4-pyridin-4-yl-quinazolin-4,6-diamine; 2- (2-fluorophenyl) -N4-pyridin-4-yl-quinazolin-4,7-diamine; 2- (2-fluorophenyl) -N6- (3-methoxy-benzyl) -N4-pyridin-4-yl-quinazolin-4,6-diamine; 2- (2-fluorophenyl) -N6- (4-methoxy-benzyl) -N4-pyridin-4-yl-quinazolin-4,6-diamine; N6-isobutyl-2- (2-fluorophenyl) -N4-pyridin-4-yl-quinazolin-4,6-diamine; 2- (2-fluorophenyl) -N6- (4-methylsulfanyl-benzyl) -N4-pyridin-4-yl-quinazolin-4,6-diamine; 4- (4-pyridylamino) -2- (4-chlorophenyl) quinazoline; 2-phenyl-4- (2-pyridylamino) quinazoline; and [2- (2-fluorophenyl) pyrido [2,3-d] pyrimidin-4-yl] pyridin-4-yl amine; or pharmaceutically acceptable salts, or derivatives, or esters, or analogs of any of the preceding compounds.

Another suitable SAPK inhibitor is BIRB796 (1- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -3- [4- (2-morpholin-4-yl-ethoxy) -naphthalen- 1-yl] urea); see US Patent No. 6,319,921. BIRB796 has the following structure:

Also suitable for use are pharmaceutically active derivatives, analogs, esters or salts of BIRB796.

Another suitable SAPK inhibitor is 2 (1H) -quinazolinone, as shown below:

Also suitable for use are pharmaceutically active derivatives, analogs, esters or salts of 2 (1H) -quinazolinone.

Additionally suitable for use is VX-745 (Pharmaceuticals and Kissei Pharmaceutical Co.). It is reported that VX-745 inhibits certain p38 isotypes, including p38 alpha, p38 beta and p38 gamma.

Pirfenidone and its analogues

Pirfenidone (5-methyl-1-phenyl-2- (1H) -pyridone) and specific analogues of pirfenidone can be used to enhance the treatment of the HCV infection mentioned here.

Pirfenidone

Analogs of Pirfenidone

Description of the substituents R ₁ , R ₂ , X

R ₁ : carbocycle (saturated and unsaturated), heterocycle (saturated or unsaturated), alkyls (saturated and unsaturated). For example phenyl, benzyl, pyrimidyl, naphthyl, indolyl, pyrrolyl, furyl, thienyl, imidazolyl, cyclohexyl, piperidyl, pyrrolidyl, morpholinyl, cyclohexenyl, butidienyl and the like.

R ₁ may further include substituents on carbocyclic or heterocyclic moieties with substituents such as halogen, nitro, amino, hydroxyl, alkoxy, carboxyl, cyano, thio, akyl, aryl, heteroalkyl, heteroaryl, and combinations thereof, for example, 4-nitrophenyl, 3 -chlorophenyl, 2,5-dinitrophenyl, 4-methoxyphenyl, 5-methylpyrrolyl, 2,5-dichlorocyclohexyl, guanidinyl-cyclohexyl and the like.

R ₂ : alkyl, carbocycle, aryl, heterocycle, hydroxyl, alkoxy, carboxyl. Examples include: methyl, ethyl, propyl, isopropyl, phenyl, 4-nitrophenyl, thienyl, hydroxyl, methoxy, carboxy, and so on.

X may be any number (from 1 to 3) of substituents on a carbocyclic or heterocyclic ring. Substituents may be the same or different. Substituents may include hydrogen, alkyl, heteroalkyl, aryl, heteroaryl, halogen, nitro, carboxyl, hydroxyl, cyano, amino, thio, alkylamino, haloaryl and the like.

Substituents may be further substituted with 1 to 3 substituents from the group consisting of alkyl, aryl, nitro, alkoxy, hydroxyl and halide groups. Examples include: methyl, 2,3-dimethyl, phenyl, p-tolyl, 4-chlorophenyl, 4-nitrophenyl, 2,5-dichlorophenyl, furyl, thienyl and the like.

Specific examples including such compounds are shown in table 10:

Table 10 IA IIB 5-Methyl-1- (2'-pyridyl) -2- (1H) pyridine, 6-Methyl-1-phenyl-3- (1H) pyridone, 6-Methyl-1-phenyl-2- (1H) pyridone, 5-Methyl-1-p-tolyl-3- (1H) pyridone, 5-Methyl-3-phenyl-1- (2'-thienyl) -2- (1H) pyridone, 5-Methyl-1- (2'-naphthyl) -3- (1H) pyridone, 5-Methyl-1- (2'-naphthyl) -2- (1H) pyridone, 5-Methyl-1-phenyl-3- (1H) pyridone, 5-Methyl-1-p-tolyl-2- (1H) pyridone, 5-Methyl-1- (5'-quinolyl) -3- (1H) pyridone, 5-Methyl-1- (1'-naphthyl) -2- (1H) pyridone, 5-Ethyl-1-phenyl-3- (1H) pyridone, 5-Ethyl-1-phenyl-2- (1H) pyridone, 5-Methyl-1- (4'-methoxyphenyl) -3- (1H) pyridone, 5-Methyl-1- (5'-quinolyl) -2- (1H) pyridone, 4-methyl-1-phenyl-3- (1H) pyridone, 5-Methyl-1- (4'-quinolyl) -2- 5-Methyl-1- (3'-pyridyl) -3- (1H) pyridone, (1H) pyridone, 5-Methyl-1- (4'-pyridyl) -2- (1H) pyridone, 5-Methyl-1- (2'-thienyl) -3- (1H) pyridone, 3-Methyl-1-phenyl-2- (1H) pyridone, 5-Methyl-1- (2'-pyridyl) -3- (1H) pyridone, 5-Methyl-1- (4'-methoxyphenyl) -2- (1H) pyridone, 5-Methyl-1- (2'-quinolyl) -3- (1H) pyridone, 1-Phenyl-2- (1H) pyridone, 1-Phenyl-3- (1H) pyridone, 1,3-Diphenyl-2- (1H) pyridone, 1- (2'-furyl) -5-methyl-3- (1H) pyridone, 1,3-diphenyl-5-methyl-2- (1H) pyridone, 1- (4'-Chlorophenyl) -5-methyl-3- (1H) pyridine. 5-Methyl-1- (3'-trifluoromethylphenyl) -2- (1H) pyridone, 3-Ethyl-1-phenyl-2- (1H) pyridone, 5-Methyl-1- (3'-pyridyl) -2- (1H) pyridone, 5-Methyl-1- (3-nitrophenyl) -2- (1H) pyridone, 3- (4'-chlorophenyl) -5-methyl-1-phenyl-2- (1H) pyridone, 5-Methyl-1- (2'-thienyl) -2- (1H) pyridone, 5-Methyl-1- (2'-thiazolyl) -2- (1H) pyridone, 3,6-Dimethyl-1-phenyl-2- (1H) pyridone, 1- (4'-chlorophenyl) -5-methyl-2- (1H) pyridone, 1- (2'-imidazolyl) -5-methyl-2- (1H) pyridone, 1- (4'-nitrophenyl) -2- (1H) pyridone, 1- (2'-furyl) -5-methyl-2- (1H) pyridone, 1-Phenyl-3- (4'-chlorophenyl) -2- (1H) pyridine.

U.S. Patent Nos. 3,974,281; 3,839,346; 4,042,699; 4,052,509; 5,310,562; 5,518,729; 5,716,632; and 6090822 describe methods for synthesizing and preparing pharmaceutical compositions of pirfenidone and specific analogues of pirfenidone for use in the methods of the present invention.

TNF antagonists

[00913] For use in the present invention, suitable TNF-α antagonists include agents that reduce TNF-α synthesis, agents that block or inhibit TNF-α binding to the TNF-α receptor (TNFR), and agents that block or inhibiting signal transduction mediated by TNFR. Unless otherwise indicated, it will be understood that here, each reference to a “TNF-α antagonist” or “TNF antagonist” means a TNF-α antagonist and not SAPK inhibitors (including pirfenidone and pirfenidone analogues).

[00914] As used herein, the terms "TNF receptor polypeptide" and "TNFR polypeptide" refer to polypeptides that are derivatives of TNFR (of any species) that are capable of binding to TNF. Two different cell surface TNFRs have been described: TNFR type II (or p75 TNFR or TNFR II) and TNFR type I (or p55 TNFR or TNFR I). Mature full-length human p75 TNFR is a glycoprotein with a molecular weight of about 75-80 kilodaltons (kD). Mature full length human p55 TNFR is a glycoprotein with a molecular weight of about 55-60 kD. Representative TNFR polypeptides are derivatives of type I TNFR and / or type II TNFR. Soluble TNFR includes p75 TNFR polypeptide; fusion of p75 TNFR with heterologous fusion partners, for example, the Fc portion of immunoglobulin.

[00915] The TNFR polypeptide may be an intact TNFR or a suitable TNFR fragment. US Pat. No. 5,605,690 provides examples of TNFR polypeptides, including soluble TNFR polypeptides, suitable for use in the present invention. In many embodiments, the TNFR polypeptide comprises the extracellular domain of TNFR. In some embodiments, the TNFR polypeptide is a fusion protein comprising an extracellular domain of TNFR linked to the constant domain of an immunoglobulin molecule. In other embodiments, the TNFR polypeptide is a fusion polypeptide comprising the extracellular p75 domain of TNFR linked to the constant domain of an IgG1 molecule. In some embodiments, when administration to humans is contemplated, the Ig used for the fusion proteins is a human immunoglobulin, for example, human IgG1.

[00916] Monovalent and multivalent forms of TNFR polypeptides can be used in the present invention. Multivalent forms of TNFR polypeptides have more than one TNF binding site. In some embodiments, implementation, TNFR is a bivalent or dimeric form of TNFR. For example, as described in US Pat. No. 5,605,690 and in Mohler et al., 1993, J. Immunol, 151: 1548-1561, a chimeric antibody polypeptide with extracellular TNFR domains substituted for the variable domains of any of the immunoglobulin heavy or light chain, or both may represent the TNFR polypeptide for the present invention. Basically, when such a chimeric TNFR: antibody polypeptide is produced by cells, it forms a bivalent molecule through disulfide bonds between immunoglobulin domains. Such a chimeric TNFR: antibody polypeptide is called TNFR: Fc.

[00917] In one embodiment, the subject process comprises administering an effective amount of a soluble TNFR ENBREL® etanercept. ENBREL® is a dimeric fusion protein consisting of the extracellular ligand binding portion of human 75 kilodaltons (p75) bound to the Fc portion of human IgG1. The Fc component of ENBREL® contains the CH2 domain, the CH3 domain, and the hinge region, but not the CH1 domain of IgG1. ENBREL® is produced in the Chinese Hamster Ovocyte cell expression system of mammals. It consists of 934 amino acids and has an average molecular weight of approximately 150 kilodaltons. Smith et al. (1990) Science 248: 1019-1023; Mohler et al. (1993) J. Immunol 151: 1548-1561; US patent No. 5395760; and US patent No. 5605690.

[00918] Monoclonal antibodies that bind to TNF-α are also suitable for use. Monoclonal antibodies include “humanized” murine monoclonal antibodies; chimeric antibodies; monoclonal antibodies that are at least about 80%, at least about 90%, at least about 95% or 100% human antibodies in the amino acid sequence; and the like. See, for example, WO 90/10077; WO 90/04036; and WO 92/02190. Suitable monoclonal antibodies include antibody fragments such as Fv, F (ab ') ₂ and Fab; synthetic antibodies; artificial antibodies; phage display antibodies; and the like.

[00919] Examples of suitable monoclonal antibodies include infliximab (REMICADE®, Centocor); and adalimumab (HUMIRA ^™ , Abbott) REMICADE® is a chimeric monoclonal antibody to TNF-α that contains about 25% of the mouse amino acid sequence and about 75% of the human amino acid sequence. REMICADE® contains the variable region of the mouse monoclonal anti-TNF-α antibody to the constant region of human IgG1. Elliott et al. (1993) Arthritis Rheum. 36: 1681-1690; Elliott et al. (1994) Lancet 344: 1105-1110; Baert et al. (1999) Gastroenterology 116: 22-28. HUMIRA ^™ is a full length human monoclonal IgG1 antibody that has been identified using phage display technology. Piascik (2003) J. Am. Pharm. Assoc. 43: 327-328.

[00920] Methods for assessing antagonistic activity against TNF are known in the art and are demonstrated here. For example, antagonistic activity against TNF can be assessed using competitive binding assays using cells. In such a study, radioactively labeled TNF was mixed with a TNF antagonist in serial dilution and cells expressing TNFR bound to the cell membrane. Portions of this suspension are centrifuged to separate free and bound TNF and the radioactivity in the free and bound fractions is determined. TNF antagonist activity is evaluated by inhibiting the binding of TNF to cells in the presence of a TNF antagonist.

[00921] As another example, the ability of TNF antagonists to neutralize TNF activity in vitro can be analyzed in a biological study using cells that suggest the presence of TNF cytotoxic activity as target cells. In such a study, target cells cultured with TNF are treated with various amounts of a TNF antagonist and sequentially examined for cytolysis. Antagonistic activity against TNF is evaluated by reducing the cytolysis of target cells induced by TNF in the presence of a TNF antagonist.

TGF-β Antagonists

[00922] TGF-β antagonists suitable for use in the subject treatment method include agents that reduce the level of TGF-β synthesis, agents that block or inhibit TGF-β binding to the TGF-β receptor, and agents that block or inhibit TGF-β receptor mediated signaling. As used herein, the term “TGF-β antagonist” refers to any agent that reduces the level of TGF-β synthesis, any agent that blocks or inhibits the binding of TGF-β to the TGF-β receptor, and any agent that blocks or inhibits signal transmission mediated by the receptor for TGF-β. Unless otherwise indicated, it will be understood here that each reference to a “TGF-R antagonist” means a TGF-β antagonist and not SAPK inhibitors (including pirfenidone and pirfenidone analogs). As used herein, the term “TGF-β” includes any subtype of TGF-β, including TGF-β 1, TGF-β2, and TGF-β3. Suitable TGF-β antagonists include, but are not limited to, antibodies specific for TGF-β (including antibodies specific for a particular subtype of TGF-β; and antibodies that cross-react with two or more subtypes of TGF-β); antibodies to the TGF-β receptor; soluble TGF-β receptor; decorin; and TGF-β signaling inhibiting agents.

[00923] Suitable TGF-β antagonists include antibodies specific for TGF-β. Antibodies specific for TGF-β are known in the art. See, for example, US patents No. 5783185, 5772998, 5674843, 5571714, 5462925 and 5426098; WO 97/13844; and US Patent Publication Nos. 3000064069 and 20030091566. Non-limiting examples of suitable anti-TGF-β antibodies include CAT-152 (lerdelibumab; Tfabio ^TM ; Cambridge Antibody Technology), a human monoclonal antibody to TGF-β2; CAT-192 (metelimumum; Cambridge Antibody Technology), human monoclonal antibody to TGF-β 1; and GC-1008 (Genzyme Corp.), a multispecific human monoclonal antibody to TGF-β1, TGF-β2 and TGF-β3.

[00924] Suitable TGF-β antagonists include soluble TGF-β receptors. Soluble TGF-β receptors lack the majority or all of the transmembrane portion of the natural TGF-β receptor, so that a non-membrane protein still retains binding to TGF-β. Soluble TGF-β receptors include soluble fusion proteins, including a portion of the TGF-β receptor fused in reading frame to a heterologous protein (not to the TGF-β receptor) ("fusion partner"). Non-limiting examples of fusion partners are immunoglobulin Fc, polyhistidine, and the like. Soluble TGF-β receptors have already been described in the art. See, for example, Wang et al. (1999) Thorax 54: 805-812; George et al. (1999) Proc. Natl. Acad. Sci. USA 96: 12719-12724; Muraoka et al. (2002) J. din. Invest. 109: 1551-1559; and Yata et al. (2002) Hepatology 35: 1022-1030.

[00925] TGF-β antagonists include Gleevec ^™ . Gleevec ^™ (also known as STI-571 or CGP57148B), chemically named 4 - [(4-methyl-1-piperazinyl) methyl] -N- [4-methyl-3 - [[4- (3-pyridinyl) - 2-pyrimidinyl] amino-phenyl] benzamide methanesulfonate, commonly known as imatinib mesylate, and sold under the trademark Gleevec ^TM . Gleevec ^TM is 2-phenylaminopyrimidine that targets the ATP binding site of the Bcr-Abl tyrosine kinase kinase domain (see, for example, Druker et al. (1996) Nature Med. 2, 561; and Buchdunger et al., (1993) Proc Natl. Acad. Sci. USA 92: 2558-2562).

In some embodiments of the invention, the agents are pyrimidine derivatives as described in US Pat. 5521184, which are incorporated herein by reference. In these embodiments of the invention, derivatives of M-phenyl-2-pyrimidinamine of formula (I) are of interest:

where R _{9 '} means hydrogen or lower alkyl,

X is oxo, thio, imino, N-lower alkyl imino, hydroxyimino or O-lower alkyl hydroxyimino,

Y is oxygen or an NH group,

k is 0 or 1 and

R ₁₀ means an aliphatic radical having at least 5 carbon atoms, or an aromatic, aromatic-aliphatic, cycloaliphatic, cycloaliphatic-aliphatic, heterocyclic or heterocyclic-aliphatic radical, and the remaining radicals R _{4 '} , R _5' , R _{6 '} , R _{7 '} and R _8' each independently of the other means hydrogen, lower alkyl, which is unsubstituted or substituted by a free or alkylated amino group, piperazinyl, piperidinyl, pyrrolidinyl or morpholinyl, or lower alkanoyl, trifluoromethyl, free a single or esterified oxy group, a free, alkylated or acylated amino group or a free or esterified carboxy group, and salts of such compounds which have at least one salt-forming group.

In these embodiments of the invention: 1-methyl-1H-pyrrolyl mainly means 1-methyl-1H-pyrrol-2-yl or 1-methyl-1H-pyrrol-3-yl.

An amino or amino lower alkyl substituted phenyl R ₁ , where the amino group is free, alkylated or acylated in each case, is phenyl substituted at any desired position (ortho, meta or pair), where the alkylated amino group is predominantly mono or a di-lower alkylamino group, for example dimethylamino, and the lower alkyl moiety of the lower alkylamino group is preferably a linear C ₁ -C ₃ alkyl, preferably such as methyl or ethyl.

The 1H-indolyl bonded to the carbon atom of the five-membered ring is 1H-indol-2-yl or IH-indol-3-yl.

Unsubstituted or substituted by lower alkyl pyridyl bonded to the carbon ring atom is lower substituted or predominantly unsubstituted 2-, or preferably 3- or 4-pyridyl, for example 3-pyridyl, 2-methyl-3-pyridyl, 4-methyl -3-pyridyl or 4-pyridyl. Pyridyl substituted at the nitrogen atom with oxygen is a radical derived from pyridine N-oxide, i.e., N-oxide-pyridyl, for example, M-oxide-4-pyridyl.

The fluoro-substituted lower alkoxy group is lower alkoxy having at least one but preferably several fluorine atoms as a substituent, especially trifluoromethoxy or preferably 1,1,2,2-tetrafluoroethoxy.

When X is oxygen, thio, imino, N-lower alkylimino, hydroxyimino or O-lower alkyl hydroxyimino, the group C = X is, in the above order, the radical C = O, C = S, C = N-H, C = N-lower alkyl, C = N-OH or CN-O-lower alkyl, respectively. X is preferably oxo. k is preferably 0, that is, the Y group is absent. Y, if present, preferably represents an NH group.

The term "lower" in the scope of this text means a radical having up to, including, 7, preferably up to, inclusive, 4, carbon atoms.

Lower alkyl R _{1 '} , R _2' , R _{3 '} and R _9' preferably means methyl or ethyl. The aliphatic radical R ₁₀ having at least 5 carbon atoms, preferably has no more than 22 carbon atoms, basically no more than 10 carbon atoms, and is a substituted or preferably unsubstituted aliphatic radical, that is, substituted or preferably unsubstituted alkynyl, alkenyl or preferably alkyl a radical, such as C ₅ -C ₇ alkyl, for example n-pentyl. The aromatic radical R ₁₀ has up to 20 carbon atoms and is unsubstituted or substituted, for example, in each case unsubstituted or substituted naphthyl, especially such as 2-naphthyl, or preferably phenyl, where the substituents are preferably selected from cyano, unsubstituted or hydroxy, amino - or 4-methyl-piperazinyl-substituted lower alkyl, especially such as methyl, trifluoromethyl, free, ether, esterified, free, alkylated or acylated amino group, and free or etherification Carboxy group ester bound. In the aromatic-aliphatic radical R _{10, the} aromatic part is as defined above, and the aliphatic part is predominantly lower alkyl, especially such as C ₁ -C ₂ alkyl, which is substituted or predominantly unsubstituted, for example benzyl. The cycloaliphatic radical R ₁₀ has mainly up to 30, more preferably up to 20, and most preferably up to 10 carbon atoms, is mono- or polycyclic and is substituted or predominantly unsubstituted, for example by a cycloalkyl radical, mainly a 5- or 6-membered cycloalkyl radical, preferably cyclohexyl. In the cycloaliphatic-aliphatic radical R _{10, the} cycloaliphatic moiety is as defined above and the aliphatic moiety is predominantly lower alkyl, especially, such as C ₁ -C ₂ alkyl, which is substituted or predominantly unsubstituted. The heterocyclic radical R ₁₀ contains mainly up to 20 carbon atoms and is predominantly saturated or unsaturated monocyclic radicals having a 5 or 6 membered ring and 1-3 heteroatoms, which are mainly selected from nitrogen, oxygen and sulfur, especially, for example, thienyl or 2 -, 3- or 4-pyridyl, or a bi- or tri-cyclic radical, in which, for example, one or two benzene radicals are condensed to form said monocyclic radical. In the heterocyclic-aliphatic radical R _{10, the} heterocyclic moiety is as defined above, and the aliphatic moiety is predominantly lower alkyl, especially, such as C ₁ -C ₂ alkyl, which is substituted or predominantly unsubstituted.

The esterified ether group preferably means lower alkoxy. The esterified ester is preferably an oxy group esterified to an ester with an organic carboxylic acid, such as a lower aliphatic acid, or a mineral acid, such as a hydrohalic acid, for example, a lower alkanoyloxy or, preferably, a halogen, such as iodine, bromine or especially fluorine or chlorine.

An alkylated amino group is, for example, a lower alkylamino group, such as methylamino, or a di-lower alkylamino group, such as dimethylamino. The acylated amino group is, for example, a lower alkanoylamino group or a benzoylamino group.

The esterified carboxy group is, for example, lower alkoxycarbonyl, such as methoxycarbonyl.

A substituted phenyl radical may have up to 5 substituents, such as fluorine, but generally, in the case of relatively large substituents, it has from 1 to 3 substituents. As examples of substituted phenyl, 4-chlorophenyl, pentafluorophenyl, 2-carbo-xiphenyl, 2-methoxyphenyl, 4-fluorophenyl, 4-cyanophenyl and 4-methylphenyl can be specifically mentioned.

Salt-forming groups in the compound of formula (I) are groups or radicals having basic or acidic properties. Compounds having at least one basic group or at least one basic radical, for example, a free amino group, a pyrazinyl radical or a pyridyl radical, can form acid addition salts, for example with inorganic acids such as hydrochloric acid, sulfuric acid or phosphoric acid or with suitable organic carboxylic or sulfonic acids, for example aliphatic mono- or di-carboxylic acids, such as trifluoroacetic acid, acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, fumaric acid, hydroxy maleic acid, malic acid, tartaric acid, citric acid or oxalic acid, or amino acids such as arginine or lysine, aromatic carboxylic acids such as benzoic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, salicylic acid, 4-aminosalicylic acid, aromatic-aliphatic carboxylic acids such as mandelic acid or cinnamic acid, heteroaromatic to arboxylic acids, such as nicotinic or isonicotinic acid, aliphatic sulfonic acids, such as methane, ethane or 2-hydroxyethane sulfonic acid, or aromatic sulfonic acids, for example, benzene, p-toluene or naphthalene-2-sulfonic acid . When several basic groups are present, mono- or poly-acid addition salts may form.

Compounds of formula (I) having acidic groups, for example a free carboxyl group in the radical R ₁₀ , can form metal or ammonium salts, such as alkali or alkaline earth metal salts, for example sodium, potassium, magnesium or calcium salts, or ammonium salts with ammonia or suitable organic amines, such as tertiary monoamines, for example triethylamine or tri- (2-hydroxyethyl) amine, or heterocyclic bases, for example N-ethylpiperidine or N, N'-dimethyl-piperazine.

Compounds of formula (I) having both acidic and basic groups can form internal salts. Of particular interest in these embodiments of the invention is a pyrimidine derivative, where R _{1 '} is 3-pyridyl, R _2' , R _{3 '} , R _5' , R _{6 '} and R _8' each is hydrogen, R _{7 '} is methyl and R _{9 '} is a group of formula (II) in which R ₁₀ is hydrogen, X is oxo, k is 0, and R ₁₀ is 4 - [(4-methyl-1-piperazinyl) methyl] phenyl. The mesylate salt of this compound, having the chemical name 4 - [(4-methyl-1-piperazinyl) meptyl] -N- [4-methyl-3 - [[4- (3-pyridinyl) -2-pyrimidinyl] amino-phenyl] benzamide methanesulfonate, commonly known as imatinib mesylate, is sold under the brand name Gleevec (TM).

Endothelin receptor antagonists

Suitable endothelin antagonists for use in the present invention include agents that reduce endothelin synthesis, agents that block or inhibit the binding of endothelin to the endothelin receptor, and agents that block or inhibit endothelin receptor-mediated signal transduction. As used here, the term "endothelin antagonist" means any agent that reduces the level of endothelin synthesis, any agent that blocks or inhibits the binding of endothelin to the endothelin receptor, and any agent that blocks or inhibits endothelin receptor-mediated signal transduction.

In some embodiments of the invention, an endothelin receptor antagonist is selective for endothelin A (ETA) receptors. In some embodiments of the invention, an endothelin receptor antagonist is selective for endothelin B (ETB) receptors. In other embodiments of the invention, an endothelin receptor antagonist is an antagonist of both ETA and ETB receptors.

Specific examples of endothelin antagonists useful in the present invention include, but are not limited to, atrasentan (ABT-627; Abbott Laboratories), Veletri ^TM (tezosentan; Actelion Pharmaceuticals, Ltd.), sitaxentan (ICOS-Texas Biotechnology), entrasentan (GlaxoSmith-Kline ), darusentan (LU1 35252; Myogen) BMS-207940 (Bristol-Myers Squibb), BMS-193884 (Bristol-Myers Squibb), BMS-182874 (Bristol-Myers Squibb), J-104132 (Banyu Pharmaceutical), VML 588 / Ro 61-1790 (Vanguard Medica), T-0115 (Tanabe Seiyaku), TAK-044 (Takeda), BQ-788 (Banyu Pharmaceutical), BQ123, YM-598 (Yamanouchi Pharma), PD 145065 (Parke-Davis), A-127722 (Abbott Laboratories), A-192621 (Abbott Laboratories), A-182086 (Abbott Laboratories), TBC3711 (ICOS-Texas Biotechnology), BSF208075 (Myogen), S-0139 (Shionogi), TBC2576 (Texas Biotechnology), TBC3214 (Texas Biot echnology), PD156707 (Parke-Davis), PD180988 (Parke-Davis), ABT-546 (Abbott Laboratories), ABT-627 (Abbott Laboratories), SB247083 (GlaxoSmithKline), SB 209670 (GlaxoSmithKline); and endothelin receptor antagonists described in the prior art, for example, Davenport and Battistini (2002) Clinical Science 103: 15-35, Wu-Wong et al. (2002) Clinical Science 103: 1075-1115, and Luescher and Barton (2000) Circulation 102: 2434-2440.

A suitable endothelin receptor antagonist is TRACLEER ^™ (bosentan; manufactured by Actelion Pharmaceuticals, Ltd.). TRACLEER ^TM is an orally active double antagonist of the endothelin receptor and blocks the binding of endothelin to both of its receptors, endothelin receptor A and endothelin receptor B. TRACLEER ^TM belongs to the class of highly substituted pyrimidine derivatives that do not have chiral centers. It has the chemical name 4-tert-butyl-N- [6- (2-hydroxy-ethoxy) -5- (2-methoxy-phenoxy) - [2,2 '] - bipyrimidin-4-yl] benzene sulfonamide monohydrate and corresponds to the following structural formula:

In some embodiments of the invention, treatment with TRACLEER ^™ is an oral administration of a dose of 62.5 mg for 4 weeks, followed by a dose of 125 mg orally.

N-Acetylcysteine (NAC)

N-acetylcysteine (NAC) is a stable form of sulfamic acid L-cysteine. NAC is an antioxidant that removes H ₂ O ₂ and other radicals. It is a precursor to glutathione (the main antioxidant), providing a cysteine substrate in the synthesis of glutathione. NAC is a commercially available approved food supplement or food product. Suitable NAC products include NAC nutrition supplements manufactured by Source Naturals (1000 mg tablets), Biochem (750 mg tablets), Twinlab (600 mg tablets), Nutricology / Allergy Research Group (500 mg tablets) and the like. Such products can be purchased at the lowest cost from health food stores and nutritional supplements and from retailers such as the General Nutrion Corporation (GNC).

Thymosin-α

Thymosin-α (Zadaxin ^TM; provided by SciClone Pharmaceuticals, Inc., San Mateo, CA) is a synthetic form of thymosin alpha 1, a hormone involved in the circulation and produced by the thymus gland. Thymosin-α increases the activity of T cells and NK cells. Zadaxin ^™ , manufactured for subcutaneous injection, is a purified sterile lyophilized preparation of chemically synthesized alpha thymosin 1 identical to human alpha thymosin 1. Alpha-thymosin 1 is an acetylated polypeptide with the following sequence: Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ue-Thr-Thr-Lys-Asp-Leu-Lys-Glu- Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH (SEQ ID NO: 103) and having a molecular weight of 3.108 daltons. The lyophilized preparation contains 1.6 mg of synthetic alpha-thymosin, 50 mg of mannitol and sodium phosphate buffer to adjust the pH to 6.8.

Ribavirin

Ribavirin, 1-β-D-ribofuranosyl-1H-1,2,4-troiazole-3-carboxamide available from ICN Pharmaceuticals, Inc., Costa Mesa, Calif, is described in the Merck Index under compound No. 8199, Eleventh Edition. Its preparation and manufacture of dosage forms are described in US Pat. No. 4,211,771. The invention also contemplates the use of ribavirin derivatives (see, for example, US Pat. No. 6,277,830). Ribavirin can be administered orally in capsule or tablet form, or in the same or different administration form and in the same or different way as IFN-α (or PEGLated or non-PEGlated form). Obviously, other types of administration of both drugs are possible if they are available, such as nasal injection, transdermal administration, suppository administration, delayed release dosage form, etc. Any form of administration will work as long as the proper dosage is delivered without destroying the active ingredient.

Ribavirin is generally administered in an amount of from about 400 mg to about 1200 mg, from about 600 mg to about 1000 mg, from about 700 mg to about 900 mg per day. In some embodiments of the invention, ribavirin is administered throughout the course of therapy with pegylated IFN-α or non-pegylated IFN-α. In other embodiments of the invention, ribavirin is administered only during the first period of time. However, in other embodiments of the invention, ribavirin is administered only during the second period of time.

Levovirin

Levovirin is the L-enantiomer of ribavirin and has the property of enhancing the ThI immune response against the Th2 immune response. Levovirin is manufactured by ICN Pharmaceuticals. Levovirin has the following structure:

Viramidine

Viramidine is a 3-carboxamidine derivative of ribavirin and acts as a prodrug of ribavirin. It is effectively converted to ribavirin by adenosine deaminase.

Viramidine has the following structure:

Nucleoside analogues

Nucleoside analogues that are suitable for use in the described method of treatment include, but are not limited to, ribavirin, levovirin, viramidine, isatoribine, L-ribofuranosyl nucleoside described in US patent No. 5559101 and covered by formula I of US patent No. 5559101 (for example, 1 β-L-ribofuranosyl uracil, 1-β-L-ribofuranosyl-5-fluorouracil, 1-β-L-ribofuranosylcytosine, 9-β-L-ribofuranosyladenine, 9-β-L-ribofuranosylhypoxanthin, 9-β-L-ribofuranosin 9-β-L-ribofuranosyl-6-thioguanine, 2-amino-α-L-ribofuran1 [1 ', 2': 4,5] oxazoline, O ^2, O ² -anhydro-1-α-L-ribofuranose luratsil, 1-α-L-ribofuranosyl-uracil, 1- (2,3,5-tri-O-benzoyl-α-ribofuranosyl) -4-thiouracil, 1-α-L-ribofuranosyl-cytosine, 1-α-L- ribofuranosyl-4-thiouracil, 1-α-L-ribofuranosyl-5-fluorouracil, 2-amino-β-L-arabinofurano [1 ', 2': 4,5] oxazoline, O ² , O ² -anhydro β-L-arabinofuranosyl uracil, 2'-deoxy-β-L-uridine, 3'5'-di-O-benzoyl-2'-deoxy-4-thio-β-L-uridine, 2'-deoxy-β- L-cytidine, 2'-deoxy-β-L-4-thiouridine, 2'-deoxy-β-L-thymidine, 2'-deoxy-β-L-5-fluoruridine, 2 ', 3'-dideoxy-β -L-uridine, 2'-deoxy-β-L-5-fluoruridine and 2'-deoxy-β-L-inosine); the compound described in US patent No. 6423695 and covered by the formula I of US patent No. 6423695; the compound described in US patent publication No. 2002/0058635 and covered by formula 1 of US patent publication No. 2002/0058635; nucleoside analogue described in WO 01/90121 A2 (Idenix); a nucleoside analogue described in WO 02/069903 A2 (Biocryst Pharmaceuticals Inc.); the nucleoside analogue described in WO 02/057287 A2 or WO 02/057425 A2 (both Merck / Isis); etc.

NS3 HCV Inhibitors

Suitable inhibitors of non-structural protein-3 (NS3) HCV include, but are not limited to, the tri-peptide described in US patent No. 6642204, 6534523, 6420380, 6410531, 6329417, 6329379 and 6323180 (Boehringer-Ingelheim); the compound described in US patent No. 6143715 (Boeh-ringer-Ingelheim); the macrocyclic compound described in US Pat. No. 6,608,027 (Boehringer-Ingelheim); the NS3 inhibitor described in US patent No. 6617309, 6608067 and 6265380 (Vertex Pharmaceuticals); the azapeptide compound described in US Pat. No. 6,624,290 (Sobering); the compound described in US patent No. 5990276 (Schering); the compound described by Pause et al. (2003) in J. Biol. Chem. 278: 20374-20380; NS3 inhibitor BILN 2061 (Boehringer-Ingelheim; Lamarre et al. (2002) Hepatology 36: 3 01 A; and Lamarre et al. (Oct. 26, 2003) Nature doi: 10.1038 / nature02099); NS3 inhibitor VX-950 (Vertex Pharmaceuticals; Kwong et al. (Oct. 24-28, 2003) 54th Ann. Meeting AASLD); NS3 SCH6 inhibitor (Abib et al. (October 24-28, 2003) Abstract 137. Program and Abstracts of the 54th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD). October 24-28, 2003. Boston, MA.); any NS3 protease inhibitors disclosed in WO 99/07733, WO 99/07734, WO 00/09558, WO 00/09543, WO 00/59929 or WO 02/060926 (for example, those described in WO 02/060926 in the table on pages 224 -226 compounds 2, 3, 5, 6, 8, 10, 11, 18, 19, 29, 30, 31, 32, 33, 37, 38, 55, 59, 71, 91, 103, 104, 105, 112 113, 114, 115, 116, 120, 122, 123, 124, 125, 126 and 127); and an NS3 protease inhibitor described in any US patent publication No. 2003019067, 20030187018 and 20030186895; etc.

Of particular interest in many embodiments of the present invention are NS3 inhibitors, which are specific NS3 inhibitors, for example, NS3 inhibitors that inhibit the activity of the NS3 serine protease, but which do not show significant inhibitory activity against other serine proteases, such as human leukocyte elastase, elastase pig pancreas or bull chymotrypsin for pancreas, or cysteine proteases such as human cathepsin B.

Of particular interest in some embodiments of the present invention are NS3 inhibitors that inhibit the helicase activity of non-structural protein-4 (NS4) HCV and which do not show significant inhibitory activity against other serine proteases, such as human leukocyte elastase, porcine pancreatic elastase or bovine pancreas chymotrypsin, or cysteine proteases such as human liver cathepsin B.

NS5B Inhibitors

Suitable non-structural protein-5 (NS5) inhibitors (NS5; RNA-dependent RNA polymerase) of HCV include, but are not limited to, the compound described in US Pat. No. 6,479,508 (Boehringer-Ingelheim); the compound disclosed in any of International Patent Applications No. PCT / CA02 / 01127, PCT / CA02 / 01128 and PCT / CA02 / 01129, filed July 18, 2002, Applicant Boehringer Ingelheim; the compound described in US patent No. 6440985 (ViroPharma); the compound disclosed in WO 01/47883, for example, JTK-003 (Japan Tobacco); dinucleotide analogue described by Zhong et al. (2003) Antimicrob. Agents Chemother. 47: 2674-2681; the benzothiadiazine compound described by Dhanak et al. (2002) in J. Biol Chem. 277 (41): 3,8322-7; an NS5B inhibitor disclosed in WO 02/100846 A1 or WO 02/100851 A2 (both Shire); an NS5B inhibitor disclosed in WO 01/85172 A1 or WO 02/098424 A1 (both Glaxo SmithKline); an NS5B inhibitor described in WO 00/06529 or WO 02/06246 A1 (both Merck); an NS5B inhibitor described in WO 03/000254 (Japan Tobacco); an NS5B inhibitor disclosed in EP 1256628 A2 (Agouron); JTK-002 (Japan Tobacco); JTK-109 (Japan Tobacco); etc.

Of particular interest in many embodiments of the present invention are NS5 inhibitors, which are specific NS5 inhibitors, for example, NS5 inhibitors that inhibit the RNA-dependent RNA polymerase of NS5 and which have insufficient inhibitory effect on other RNA-dependent RNA polymerases and on DNA-dependent RNA polymerases.

Alpha Glucosidase Inhibitors

Alpha glucosidase inhibitors are a class of oral medications for the treatment of type 2 diabetes, which lower the absorption of carbohydrates in the intestines, which slows the increase in blood glucose levels throughout the day, especially after meals, in patients with type 2 diabetes. Suitable alpha-glucosidase inhibitors for use in the combination treatment described herein include, but are not limited to, n- (n-nonyl) -deoxyhalactonojirimycin (n, n-DGJ); N-nonyl-deoxynojirimycin (N-nonyl-DNJ); N-butyl-deoxynojirimycin (NB-DNJ); 1-deo-xyno-jirimycin (DNM); perbutylated-N-butyl-1-deoxynogyromycin (p-N-butyl-DNJ); and 6-O-butanoyl castanospermine; etc.

Additional antiviral therapeutic agents

Additional antiviral therapeutic agents that may be administered in the combination treatment described herein include, but are not limited to, alpha glucosidase inhibitors; Inosine Monophosphate Dehydrogenase Inhibitors (IMPDH); ribozymes that are complementary to viral nucleotide sequences; antisense RNA inhibitors; etc.

Alpha Glucosidase Inhibitors

Alpha glucosidase inhibitors are a class of oral medications for the treatment of type 2 diabetes, which lower the absorption of carbohydrates in the intestines, which slows the increase in blood glucose levels throughout the day, especially after meals, in patients with type 2 diabetes. Suitable alpha-glucosidase inhibitors for use in the combination treatment described herein include, but are not limited to, n- (n-nonyl) -deoxyhalactonojirimycin (n, n-DGJ); N-nonyl-deoxynojirimycin (N-nonyl-DNJ); N-butyl-deoxynojirimycin (NB-DNJ); 1-deoxyno-jirimycin (DNM); perbutylated-N-butyl-1-deoxynogyromycin (p-N-butyl-DNJ); and 6-O-butanoyl castanospermine; etc.

IMPDH Inhibitors

Suitable IMPDH inhibitors for use in the described combination treatment include, but are not limited to, VX-497 (tetrahydrofuran-3-yl (5) -N-3- [3- (3-methoxy-4-oxazol-5- ester il-phenyl) -ureido] benzylcarbamic acid); Vertex Pharmaceuticals; see, for example, Markland et al. (2000) Antimicrob. Agents Chemother. 44: 859-866); ribavirin; levovirin (Ribapharm; see, for example, Watson (2002) Curr Opin Investig Drugs 3 (5): 680-3); Viramidine (Ribapharm); etc.

Ribozyme and antisense agents

Ribozyme and antisense antiviral agents that are suitable for use in the described combination treatment method include, but are not limited to, ISIS 14803 (ISIS Pharmaceuticals / Elan Corporation; see, for example, Witherell (2001) Curr Opin Investig Drugs. 2 (11): 1523-9); Heptazyme ^TM; etc.

Side Effects

In some embodiments of the invention, the combination treatment comprises administering a palliative agent (for example, an agent that reduces the discomfort caused by a therapeutic agent in a patient) or another agent for preventing, treating, or reducing a side effect of the therapeutic agent. Such agents are also referred to as “side effect control agents”.

Suitable side effect control agents include those that are effective in treating pain; agents that reduce gastrointestinal discomfort; painkillers, anti-inflammatory, antipsychotic, antineuropathic, sedative and hematopoietic agents. In addition, the invention contemplates the use of any substance that alleviates the condition of patients suffering from pain or from any other side effect during the treatment method described in the invention. Typical palliative agents include acetaminophen, ibuprofen, and other NSAIDs, H2 blockers, and antacids.

Painkillers that can be used to relieve pain in the methods of the present invention include non-narcotic painkillers such as non-steroidal anti-inflammatory drugs (NSAIDs) acetaminophen, salicylate, acetylsalicylic acid (aspirin, diflunisal), ibuprofen, Motrin, Nrosrosin, Nrosrosin, Nrosrosin, Trisin indomethacin, glucamethacin, acemetacin, sulindac, naproxen, piroxicam, diclofenac, benoxaprofen, ketoprofen, oxaprozin, etodolac, ketorolac tromethamine, ketorolac, nabumeton and the like and mixtures of yx or more of said agents above.

Other suitable painkillers include fentanyl, buprenorphine, codeine sulfate, morphine hydrochloride, codeine, hydromorphone (Dilaudide), levorphanol (Levo-Dromoran), methadone (Dolofin), morphine, oxycodone (Percodan) and oxymorphone (Numorphan). Also suitable are benzodiazepines, including, but not limited to, flurazepam (Dalman), diazepam (Valium) and Versed, etc.

Anti-inflammatory drugs

Suitable anti-inflammatory drugs include, but are not limited to, steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs.

Suitable steroidal anti-inflammatory drugs include, but are not limited to, hydrocortisone, hydroxyltriamcinolone, alpha-methyl dexamethasone, dexamethasone phosphate, beclomethasone dipropionate, clobetasone valerate, desonide, deoxymethasone, deoxycorticosterone done acetone dialodenone done acetone done acetone dano-acetone-di-acetone-diafluoroacetamide fludrocortisone, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortin butyl ether, fluocortolone, flupredniden (fluprednilidene) acetate, flurandr nolon, haltsinonid, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, konizon, kortodokson, flutsetonid, fludrocortisone, diflurozon diacetate, fluradrenolon acetonide, medrysone, amtsinafel, amcinafide, betamethasone and its esters, chloroprednisone, hlorprednizon acetate klokortelon, klestsinolon, dihlorizon , difluprednat, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentyl propionate, hydrocortamate, meprednisone, parametasone, prednisolone, prednisone, b eclomethasone dipropionate, triamcinolone and mixtures of two or more of the above.

Suitable non-steroidal anti-inflammatory drugs include, but are not limited to, 1) oxicam, such as piroxicam, isoxicam, tenoxicam and sudoxicam; 2) salicylates, such as aspirin, disalcid, benorylate, trilizate, safaprine, solprin, diflunisal and fendosal; 3) acetic acid derivatives such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, flurofenac, thiopinac, zidomethacin, asemethacin, fentiazac, zomepirakt, clidanac, oxepinac and felbinak; 4) phenamates such as mefenamic, meclofenamic, flufenamic, niflumic and tolfenamic acids; 5) derivatives of propionic acid, such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, phenoprofen, fenbufen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, thioxaprofen, suprofen and tyminopen; and 6) pyrazoles, such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone and trimethasone .; mixtures of these non-steroidal anti-inflammatory drugs may also be used, as well as their pharmaceutically acceptable salts and esters.

Suitable anti-inflammatory drugs include, but are not limited to, Alclofenac; Alclomethasone dipropionate; Algeston Acetonide; Alpha amylase; Amcinafal; Amcinafid; Amfenac Sodium; Amiprilosis hydrochloride; Anakinra Anirolac; Anitrazafen; Apazon; Balsalazide disodium; Bendazak; Benoxaprofen; Benzidamine hydrochloride; Bromeline; Broperamol; Budesonide; Carprofen; Cycloprofen; Cintazone; Cliprofen; Clobetasol propionate; Clobetasone butyrate; Klopirak; Clotikazone propionate; Cormetasone Acetate; Cortodoxone; Deflazacort; Desonide; Desoxymethasone; Dexamethasone dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diflorazon diactate; Diflumidone Nitrium; Diflunisal; Difluprednat; Diffalon; Dimethyl sulfoxide; Drocinonide; Etdrizone; Enlimomab; Enolikam sodium; Epirizole; Etodolac; Etofenamate; Felbinak; Phenamol; Fenbufen; Fenclofenac; Fenclorek; Fendozal; Fenpipalon; Fentyazak; Flazalon; Fluazacort; Flufenamic acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin meglumin; Fluocortin butyl; Fluorometholone Acetate; Fluoquasone; Flurbiprofen; Fluuretophen; Fluticasone propionate; Furaprofen; Furobufen; Galcinonide; Halobetazole propionate; Halopredone acetate; Ibufenac; Ibuprofen; Ibuprofen aluminum; Ibuprofen piconol; Ilonidap; Indomethacin; Indomethacin sodium; Indoprofen; Indoxol; Intrazole; Isoflupredone acetate; Isoxepack; Isoxicam; Ketoprofen; Lofemizole hydrochloride; Lornoxicam; Loteprednol etabonate; Meclofenamate sodium; Meclofenamic acid; Meclarizon Dibutyrate; Mefenamic acid; Mesalamine; Mezeklazone; Methylprednisolone suleptanate; Morniflumat; Nabumeton; Naproxen; Naproxen sodium; Naproxol; Nimazon; Olsalazine sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone; Paraniline hydrochloride; Pentosan Sodium Polysulfate; Fenbutazone sodium glycerate; Pirfenidone; Piroxicam; Piroxicam cinnamate; Piroxicam olamine; Pirprofen; Prednaznat; Prifelon; Procuazone; Proxazole; Proxazole citrate; Rimexolone; Romazarite; Salcolex; Salnatsedin; Salsalat; Sanguinarium chloride; Seclason; Sermethacin; Sudoxicam Sulindak; Suprofen; Talmetacin; Talliflumat; Talosalat; Tebufelon; Tenidap; Tenadap Sodium; Tenoxicam; Tesikam; Tesimide; Tetridamine; Tiopinac; Thixocortol pivalate; Tolmethine; Tolmethine sodium; Triclonide; Triflumidate; Zidomethacin; Zomepirac sodium.

Antipsychotic and antineuropathic drugs that can be used to alleviate the side psychiatric effect in the methods of the present invention include any all selective serotonin receptor inhibitors (SSRIs) and other antidepressants, anxiolytics (e.g. alprazolam), etc. Antidepressants include, but are not limited to, serotonin reuptake inhibitors such as Celexa ^® , Desyrel ^® , Effexor ^® , Luvox ^® , Paxil ^® , Prozac ^® , Zoloft ^® and Serzone ^® ; tricycles such as Adapin ^® , Anafrinil ^® , Elavil ^® , Janimmine ^® , Ludiomil ^® , Pamelor ^® , Tofranil ^® , Vivactil ^® , Sinequan ^® and Surmontil ^® ; monoamine oxidase inhibitors such as Eldepryl ^® , Marplan ^® , Nardil ^® and Pamate ^® . Anti-anxiolytic agents include, but are not limited to, azaspirones such as BuSpar ^® , benzidiazepines such as Ativan ^® , Librium ^® , Tranxene ^® , Centrax ^® , Klonopin ^® , Paxipam ^® , Serax ^® , Valium ^® and Xanax ^® ; and beta-blockers such as Inderal ^® and Tenormin ^®.

Agents that reduce gastrointestinal discomfort, such as nausea, diarrhea, gastrointestinal cramping and the like, are acceptable palliative agents for use in the combination therapy of the present invention. Suitable agents include, but are not limited to, antiemetic, antidiarrheal agents, H2 blockers, antacids, and the like.

Suitable H2 blockers (type 2 histamine receptor antagonists) that are suitable for use as a palliative in the methods of treatment described herein include, but are not limited to, cimetidine (e.g. Tagamet, Peptol, Nucimet, apo-cimetidine, non-cimetidine); Ranitidine (e.g., Zantak, Nuranit, Novo-randin and apo-ranitidine); and Famotidine (Peptsid, Apo-Famotidine and Novo-Famotidine).

Suitable antacids include, but are not limited to, aluminum hydroxide and magnesium hydroxide (Maalox, Mylanta ^®); aluminum carbonate gel (Basajel ^® ); aluminum hydroxide (Amphojel ^® , AltemaGEL ^® ); calcium carbonate (Turns ^® , Titralac ^® ); magnesium hydroxide and sodium bicarbonate.

Antiemetics include, but are not limited to, 5-hydroxytryptophan-3 (5HT3) inhibitors; corticosteroids such as dexamethasone and methylprednisolone; Marinol ^® (dronabinol); prochlorperazine; Benzidiazepines; promethazine; and metoclopramide cisapride; Alosetron hydrochloride; Batanopride hydrochloride; Bemesetron; Benzquinamide; Chlorpromazine; Chlorpromazine hydrochloride; Kleboprid; Cyclizine hydrochloride; Dimenhydrinate; Diphenidol; Diphenidol hydrochloride; Diphenidol pamoate; Dolasetron mesylate; Domperidone; Dronabinol; Floodorex; Flumeridone; Galdansetron hydrochloride; Granisetron; Granisetron hydrochloride; Lusetron Mesylate; Meclizine hydrochloride; Metoclopromide hydrochloride; Metopimazine; Ondansetron hydrochloride; Pankoprid; Prochlorperazine; Prochlorperazine Edisilate; Prochlorperazine maleate; Promethazine hydrochloride; Thiethylpyrazine; Thiethylpyrazine Malate; Thiethylpyrazine maleate; Trimethobenzamide hydrochloride; Zakoprid hydrochloride.

Antidiarrheal agents include, but are not limited to, Rolgamidine, Diphenoxylate Hydrochloride (Lomotil), Metronidazole (Flagyl), Methylprednisolone (Medrol), Sulfasalazine (Azulfinide) and the like.

Suitable hematopoietic agents that can be used to prevent or restore a reduced hemocyte count in the methods of the present invention include erythropoietins such as EPOGEN ^® epoetin alfa, granulocyte colony stimulating factors (G-CSFs) such as NEUPOGEN ^® filgrastim, factors stimulating granulocyte colonies macrophages (GM-CSFs), thrombopoietins, etc.

METHODS OF TREATMENT

[00996] The present invention relates to a method for treating a disease, disorder or pathological condition of a patient. In one aspect, the method of treating a patient uses an oral pharmaceutical composition comprising a known hyperglycosylated, protease-resistant, polypeptide variant of the parent therapeutic protein, said known protease-resistant polypeptide variant, containing at least one mutant protease cleavage site instead of the native site protease cleavage, which is present in the parent therapeutic protein; and including: i) a carbohydrate moiety covalently attached to at least one non-native glycosylation site that is not present in the parent therapeutic protein; or ii) a carbohydrate moiety covalently attached to at least one native glycosylation site that is present but not glycosylated in the parent therapeutic protein. The oral pharmaceutical composition is administered orally to the patient in an amount in which the patient receives the first number of moles of the known hyperglycosylated, protease-resistant, variant polypeptide in the first dosage interval. The first number of moles of the known hyperglycosylated, resistant to the action of proteases variant exceeds the second number of moles of the parent therapeutic protein in the parenteral pharmaceutical composition. A parenteral pharmaceutical composition is an immediate release formulation suitable for subcutaneous bolus injection, and the parent polypeptide has been proven to be effective in treating a patient's disease, disorder or pathological condition by subcutaneous bolus administration to a patient in a parenteral pharmaceutical composition in an amount in which the patient receives a second number moles of the parent therapeutic protein in the second dosing interval. In the present method of treating a patient’s disease, the method includes orally administering to the patient an oral pharmaceutical composition in an amount in which the patient receives the first number of moles of the known hyperglycosylated variant of the protease resistant polypeptide in the first dosage interval, the same as the second dosage interval, or shorter.

[00997] Thus, a known hyperglycosylated, protease-resistant, variant polypeptide is effective in treating a disease, disorder or pathological condition in a patient when orally administered to a patient at a dose of the first number of moles in the first dosing interval during the required treatment period.

[00998] In another aspect, the invention relates to a modification of the above method for treating a patient’s disease, disorder or pathological condition, wherein an oral pharmaceutical composition comprising a known hyperglycosylated, protease-resistant, polypeptide variant is administered orally to a patient in a first dose with a first dosing frequency, where the parenteral pharmaceutical composition has been proven to be effective in treating a patient’s disease when administered to the patient by subcutaneous bolus injection of a second doses of the parent treatment protein with a second dosing frequency, where the first dose of the known hyperglycosylated, protease-resistant protease variant of the polypeptide in moles per kilogram of the patient’s body weight exceeds the second dose of the parent treatment protein in moles per kilogram of the patient’s body weight, while the first and second doses are calculated on the body weight of the same patient, and where when the patient is orally administered the first dose of a known glycosylated, protease-resistant, variant polypeptide, all is known th hyperglycosylated resistant to protease polypeptide variant in the first dose is released over a period of no longer than the time period between doses in the second dosing frequency. In some embodiments, the parenteral pharmaceutical composition is proven to be effective in treating a disease, disorder or pathological condition in a patient when administered to the patient at a dose, based on body weight, of the parent therapeutic protein in a second dosage interval, i.e. the second dose is a dose based on body weight, and the parenteral pharmaceutical composition is in a form that allows dose selection based on body weight. In some of the above embodiments, the first dose is a dose based on body weight of a known hyperglycosylated, protease-resistant, variant polypeptide, and the oral pharmaceutical composition is in a form that allows dose selection based on body weight.

[00999] In another aspect, the invention relates to a modification of the above method for treating a patient’s disease, disorder or pathological condition, in which an oral pharmaceutical composition comprising a known hyperglycosylated, protease-resistant, variant polypeptide is administered orally to a patient in a first dose with a first dosing frequency, moreover, it is proved that the parenteral pharmaceutical composition is effective in the treatment of a disease, disorder or pathological condition of a patient administering to this patient a subcutaneous bolus injection of a second dose of the parent treatment protein with a second dosing frequency, where the first dose of the known hyperglycosylated, protease-resistant, variant polypeptide in moles per kilogram of the patient’s body weight exceeds the second dose in moles per kilogram of the patient’s body parent protein, when the first and second doses are calculated for the body weight of the same patient, and where the period of time between doses at the first dosing frequency is the same or shorter than the per time between doses at the second dosing frequency. In some embodiments, the parenteral pharmaceutical composition has been proven to be effective in treating a patient’s disease, disorder or pathological condition by administering to the patient a parental treatment protein in a dose calculated on a body weight basis in a second dosage interval, i.e. the second dose is a dose depending on body weight, and the parenteral pharmaceutical composition is in a form that allows dosing based on body weight. In some of the embodiments set forth above, the first dose is determined based on body weight, and the oral pharmaceutical composition is in a form allowing for such a dosage based on body weight.

[001000] The choice of a hyperglycosylated, protease-resistant protease variant of the polypeptide will partially depend on the disease, disorder or pathological condition being treated. As noted above, the desired hyperglycosylated, protease-resistant, variant polypeptide is effective for treating a disease, disorder or pathological condition that can be treated with the parent therapeutic protein. The following are non-limiting examples.

Treatment Methods Using IFN-α

[001001] In one aspect, wherein a known hyperglycosylated, protease-resistant protease, a polypeptide variant is a known hyperglycosylated, protease-resistant, IFN-α, the subject process relates to administering to a subject in need thereof a therapeutically effective amount of a known hyperglycosylated, stable to the action of proteases, IFN-α in the treatment of a viral infection, for example, an infection caused by hepatitis C virus (HCV). In some embodiments, the implementation of this method mainly includes oral administration to an individual in need of a known hyperglycosylated, protease-resistant, variant of the parent IFN-α 2 polypeptide in a first dose with a first dosing frequency, at least also or more often than at the second dosing frequency, the effectiveness of which has been confirmed in the treatment of HCV infection, including the administration to the individual in need of this parenteral composition of parent IFN-α 2 in the second dose with a second dosing frequency, where the first dose contains the first number of moles of the hyperglycosylated form resistant to protease, the polypeptide variant, which is greater than the second number of moles of the parent IFN-α 2 in the second dose.

[001002] In one non-limiting example, parental IFN-α 2 is proven to be effective in treating HCV infection in a method comprising administering to a patient in need thereof 3 million units (or 15 micrograms) of IFN-α 2 by subcutaneous bolus injection three times a week for 48 weeks. In some of these embodiments, a known, protease-resistant or protease-resistant hyperglycosylated variant of the parent IFN-α 2 polypeptide may be selected, for example, from the group of glycopeptide variants [D99N] IFN-α 2a, [D99N, D105N] IFN-α 2a, [D99N] IFN-α 2b and [D99N, D105N] IFN-α 2b (where amino acid numbering is shown in Figure 24); where this option further comprises one or more single amino acid substitutions at one or more predetermined positions corresponding to any of the amino acid positions: 41, 58, 78, 107, 117, 125, 133 and 159 (where the numbering of amino acids is shown in Figure 2), or any mutation shown in Table 1, such that the variant contains at least one mutant protease cleavage site instead of the native protease cleavage site located in the parent IFN-α 2. This variant is administered orally to the patient in a first dose containing ne the number of moles of this variant with the first dosing frequency, where the first number of moles exceeds the number of moles of the parent IFN-α 2, in 3 million units (or 15 micrograms) of the parent IFN-α 2, and where the first dosage frequency is at least three times a week. Alternatively, the first dosing frequency is four times a week, five times a week, six times a week, once a day, twice a day, or three times a day.

[001003] In some embodiments, the invention relates to any of the foregoing methods for treating HCV infection using a known, protease-resistant or protease-resistant hyperglycosylated variant IFN-α 2 polypeptide, in which a known protease-resistant or resistant to the action of proteases, the hyperglycosylated version of the polypeptide contains any of the carrier peptides described in Table 9 above, in covalent or non-covalent association with the desired variant m polypeptide. In some of these embodiments, a known, protease-resistant, or protease-resistant hyperglycosylated version of the polypeptide comprises any such carrier peptide in direct or indirect covalent association with the desired polypeptide, including, without limitation, any such peptide - a carrier fused to the N-terminus of the desired polypeptide variant.

[001004] In another non-limiting example, parental IFN-α 2 is proved to be effective in treating HCV infection in a method comprising administering to a patient in need thereof 15 micrograms (or 8.0 × 10 ^-10 mol) of IFN-α 2 by subcutaneous bolus injection three times a week for 48 weeks. In some of these embodiments, a known, protease-resistant or protease-resistant hyperglycosylated variant of the parent IFN-α 2 polypeptide may be selected, for example, from the glycopeptide variant group [D99N] IFN-α 2a, [D99N, D105N] IFN-α 2a, [D99N] IFN-α 2b and [D99N, D105N] IFN-α 2b (where amino acid numbering is shown in Figure 24); where this option further comprises one or more single amino acid substitutions at one or more predetermined positions corresponding to any of the amino acid positions: 41, 58, 78, 107,117, 125, 133 and 159 (where the numbering of amino acids is shown in Figure 2), or one of mutations shown in Table 1, such that the variant contains at least one mutant protease cleavage site instead of the native protease cleavage site located in the parent IFN-α 2; and this option is administered orally to the patient in a first dose containing the first number of moles of this option with a first dosing frequency, where the first number of moles exceeds 8.0 × 10 ^-10 mol, or at least 1.6 × 10 ^-9 mol, or at least about 2.4 × 10 ^-9 mol, or at least about 3.2 × 10 ^-9 mol, or at least about 4.0 × 10 ^-9 mol, or at least about 4.8 × 10 ^-9 mol or at least about 5,6 × 10 ^-9 mol or at least about 6,4 × 10 ^-9 mol or at least about 7,2 × 10 ^-9 mol or at at least about 8.0 × 10 ⁻⁹ mol, and whether at least about 8.0 x 10 ^-8 mol, and where the first dosing frequency is at least three times a week. Alternatively, the first dosing frequency is four times a week, five times a week, six times a week, once a day, twice a day, or three times a day.

[001005] In another aspect, where a known, protease-resistant, or protease-resistant, hyperglycosylated variant of the polypeptide is a known, protease-resistant, or protease-resistant, hyperglycosylated consensus IFN-α, the subject process comprises administering to an individual, in need of this, therapeutics of an effective amount of a known, protease-resistant, or protease-resistant hyperglycosylated consensus IFN-α when treated and a viral infection, for example, an infection caused by hepatitis C virus. In some embodiments, the method mainly includes oral administration to an individual in need of a known, protease resistant, or protease resistant, hyperglycosylated variant of the parental consensus polypeptide IFN-α in the first dose and with the first dosing frequency, at least also often, or more often than with the second dosing frequency, the effectiveness of which is confirmed in the treatment of HCV inf a dose that includes administering to an individual in need of a parenteral composition of parental consensus IFN-α in a second dose with a second dosage frequency, where the first dose contains the first number of moles of a known, protease-resistant, or protease-resistant, hyperglycosylated variant a polypeptide that exceeds the second mole of parental consensus IFN-α in a second dose.

[001006] In one non-limiting example, parental interferon alfacon-1 is proved to be effective in treating HCV infection in a method comprising administering to a patient in need thereof 9 million units (or 9 micrograms) of interferon alfacon-1 INFERGEN® by subcutaneous a bolus injection three times a week for 48 weeks. In some of these embodiments, a known, protease-resistant, or protease-resistant, hyperglycosylated version of the parent interferon alfacon-1 polypeptide may be selected, for example, from the group of glycopeptides | T) 99M] interferon alfacon-1, [D99N, 0105M] interferon alfacon-1, [D99N, D105N, E134M] interferon alfacon-1, [D105N, E134N] interferon alfacon-1, [E134M] interferon alfacon-1 and [D99N, E134N] -interferon alfacon-1 (where amino acid numbering is shown in Figure 24); where this glycopeptide further comprises one or more single amino acid substitutions at one or more predetermined positions corresponding to any of the amino acid positions: 41, 58, 78, 107, 117, 125, 133 and 159 (where the numbering of amino acids is shown in Figure 9), so that said variant comprises at least one mutant protease cleavage site instead of the native cleavage site located in the parent consensus IFN-α; and administered orally to a patient in a first dose containing the first number of moles of a known, protease-resistant, or protease-resistant hyperglycosylated version of the polypeptide with a first dosing frequency, where the first number of moles exceeds the number of moles of interferon alfacon-1 in 9 million units (or 9 micrograms) of interferon alfacon-1 and where the first dosing frequency is at least three times a week. Alternatively, the first dosing frequency is four times a week, five times a week, six times a week, once a day, twice a day, or three times a day.

[001007] In another non-limiting example, parental interferon alfacon-1 is proved to be effective in treating HCV infection in a method comprising administering to a patient in need thereof 15 million units (or 15 micrograms) of interferon alfacon-1 INFERGEN® by subcutaneous a bolus injection three times a week for 48 weeks. In some of these embodiments, a known, protease-resistant or protease-resistant hyperglycosylated variant of the parent interferon alfacon-1 polypeptide, for example, may be selected from the group of glycopeptides [D99N] interferon alfacon-1, [D99N, 0105M] interferon alfacon-1, [D99N, D105N, E134K] interferon alfacon-1, [D105N, E134M] interferon alfacon-1, [E134 "K] interferon alfacon-1 and [D99N, E134M] interferon alfacon-1 (where amino acid numbering is given 24); wherein the glycopeptide further comprises one or how many single amino acid substitutions at one or more predetermined positions corresponding to any of the amino acid positions: 41, 58, 78, 107, 117, 125, 133, and 159 (where the numbering of amino acids is shown in Figure 9), so that this variant contains at least at least one mutant protease cleavage site instead of the native cleavage site located in the parental consensus IFN-α; and administered orally to the patient in a first dose containing the first number of moles of a known, protease-resistant, or protease-resistant a perglycosylated version of the polypeptide with a first dosing frequency, where the first number of moles exceeds the number of moles of interferon alfacon-1 in 15 million units (or 15 micrograms) of interferon alfacon-1 and where the first dosing frequency is at least three times a week. Alternatively, the first dosing frequency is four times a week, five times a week, six times a week, once a day, twice a day, or three times a day.

[001008] In another non-limiting example, parental interferon alfacon-1 is proven to be effective in treating HCV infection in a method comprising administering to a patient in need thereof 9 micrograms (or 4.6 x 10'10 mol) of interferon alfacon- 1 by subcutaneous bolus injection three times a week for 48 weeks. In some of these embodiments, a known, protease-resistant or protease-resistant hyperglycosylated variant of the parent interferon alfacon-1 polypeptide may be selected, for example, from the group of glycopeptides [099M] interferon alfacon-1, [D99N, D105N] interferon alfacon-1, [D99N, D105N, E134M] interferon alfacon-1, [D105N, E134N] interferon alfacon-1, [E134N] interferon alfacon-1 and [D99N, E134N] interferon alfacon-1 (where the numbering of amino acids is shown in the Figure 24); where this glycopeptide further comprises one or more amino acid substitutions at one or more predetermined positions corresponding to any of the amino acid positions: 41, 58, 78, 107, 117, 125, 133 and 159 (where the numbering of amino acids is shown in Figure 9), so that said variant comprises at least one mutant protease cleavage site instead of the native cleavage site located in the parent consensus IFN-α; and orally administered to the patient in a first dose containing the first number of moles of a known, resistant to the action of proteases, or resistant to the action of proteases hyperglycosylated variant of the polypeptide with a first dosing frequency, where the first number of moles exceeds 4.6 × 10 ^-10 mol, or at least about 9.2 x 10 ^-10 mol, or at least about 1.4 x 10 ^-9 mol, or at least about 1.8 x 10 ^-9 mol, or at least about 2.3 x 10 ^-9 mol, or at least about 2.8 × 10 ^-9 mol, or at least about 3.2 × 10 ^-9 mol, or at least approximately 3.7 x 10 ^-9 mol, or at least about 4.1 x 10 ^-9 mol, or at least about 4.6 x 10 ^-9 mol, or at least about 4.6 x 10 ^-8 mole, and where the first dosing frequency is at least three times a week, Alternatively, the first dosing frequency is four times a week, five times a week, six times a week, once a day, twice a day, or three times in a day.

[001009] In another non-limiting example, parental interferon alfacon-1 is proven to be effective in the treatment of HCV infection in a method comprising administering to a patient in need thereof 15 micrograms (or 7.6 x 10 ^-10 mol) of interferon alfacon- 1 by subcutaneous bolus injection three times a week for 48 weeks. In some of these embodiments, a known, protease-resistant, or protease-resistant hyperglycosylated variant of the parent interferon alpha-con-1 polypeptide, for example, may be selected from the group of glycopeptides [D99N] interferon alfacon-1, [D99N, D105N] interferon alfacon-1, [D99N, D105N, E134N] interferon alfacon-1, [D105N, E134N] interferon alfacon-1, [E134N] interferon alfacon-1 and [D99N, E134N] interferon alfacon-1, where this glycopeptide further comprises one or more amino acid substitutions in one or more preset positions corresponding to any of the amino acid positions: 41, 58, 78, 107, 117, 125, 133 and 159, such that the variant contains at least one mutant protease cleavage site instead of the native cleavage site located in the parental consensus IFN-γ α; and orally administered to the patient in a first dose containing the first number of moles of a known, protease-resistant, or protease-resistant hyperglycosylated version of the polypeptide with a first dosing frequency, where the first number of moles exceeds 7.6 × 10 ^-10 mol, or at least about 1.5 × 10 ^-9 mol, or at least about 2.3 × 10 ^-9 mol, or at least about 3.0 × 10 ^-9 mol, or at least about 3.8 × 10 ^-9 mole, or at least about 4,6 × 10 ^-9 mol or at least about 5,3 × 10 ^-9 mol or at least at ERNO 6,1 × 10 ^-9 mol or at least about 6,8 × 10 ^-9 mol or at least about 7,6 × 10 ^-9 mol or at least about 7,6 × 10 ^-8 mole, and where the first dosing frequency is at least three times a week. Alternatively, the first dosing frequency is four times a week, five times a week, six times a week, once a day, twice a day, or three times a day.

[001010] In another non-limiting example, parental interferon alfacon-1 is proven to be effective in treating HCV infection in a method comprising administering to a patient in need thereof 9 micrograms (or 4.5 × 10 ^-8 mol) of interferon alfacon- 1 by subcutaneous bolus injection once a day for 48 weeks. In some of these embodiments, a known, protease-resistant or protease-resistant hyperglycosylated variant of the parent interferon alfacon-1 polypeptide may be selected, for example, from the group of glycopeptides [D99N] interferon alfacon-1, [D99N, D105N] interferon alfacon-1, [D99N, D105N, E134N] -interferon alfacon-1, [D105N, E134N] interferon alfacon-1, [E134N] -interferon alfacon-1 and [D99N, E134M] interferon alfacon-1, where this glycopeptide is additional contains one or more amino acid substitutions in one or more preset positions corresponding to any of the amino acid positions: 41, 58, 78, 107, 117, 125, 133 and 159, such that the variant contains at least one mutant protease cleavage site instead of the native cleavage site located in the parental consensus IFN-γ α; and orally administered to the patient in a first dose containing the first number of moles of a known, resistant to the action of proteases, or resistant to the action of proteases hyperglycosylated variant of the polypeptide with a first dosing frequency, where the first number of moles exceeds 4.6 × 10 ^-10 mol, or at least about 9.2 x 10 ^-10 mol, or at least about 1.4 x 10 ^-9 mol, or at least about 1.8 x 10 ^-9 mol, or at least about 2.3 x 10 ^-9 mol, or at least about 2.8 × 10 ^-9 mol, or at least about 3.2 × 10 ^-9 mol, or at least approximately 3.7 x 10 ^-9 mol, or at least about 4.1 x 10 ^-9 mol, or at least about 4.6 x 10 ^-9 mol, or at least about 4.6 x 10 ^-9 mole, and where the first dosing frequency is at least once a day. Alternatively, the first dosing frequency is twice a day or three times a day.

[001011] In another non-limiting example, parental interferon alfacon-1 is proved to be effective in treating HCV infection in a method comprising administering to a patient in need thereof 15 micrograms (or 7.5 × 10 ^-8 mol) of interferon alfacon- 1 by subcutaneous bolus injection once a day for 48 weeks. In some of these embodiments, a known, protease-resistant or protease-resistant hyperglycosylated variant of the parent interferon alfacon-1 polypeptide may be selected, for example, from the group of glycopeptides [D99N] interferon alfacon-1, [D99N, D105N] interferon alfacon-1, [D99N, D105N, E134N] interferon alfacon-1, [D105N, E134N] interferon alfacon-1, [E134N] interferon alfacon-1 and [D99N, E134N] interferon alfacon-1, where this glycopeptide further comprises one or more amino acid substitutions in one or more preset positions corresponding to any of the amino acid positions: 41, 58, 78, 107, 117, 125, 133 and 159, such that the variant contains at least one mutant protease cleavage site instead of the native cleavage site located in the parental consensus IFN-γ α; and orally administered to the patient in a first dose containing the first number of moles of a known, protease-resistant, or protease-resistant hyperglycosylated version of the polypeptide with a first dosing frequency, where the first number of moles exceeds 7.6 × 10 ^-10 mol, or at least about 1.5 × 10 ^-9 mol, or at least about 2.3 × 10 ^-9 mol, or at least about 3.0 × 10 ^-9 mol, or at least about 3.8 × 10 ^-9 mole, or at least about 4,6 × 10 ^-9 mol or at least about 5,3 × 10 ^-9 mol or at least at ERNO 6,1 × 10 ^-9 mol or at least about 6,8 × 10 ^-9 mol or at least about 7,6 × 10 ^-9 mol or at least about 7,6 × 10 ^-8 mole, and where the first dosing frequency is at least once a day. Alternatively, the first dosing frequency is twice a day or three times a day.

[001012] In some embodiments, the invention relates to any of the foregoing methods for treating HCV infection using a known, protease-resistant or protease-resistant hyperglycosylated variant consensus IFN-α polypeptide, in which a known, protease-resistant or resistant to the action of proteases, the hyperglycosylated version of the polypeptide contains any of the carrier peptides described in Table 9 above, in covalent or non-covalent association with the desired m variant of the polypeptide. In some of these embodiments, a known, protease-resistant or protease-resistant hyperglycosylated version of the polypeptide comprises such a carrier peptide in direct or indirect covalent association with the desired polypeptide variant, including, without limitation, any such carrier peptide, fused to the N-terminus of the desired polypeptide variant.

Treatment Methods Using IFN-γ

[001013] In another aspect, wherein a known, protease-resistant or protease-resistant hyperglycosylated variant of a polypeptide is a hyperglycosylated IFN-γ, the subject process provides for the administration to an individual in need of a therapeutically effective amount of a known, protease-resistant hyperglycosylated IFN-γ in a method of treating a viral infection, for example, HCV infection. In some embodiments, this method mainly involves the oral administration of a known, protease-resistant, or protease-resistant hyperglycosylated variant of the parent IFN-γ polypeptide to an individual in need thereof, in a first dose with a first dosing frequency, also often, or more often than at the second dosing frequency, the effectiveness of which has been confirmed in the treatment of HCV infection, which includes the introduction to the individual in need of a therapeutically effective of IFN-α and co-administering to this individual the parenteral composition of parent IFN-γ in a second dose with a second dosing interval, where the first dose includes the first number of moles of a known, protease-resistant, or protease-resistant hyperglycosylated polypeptide that exceeds the second number of moles of parent IFN-γ in a second dose.

[001014] In one non-limiting example, the parent IFN-γ is IFN-γ 1b, the effectiveness of which has been proven in the treatment of HCV infection in a method comprising administering to a patient in need thereof a therapeutically effective amount of IFN-α and co-administering to this patient 100 micrograms (6.0 × 10 ^-9 mol) of IFN-γ 1b by subcutaneous bolus injection three times a week for 48 weeks. In some of these embodiments, a known, protease-resistant or protease-resistant hyperglycosylated variant of the parent IFN-γ 1b polypeptide may be selected, for example, from the group of glycopeptides [S99T] IFN-γ, [E38N] IFN-γ, [E38N, S40T] IFN-γ, [E38N, S99T] IFN-γ and [E38N, S40T, S99T] IFN-γ, wherein said glycopeptide variant further comprises one or more amino acid substitutions shown in Table 3 (IFN-γ) so that said glycopeptide variant contains at least one mutant protease cleavage site instead of nat vnogo cleavage site located in the parent IFN-γ polypeptide; and administered orally to a patient in a first dose containing the first number of moles of a known, protease-resistant, or protease-resistant hyperglycosylated version of the polypeptide with a first dosing frequency, where the first number of moles exceeds the number of moles of IFN-γ 1b in 100 micrograms (6.0 × 10 ^-9 mol) IFN-γ 1b and where the first dosing frequency is at least three times a week. Alternatively, the first dosing frequency is four times a week, five times a week, six times a week, once a day, twice a day, or three times a day.

[001015] In another non-limiting example, the parent IFN-γ is IFN-γ 1b and has been shown to be effective in treating HCV infection in a method comprising administering to a patient in need thereof a therapeutically effective amount of IFN-α and co-administering to that patient 50 micrograms (3.0 × 10 ^-9 mol) of IFN-γ 1b by subcutaneous bolus injection three times a week for 48 weeks. In some of these embodiments, a known, protease-resistant or protease-resistant hyperglycosylated variant of the parent IFN-γ 1b polypeptide may be selected, for example, from the group of glycopeptides [S99T] IFN-γ, [E38N] IFN-γ, [E38N, S40T] IFN-γ, [E38N, S99T] IFN-γ and [E38N, S40T, S99T] IFN-γ, wherein said glycopeptide variant further comprises one or more amino acid substitutions shown in Table 3 (IFN-γ) so that said glycopeptide variant contains at least one mutant protease cleavage site instead of nat vnogo cleavage site located in the parent polypeptide IFN-γ; and orally administered to a patient in a first dose containing the first number of moles of a known, protease-resistant or protease-resistant hyperglycosylated version of the polypeptide with a first dosing frequency, where the first number of moles exceeds the number of moles of IFN-γ 1b in 50 micrograms (3.0 × 10 ^-9 mol) IFN-γ 1b and where the first dosing frequency is at least three times a week. Alternatively, the first dosing frequency is four times a week, five times a week, six times a week, once a day, twice a day, or three times a day.

[001016] In another non-limiting example, the parent IFN-γ is IFN-γ 1b and has been shown to be effective in treating HCV infection in a method comprising administering to a patient in need thereof a therapeutically effective amount of IFN-α and co-administering to that patient 100 micrograms (6.0 × 10 ^-9 mol) of IFN-γ 1b by subcutaneous bolus injection three times a week for 48 weeks. In some of these embodiments, a known, protease-resistant, or protease-resistant hyperglyzed variant of the parent IFN-γ 1b polypeptide may be selected, for example, from the group of glycopeptides [S99T] IFN-γ, [E38N] IFN-γ, [E38N, S40T] IFN-Y, [E38N, S99T] IFN-γ and [E38N, S40T, S99T] IFN-γ, wherein said glycopeptide variant further comprises one or more amino acid substitutions shown in Table 3 (IFN-γ) , so that the indicated variant of the glycopeptide contains at least one mutant protease cleavage site instead of native a cleavage site located in the parent IFN-γ polypeptide; and orally administered to the patient in a first dose containing the first number of moles of a known, protease-resistant or protease-resistant hyperglycosylated version of the polypeptide with a first dosing frequency, where the first number of moles exceeds 6.0x10 ^-9 mol, or at least about 1 , 2 × 10 ^-8 mol, or at least about 1.8 × 10 ^-8 mol, or at least about 2.4 × 10 ^-8 mol, or at least about 3.0 × 10 ^-8 mol, or at least about 3.6 × 10 ^-8 mol, or at least about 4.2 × 10 ^-9 mol, or at least about typically 4.8 x 10 ^-9 mol, or at least about 5.4 x 10 ^-8 mol, or at least about 6.0 x 10 ^-8 mol, or at least about 6.0 x 10 ^-7 mole, and where the first dosing frequency is at least three times a week. Alternatively, the first dosing frequency is four times a week, five times a week, six times a week, once a day, twice a day, or three times a day.

[001017] In another non-limiting example, the parent IFN-γ is IFN-γ 1b and has been shown to be effective in treating HCV infection in a method comprising administering to a patient in need thereof a therapeutically effective amount of IFN-α and co-administering to that patient 50 micrograms (3.0 × 10 ^-9 mol) of IFN-γ 1b by subcutaneous bolus injection three times a week for 48 weeks. In some of these embodiments, a known, protease-resistant or protease-resistant hyperglycosylated variant of the parent IFN-γ 1b polypeptide may be selected, for example, from the group of glycopeptides [S99T] IFN-γ, [E38N] IFN-γ, [E38N, S40T] IFN-γ, [E38N, S99T] IFN-γ and [E38N, S40T, S99T] IFN-γ, wherein said glycopeptide variant further comprises one or more amino acid substitutions shown in Table 3 (IFN-γ) so that said glycopeptide variant contains at least one mutant protease cleavage site instead of nat vnogo cleavage site located in the parent polypeptide IFN-γ; and orally administered to a patient in a first dose containing a first number of moles of a known, protease-resistant, or protease-resistant hyperglycosylated version of the polypeptide with a first dosing frequency, where the first number of moles exceeds 3.0 × 10 ^-9 mol, or at least about 6.0 x 10 ^-9 mol, or at least about 9.0 x 10 ^-9 mol, or at least about 1.2 x 10 ^-8 mol, or at least about 1.5 x 10 ^-8 mol, or at least about 1.8 × 10 ^-8 mol, or at least about 2.1 × 10 ^-8 mol, or at least about erno 2.4 × 10 ^-8 mol, or at least about 2.7 × 10 ^-8 mol, or at least about 3.0 × 10 ^-8 mol, or at least about 3.0 × 10 ^-7 mole, and where the first dosing frequency is at least three times a week. Alternatively, the first dosing frequency is four times a week, five times a week, six times a week, once a day, twice a day, or three times a day.

[001018] In some embodiments, the invention relates to any of the foregoing methods for treating HCV infection with a known, protease-resistant, or protease-resistant hyperglycosylated version of an IFN-γ polypeptide, in which a known protease-resistant or protease-resistant the hyperglycosylated polypeptide variant contains any of the carrier peptides described in Table 9 above, in covalent or non-covalent association with the desired polypeptide variant. In some of these embodiments, a known, protease-resistant, or protease-resistant hyperglycosylated version of the polypeptide contains any of these carrier peptides in direct or indirect covalent association with the desired polypeptide variant, including, without limitation, any such peptide - carrier fused to the N-terminus of the desired polypeptide variant.

[001019] Therapeutically effective amounts of IFN-α suitable for use in the present methods for treating HCV infection using IFN-α and IFN-γ combination therapy are provided in the methods for treating HCV infection using IFN-α therapy described above under the heading "Treatment Methods using IFN-α. " In addition, therapeutically effective amounts of IFN-α suitable for use in the present methods for treating HCV infection using IFN-α and IFN-γ combination therapy are provided in the methods for treating HCV infection using IFN-α and IFN-γ combination therapy described in WO 03/030613.

[001020] In another aspect, where a known, protease-resistant or protease-resistant hyperglycosylated version of a polypeptide is a hyperglycosylated IFN-γ, the subject process provides for the administration to an individual in need of a therapeutically effective amount of a known, protease-resistant, or protease-resistant hyperglycosylated IFN-γ in a method for treating fibrosis, for example, pulmonary fibrosis or liver fibrosis. In some embodiments, the method mainly involves the oral administration of a known, protease-resistant, or protease-resistant hyperglycosylated variant of the parent IFN-γ polypeptide to an individual in need of it, in a first dose with a first dosing frequency, also often, or more often than at the second dosing frequency, the effectiveness of which has been proven in the treatment of fibrosis, which includes the administration to an individual in need of this parenteral pharmaceutical composition ozition of parent IFN-γ in a second dose with a second dosing frequency, where the first dose contains the first number of moles of a known, protease-resistant or protease-resistant hyperglycosylated version of the polypeptide that exceeds the second number of moles of parent IFN-γ in a second dose.

[001021] In one non-limiting example, the parent IFN-γ is IFN-γ 1b and has been proven effective in treating fibrosis in a method comprising administering to a patient in need thereof 200 micrograms (1.2 × 10 ⁻⁸ mol) IFN-γ 1b by subcutaneous bolus injection three times a week for 1 year or more. In some of these embodiments, a known, protease-resistant or protease-resistant hyperglycosylated variant of the parent IFN-γ 1b polypeptide may be selected, for example, from the group of glycopeptides [S99T] IFN-γ, [E38N] IFN-γ, [E38N, S40T] IFN-γ, [E38N, S99T] IFN-γ and [E38N, S40T, S99T] IFN-γ, wherein said glycopeptide variant further comprises one or more amino acid substitutions shown in Table 3 (IFN-γ) so that said glycopeptide variant contains at least one mutant protease cleavage site instead of nat vnogo cleavage site located in the parent polypeptide IFN-γ; and orally administered to a patient in a first dose containing the first number of moles of a known, protease-resistant, or protease-resistant hyperglycosylated version of the polypeptide with a first dosing frequency, where the first number of moles exceeds 1.2 × 10 ^-8 mol, or at least about 2.4 x 10 ^-8 mol, or at least about 3.6 x 10 ^-8 mol, or at least about 4.8 x 10 ^-8 mol, or at least about 6.0 x 10 ^-8 mol, or at least about 7.2 × 10 ^-8 mol, or at least about 8.4 × 10 ^-8 mol, or at least about erno 9.6 × 10 ^-8 mol, or at least about 1.1 × 10 ^-7 mol, or at least about 1.2 × 10 ^-7 mol, or at least about 1.0 × 10 ^-6 mole, and where the first dosing frequency is at least three times a week. Alternatively, the first dosing frequency is four times a week, five times a week, six times a week, once a day, twice a day, or three times a day.

[001022] In some embodiments, the invention relates to any of the foregoing methods for treating fibrosis with a known, protease-resistant, or protease-resistant hyperglycosylated version of the IFN-γ polypeptide, where the fibrosis is pulmonary fibrosis, such as idiopathic pulmonary fibrosis, or fibrosis the liver. In some of the considered methods for treating idiopathic pulmonary fibrosis, the patient's initial forced lung capacity (FVC) is> 55% of the expected normal FVC. In other methods of the invention for the treatment of idiopathic pulmonary fibrosis, the initial diffusion capacity of the lungs for carbon monoxide (DL _CO ) in a patient is> 35% of the expected normal value of DL _CO . In other methods of the invention for the treatment of idiopathic pulmonary fibrosis, the patient's initial forced lung capacity (FVC) is> 55% of the expected normal FVC, the initial diffusion capacity of the lungs for carbon monoxide (DL _CO ) ≥35% of the expected normal DL _CO .

[001023] In some embodiments, the invention relates to any of the above methods for treating fibrosis with known, resistant to proteases, or resistant to proteases known, resistant to proteases, or resistant to proteases hyperglycosylated variant of the polypeptide contains any of the transfer peptides, described in Table 9 above, in covalent or non-covalent association with the desired polypeptide variant. In some of these embodiments, the known, protease-resistant, or protease-resistant hyperglycosylated polypeptide comprises any such carrier peptide in direct or indirect covalent association with the desired polypeptide, including, without limitation, any such carrier peptide fused to the N-terminus of the desired polypeptide variant.

[001024] In some embodiments, the invention relates to any of the methods for treating a disease in a patient with a known, protease-resistant, or protease-resistant hyperglycosylated variant of the treatment parental IFN-γ 1b polypeptide, where the method is modified to use any resistant to proteases of a variant of glycosylated native (wild type) human IFN-γ (described in WO 02/081507) as a known, resistant to the action of proteases, or resistant to the action of thease hyperglycosylated variant of the polypeptide of the therapeutic parent IFN-γ 1b. In one non-limiting example, protease-resistant, a variant of a glycosylated native human IFN-γ includes one or more amino acid substitutions shown in Table 3 (IFN-γ), so that the variant contains at least one mutant protease cleavage site instead native cleavage site in the parent IFN-γ 1b polypeptide.

Treatment Methods Using IFN-β

[001025] In some embodiments, where the known protease-resistant or protease-resistant hyperglycosylated variant of the polypeptide is a known protease-resistant or protease-resistant hyperglycosylated IFN-β, the subject process comprises administering to an individual in need in this, a therapeutically effective amount of a known protease-resistant or protease-resistant hyperglycosylated IFN-β in a method of treatment asseyannogo sclerosis. This method mainly involves the oral administration of a known, protease-resistant, or protease-resistant hyperglycosylated variant of the parent IFN-β polypeptide to an individual in need thereof, in a first dose with a first dosing frequency of at least the same, or more often than the second dosing frequency, the effectiveness of which has been proven in the treatment of multiple sclerosis, including the introduction to the individual in need of this parenteral composition of parent IFN-β in a second dose with a second dosing frequency, where the first dose includes the first number of moles of a known, resistant to the action of proteases, or resistant to the action of proteases hyperglycosylated variant of the polypeptide, which exceeds the second number of moles of the parent IFN-β in the second dose.

[001026] In one non-limiting example, the therapeutic parent IFN-β1 is IFN-β 1b, and has been proven to be effective in treating multiple sclerosis in a method comprising administering to a patient in need thereof 0.25 mg (or 1.35 × 10 ^-8 mol) IFN-β 1b (BETASERON®) by subcutaneous bolus injection every other day for the required treatment period. In some of these embodiments, a known protease-resistant or protease-resistant hyperglycosylated variant of the polypeptide is a known protease-resistant variant of the active ingredient IFN-β 1a AVONEX® (for example, where the variant is resistant to protease contains one or more amino acid substitutions shown in Table 2) and is administered orally to a patient in a first dose containing the first number of moles of this variant with the first dosing frequency, where the first number of moles it exceeds 1.35 × 10 ^-8 mol, or at least about 2.7 × 10 ^-8 mol, or at least about 4.0 × 10 ^-8 mol, or at least about 5.4 × 10 ^{- 8} mol, or at least about 6.75 × 10 ^-8 mol, or at least about 8.1 × 10 ^-8 mol, or at least about 9.45 × 10 ^-8 mol, or at least about 1.1 × 10 ^-7 mol, or at least about 1.2 × 10 ^-7 mol, or at least about 1.35 × 10 ^-7 mol, or at least about 1.35 × 10 ^-6 mol and where the first dosing frequency is at least once every two days. Alternatively, the first dosing frequency is four times a week, five times a week, six times a week, once a day, twice a day, or three times a day.

Methods of treatment using erythropoietin (EPO)

[001027] In some embodiments, where the known protease-resistant or protease-resistant hyperglycosylated version of the polypeptide is a known protease-resistant or protease-resistant hyperglycosylated EPO, the subject process provides administration to an individual in need thereof , a therapeutically effective amount of a known, resistant to protease, or resistant to protease hyperglycosylated EPO in the method of treatment nemii. This method mainly involves oral administration to an individual in need of a known, protease-resistant or protease-resistant hyperglycosylated variant of the parent EPO polypeptide in a first dose with a first dosing frequency of at least the same or more often than the second dosing frequency, the effectiveness of which has been proven in the treatment of anemia, which includes the administration to the individual in need of this parenteral composition of the parent EPO in a second dose with a second often that dosage, where the first dose contains the first number of moles of a known, resistant to the action of proteases, or resistant to the action of proteases hyperglycosylated variant of the polypeptide, exceeding the second number of moles of the parent EPO in the second dose.

[001028] In one non-limiting example, parental EPO is proven to be effective in treating anemia in a method comprising administering to a patient in need thereof 100 Units (770 micrograms or 2.5 × 10 ^-8 mol) of EPOGEN® erythropoietin alpha per kilogram of patient’s body weight, by subcutaneous bolus injection three times a week for the required treatment period. In some of these embodiments, a known, protease-resistant, or protease-resistant hyperglycosylated variant of the polypeptide is a protease-resistant variant of the active ingredient darbepoetin alpha ARANESP® (for example, where a protease-resistant or protease-resistant hyperglycosylated variant contains one or more amino acid substitutions shown in Table 4), and is administered orally to the patient in a first dose containing the first number of moles of this a variant with a first dosing frequency, where the first number of moles is a product of a value in excess of 2.5 × 10 ^-8 mol, or at least about 5.0 × 10 ^-8 mol, or at least about 7.5 × 10 ^-8 mol, or at least about 1.0 × 10 ^-7 mol, or at least about 1.25 × 10 ^-7 mol, or at least about 1.5 × 10 ^-7 mol, or at least about 1 75 × 10 ^-7 mol, or at least about 2.0 × 10 ^-7 mol, or at least about 2.25 × 10 ^-7 mol, or at least about 2.5 × 10 ^-7 mol, or at least about 2.5 × 10 ^-6 mol, per kilogram of mass the patient’s body multiplied by the patient’s body weight, and where the first dosing frequency is at least three times a week. Alternatively, the first dosing frequency is four times a week, five times a week, six times a week, once a day, twice a day, or three times a day.

[001029] In another non-limiting example, parental EPO is effective in treating anemia in a method comprising administering to a patient in need thereof 50 Units (385 micrograms or 1.25 × 10 ^-8 mol) of EPOGEN® epoetin alpha per kilogram of patient’s body weight, by subcutaneous bolus injection three times a week for the required treatment period. In some of these embodiments, a known protease-resistant or protease-resistant hyperglycosylated variant of the polypeptide is a known protease-resistant variant of the active ingredient darbepoetin alpha ARANESP® (for example, where protease-resistant or resistant the action of proteases, the hyperglycosylated version contains one or more amino acid substitutions shown in Table 4) and is administered orally to the patient in a first dose containing the first number moles of this embodiment from the first dosing frequency, where the first number of moles is the product of a value exceeding 1,25 × 10 ^-8 mole, or at least about 2,5 × 10 ^-8 mole, or at least about 3,75 × 10 ^-8 mol, or at least about 5.0 × 10 ^-8 mol, or at least about 6.25 × 10 ^-8 mol, or at least about 7.5 × 10 ^-8 mol, or at least at least about 8.75 × 10 ^-8 mol, or at least about 1.0 × 10 ^-7 mol, or at least about 1.125 × 10 ^-7 mol, or at least about 1.25 × 10 ^-7 mol , or at least about 1.25 × 10 ^-6 mol, per kilogram of patient’s body weight, multiplied by the patient’s body weight, and where the first dosing frequency is at least three times a week. Alternatively, the first dosing frequency is four times a week, five times a week, six times a week, once a day, twice a day, or three times a day.

KITS AND CONTAINERS

[001030] The present invention relates to a container containing a known, resistant to the action of proteases, or resistant to the action of proteases hyperglycosylated variant of the polypeptide. The invention further relates to a kit containing a composition comprising a unit dosage form of a known, protease-resistant, or protease-resistant hyperglycosylated version of a polypeptide in a container, and a label with instructions for using the kit.

[001031] Suitable containers include containers adapted for subcutaneous injection, including a syringe (for use with a needle), a syringe, and the like. In some embodiments, the agonist in question is administered via a pencil injector (eg, a pencil device for drug delivery), a number of which are known in the art. Typical devices that can be adapted for use in the present methods are any of the various devices in the form of pencil injectors from Becton Dickinson, for example, BD ^TM Pen, BD ^TM Pen II, BD ^TM Auto-Injector; injector pencil from Innoject, Inc .; any device in the form of pencils for drug delivery, described in US patent No. 5728074, 609-6010, 6,146,361, 6248095, 6277099 and 6221053; and the like. Pencil devices for drug delivery can be disposable or reusable and rechargeable. Intraject® needleless injection systems (Aradigm Corp.) are also suitable for use.

[001032] The invention further relates to a drug delivery device comprising (for example, pre-filled) a reservoir containing a liquid composition that contains a single dose of a glycosylated synthetic type I interferon receptor polypeptide agonist in accordance with the present invention. In some embodiments, the present invention relates to a pre-filled syringe containing a pharmaceutical composition comprising a glycosylated synthetic type I interferon receptor polypeptide agonist in accordance with the present invention.

[001033] The present invention relates to compositions comprising a glycosylated synthetic Type I interferon receptor polypeptide agonist and glycosylated IFN-γ, in accordance with the present invention, in a unit dose of a liquid composition contained in a separate reservoir for use in a drug delivery device. In some aspects, the present invention relates to a drug reservoir or other container containing a glycosylated synthetic polypeptide agonist of an interferon type I receptor and glycosylated IFN-γ in accordance with the present invention, co-mixed in liquids, where a glycosylated synthetic polypeptide agonist of an interferon I receptor of the type and glycosylated IFN-γ of the present invention are present in the composition in an amount suitable for one dose of each. The reservoir can be in the form of any of a variety of forms, including, but not limited to, in the form of a cartridge, syringe, reservoir for a device providing continuous drug delivery, and the like.

[001034] The invention further relates to a drug delivery device comprising (for example, a pre-filled) reservoir containing a liquid composition that contains a single dose of a glycosylated synthetic type I interferon receptor polypeptide agonist in accordance with the present invention, and a single dose of glycosylated IFN-γ γ. Exemplary drug delivery devices include, but are not limited to, injection devices such as pencil injectors, needles / syringes, continuous delivery devices, and the like. Any dose amounts, including synergistically effective amounts described herein, may be used in a pharmaceutical composition, in a reservoir, or in a drug delivery device.

[001035] In some embodiments, the present invention relates to a pre-filled syringe containing a pharmaceutical composition comprising a glycosylated synthetic type I interferon receptor polypeptide agonist, glycosylated IFN-γ, in accordance with the present invention, and a pharmaceutically acceptable excipient, where the glycosylated synthetic an interferon type I receptor polypeptide agonist and glycosylated IFN-γ, in accordance with the present invention, comprise a composition together.

[001036] In other embodiments, the present invention relates to a syringe comprising (a) a first cylinder pre-filled with a pharmaceutical composition comprising a glycosylated synthetic type I interferon receptor polypeptide agonist in accordance with the present invention; (b) a second cylinder pre-filled with a pharmaceutical composition comprising glycosylated IFN-γ.

[001037] In some embodiments, the present invention relates to a container containing a known, protease-resistant or protease-resistant hyperglycosylated version of a polypeptide in a composition suitable for oral delivery. Compositions suitable for oral delivery include liquid compositions, solid compositions (e.g., tablets, capsules, and the like) and semi-solid compositions (e.g., gels, gel capsules, etc.).

PATIENTS WHO SUITABLE FOR THESE TREATMENTS

[001038] The methods of the present invention are suitable for treating patients suffering from various diseases or at risk of developing such diseases. In most embodiments, the patient is a human.

Fibrosis

[001039] The methods of the present invention for treating fibrosis are suitable for treating patients diagnosed with fibrosis. Fibrosis includes, but is not limited to, pulmonary fibrosis, including idiopathic pulmonary fibrosis (ILF) and pulmonary fibrosis of known etiology, liver fibrosis, and renal fibrosis. Other exemplary fibrotic conditions include skeletal muscle fibrosis, cardiac fibrosis, adhesions after surgery, scleroderma, glaucoma, and skin lesions such as keloids.

Malignant tumors

[001040] Patients who are suitable for treatment with the cancer treatment method of the present invention include patients suffering from any type of cancer. This treatment is also suitable for patients in whom previous treatment of a malignant tumor with a standard chemotherapeutic agent was unsuccessful. This treatment is also suitable for patients who previously received standard chemotherapy treatment who first responded to such treatment and who subsequently developed relapse. This treatment is also suitable for patients in whom treatment with another agent for the treatment of malignant tumors was unsuccessful.

HCV infection

[001041] Patients who are treated with the methods of the invention for the treatment of HCV infection include patients who have been clinically diagnosed with HCV infection. In patients infected with HCV, serum HCV RNA and / or anti-HCV antibodies are detected in the blood.

[001042] Patients diagnosed with HCV infection include intact patients (for example, patients who have not previously received treatment for HCV, in particular those patients who have not previously received treatment based on IFN-α and / or based on ribavirin ) and patients whose previous HCV treatment was unsuccessful (“failed treatment” patients). Patients whose treatment outcome was unsuccessful include non-responders (i.e., patients in whom the HCV titer was not significantly or sufficiently reduced by previous HCV treatment, for example, previous monotherapy using IFN-α, previous combination therapy using IFN-α and ribavirin, or previous combination therapy using pegylated IFN-α and ribavirin); and relapsing (i.e., patients who have previously received HCV treatment, for example, who have previously received monotherapy using IFN-α, combination therapy using IFN-α and ribavirin, or combination therapy using pegylated IFN-α and ribavirin, which HCV titer decreased and subsequently increased).

[001043] In specific embodiments of interest, the HCV titer in patients is at least about 10 ⁵ , at least about 5 × 10 ^5, or at least about 10 ⁶ , or at least about 2 × 10 ⁶ genomic copies HCV per milliliter of serum. The patient can be infected with any HCV genotype (genotype 1, including 1a and 1b, 2, 3, 4, 6, etc., and subtypes (e.g. 2a, 2b, 3a, etc.)), HCV genotypes such as genotype 1 and especially HCV subtypes and quasi-species are particularly difficult to treat.

[001044] Also of interest are HCV-positive patients (described above) who have severe fibrosis or early cirrhosis (compensated, Child-Pugh class A or less), or more common cirrhosis (decompensated, class B or C system Child-Pugh) due to chronic HCV infection and which are infected with the virus despite previous antiviral treatment based on the use of IFN-α, or which cannot tolerate treatment based on IFN-α, or which have contraindications for such treatment. In specific embodiments of interest, HCV-positive patients with stage 3 or 4 liver fibrosis according to the METAVIR scale are suitable for treatment by the methods of the present invention. In other embodiments, patients who are suitable for treatment with the methods of the present invention are patients with decompensated cirrhosis with clinical manifestations, including patients with end-stage cirrhosis, including those awaiting liver transplantation. In other embodiments, patients who are suitable for the treatment methods of this invention include patients with moderate fibrosis, including patients with early fibrosis (stages 1 and 2 for METAVIR, Ludwig and Scheuer; or stages 1, 2 or 3 for Ishak system.).

EXAMPLES

[001045] The following examples provide those skilled in the art with a full disclosure and description of the implementation and use of the present invention and are not intended to limit the scope of the present invention and the experiments below do not represent all of the experiments performed. Attempts have been made to ensure accuracy with respect to the quantitative parameters used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, the parts are mass parts, the molecular weight is the average molecular weight, the temperature is given in degrees Celsius, and the pressure is atmospheric or close to atmospheric. Standard abbreviations, for example, bp, pairs of nucleotide bases can be used; TPN, thousand pairs of nucleotides; pl, picoliter (s); s or sec, second (s); min, minute (s); h or hour, hour (s); a.k., amino acid (s); nt, nucleotide (s); im, intramuscular (intramuscularly); I.P., intraperitoneally (intraperitoneally); SC, subcutaneous (subcutaneous); and the like.

Example 1: Obtaining type I interferon receptor polypeptide agonists with non-native glycosylation sites

[001046] Among the type I interferons, two subtypes of interferon alpha (IFN alpha 2b and 14), IFN beta 1 and IFN omega 1 are inherently glycosylated in mammalian cells (Figure 24). The Figure 24 presents a comparison of the amino acid sequences of Infergen (SEQ ID NO: **) and varieties of interferon type I (SEQ ID NOs: ** - **) which are reported to be glycosylated in nature. Amino acid residues along which glycosylation occurs are marked in bold. The asparagine residues represent the N-glycosylation anchor site, and the threonine residue represents the O-glycosylation anchor site. Most of the sequences shown above (SEQ ID NO: **).

[001047] Based on the high degree of identity of the sequences of Infergen and other type I interferons, glycerylation sites in Infergen were created based on alignment of the Inergen amino acid sequence with naturally glycosylated interferons (Figure 25). The Figure 25 presents a comparison of the amino acid sequences of amino acids 61-120 of Infergen (SEQ ID NO: **) and various glycosylated mutant variants (SEQ ID NOs: ** - **). Sites 1, 2, and 3 are examples of positions in which glycosylation sites are formed. Sites 1 and 2 only form N-glycosylation sites. Site 3 forms both N-glycosylation and O-glycosylation sites. In Infergen, N-glycosylation and O-glycosylation sites were created. N-glycosylation includes a unique branched oligosaccharide structure which is attached to the asparagine residue located in the Asn-X-Ser / Thr motif. O-glycosylation consists of an oligosaccharide chain or chains of glycosaminoglycans attached to the OH group of a serine or threonine residue. Most of the sequences shown above (SEQ ID NO: **).

Experiment plan

[001048] Obtaining an Infergen gene optimized for expression in human cells.

Infergen is currently produced in E. coli and therefore contains codons optimized for bacterial expression. Glycosylated Infergen should be produced in mammalian cell lines. To increase the level of protein expression in mammalian cells, a new Infergen gene was created and synthesized with the preferred use of human codons (Table 8) using the most common codon for each selected amino acid (Figure 26). Figure 26 depicts a representative synthetic mammalian Inhergen nucleotide sequence (SEQ ID NOs: ** H **) using a preferred human codon. The open reading frame is indicated by the translated Infergen amino acid sequence (SEQ ID NO: **). Six pairs of complementary primers A to F are shown alternately in italics and bold text. The upper sense strands of the primer pair are indicated by odd numbers, and the lower sense strands are indicated by even numbers. In the region located in the opposite direction from the ATQ start codon, the short GCCACC sequence, the Kozak consensus sequence, are designed to increase the efficiency of eukaryotic translation. Two consecutive stop codons - TAA and TGA - are used to ensure complete broadcasting. Table 8 (above) shows the preferably used human codon. From the book "Molecular Cloning: A Laboratory Manual" by Sambrook J. and Russell D. W. Third Edition (2001) Cold Spring Harbor Press.

[001049] Methods for producing the mammalian synthetic Infergen gene.

Since the new DNA sequence of the mammalian Infergen is synthetic and not natural, the chemical pathway is an appropriate way for gene synthesis. Typically, chemical gene synthesis involves the synthesis of short oligonucleotides, their annealing, ligation, and cloning into a plasmid. A total of 6 oligonucleotides were used to obtain the synthetic mammalian Infergen gene (Figure 26).

[001050] Each pair of primers has a terminal sequence that allows them to bind to an adjacent oligonucleotide after annealing. The 5 'and 3' ends of the synthetic gene contain Hind III and Eco RI endonuclease restriction sites, respectively, which allow ligation into the plasmid. Detailed information on the sequences of these primers is given (Table 11).

Table 11
Primer sequences for the chemical synthesis of the mammalian Infergen gene Prime Meaning Sequence (from 5 'to 3') Yes [Phosph] AOSTTOSSASSASSATOTOSAASSTSSSSAOASSA CAGCCTGGGCAACCGCCGCGCCCTGATCCTGCTGGCCC AGATGCGCCGCATCAGCCCC (SEQ ID NO: **) No [Phosph] SSSSSSSATSTSSSSSSASSASSATSSASSSSSSSSSSGGTTGCCCAGGCTGTGGGTCTGGGGCAGGTCGCACAT GGTGGCA (SEQ ID NO: **) Yes [Phosph] TTSAOSTOSSTOAA (EOASSOSASASOASTSTSOSST CtCAGGAGGAGTTCGACGGCAACCAGTTCCAGAAG GCCCAGGCCATCAGCG (SEQ ID NO: **) No [Phosph] SSTSSSSSTTSTSSAASTSSTTSSSSSTSSAASTSS TCCTGGGGGAGAGCCGAAGTCGTGGCGGTCCTTCAGGC AGCTGAAGGGGGCTGAT (SEQ ID NO: **) Yes [Phosph] TGCTGCACGAGATGATCCAGCAGACCTTCAACCTGT TCAGCACCAAGGACAGCAGCGCCGCCTGGGACGAGA GCCTGCTGGAGAAG (SEQ ID NO: **) No [Phosph] AGGCTCTCGTCCCAGGCGGCGCTGCTGTCCTTGGTG CTGAACAGGTTGAAGGTCTGCTGGATCATCTCGTGC AGCACGCTGATGG (SEQ ID NO: **) Yes [Phosph] TTSTASASSOAOOSTOTASSAOOSAOOSTOAASOASSTO GAGGCCTGCGTGATCCAGGAGGTGGGCGTGGAGGAG ACCCCCCTGATGAACGTGG (SEQ ID NO: **) No [Phosph] TSASSSSSTSTSTSTSSASSSSSSASSTSTSSATSA CGCAGGCCTCCAGGTCGTTCAGCTGCTGGTACAGCT CGGTGTAGAACTTCTCCAGC (SEQ ID NO: **) Yes [Phosph] ACAGCATCCTGGCCGTGAAGAAGTACTTCCAGCGC ATCACCCTGTACCTGACCGAGAAGAAGTACAGCCCCT GCGCCTGGGAGGTGG (SEQ ID NO: **) No [Phosph] AGGCG SAOOSOOSTOTTSTSTSTSTSOOTSAOOTAS AGGGTGATGCGCTGGAAGTACTTCTTCACGGCCAGGA TGCTGTCCACGTTCA (SEQ ID NO: **) Yes [Phosph] TOSOSOSSOAOATSATOS (KHAOSTTSAOOSSTAOOS ACCAACCTGCAGGAGCGCCTGCGCCGCAAGGAGTAAT GAG (SEQ ID NO: **) No [Phosph] AATTSTSATTASTSTSTOSOOSOSAOSOSTOSSTO CAGGTTGGTGCTCAGGCTGAAGCTGCGCATGATCTCG GCGCGCACCACCTCCC (SEQ ID NO: **)

[001051] Methods for the production of glycosylated mutant mammalian Infergenes.

Sequence changes necessary to obtain a glycosylated, compliant, Infergen are minimal and can be introduced into a synthetic gene using standard site-directed mutagenesis techniques. (Figure 27). Figure 27 shows a comparison of the mammalian Infergen nucleic acid sequences (SEQ ID NO: **) and its glycosylated mutants (SEQ ID NOs: ** - **); with the dominant sequence shown (SEQ ID NO: **). Nucleotides that differ are shown in frames. The codons used are based on codons, preferably used in mammals (Table 8). Nucleotides that differ from the dominant sequence are shown in frames.

[001052] Figure 28 shows a comparison of the amino acid sequence of human interferon beta 1 (SEQ ID NO: **) and exemplified mutant glycosylated IFN-β1 (SEQ ID NOs: ** - **); the dominant sequence (SEQ ID NO: **) is shown above. Sites 1 and 2 represent the positions in which glycosylated mutants are formed. Basically, only N-glycosylation sites are formed at site 1. Both N-glycosylation and O-glycosylation sites are formed at site 2. Natural N-glycosylation sites in human IFN-β1 and mutant are shown in the frame.

[001053] Figure 29 shows a comparison of the amino acid sequence of human interferon omega-1 (SEQ ID NO: **) and exemplified glycosylated mutants (SEQ ID NOs: ** - **); with the dominant sequence (SEQ ID NO: **) shown above. Sites 1 and 2 represent the positions in which glycosylated mutants are formed. Basically, only N-glycosylation sites are formed at site 1. Both N-glycosylation and O-glycosylation sites are formed at site 2. Natural N-glycosylation sites in human IFN-ω and mutant are shown in the frame.

Example 2: Development, creation, expression and preparation of glycosylation sites for fusion mammalian Infergen constructs with other type I interferon signal peptides.

Materials and methods

Construction of fusion genes

[001054] Infergen Amino Acid Alignments and Infergen Fusion Proteins Exemplified by Human Interferon Alfa-14 and Beta are shown in Figure 30. A two-step polymerase chain reaction has been developed to synthesize fusion genes to produce putative fusion proteins. Primers used in PCR reactions are listed in Table 12 below.

Table 12 Primer Name Sequence (5 'to 3') IFNa14_Inner GCCCTGGTGGTGCTGAGCTGCAAGAGCAGC-TGCAGCCTGGGCTGCGACCTGCCCCAGACCCACAGC (SEQ IDNO: 1350) IFNa14_Outer TATAAAGCTTGCCACCATGGCCCTGCCCTTC-GCCCTGATGATGGCCCTGGTGGTGCTGAGCTGCAAG (SEQ ID NO: 1351) NO: 1351) IFNb_Inner GCCCTGCTGCTGTGCTTCAGCACCACCGGCCC-TGAGCATGAGCTGCGACCTGCCCCAGACCCACAGC (SEQ IDN0: 1352) IFNb_Outer TATAAAGCTTGCCACCATGACCAACAAGTGC-CTGCTGCAGATCGCCCTGCTGCTGTGCTTCAGCACC (SEQ ID NO: 1353) INFERGEN_End TATAGAATTCTCATTACTCCTTGCGGCCCCGGCG (SEQ ID NO: 1354)

[001055] The mammalian Infergen gene was synthesized and cloned into a pcDNA3.1 (Invitrogen, Carlsbad, CA) plasmid and was used as a template. For the first PCR cycle to obtain the fusion of the Infergen gene with the signal peptide of human interferon alpha 14, the IFNa14_Inner primer was combined with the INFERGEN_End primer to obtain PCR templates for the second PCR cycle, which was performed using the IFNa14_Outer primer in combination with the INFERGEN_END primer. The final PCR product was digested with both Hind III and EcoRI and was cloned into a pre-digested pcDNA3.1 vector. The same procedure was used to obtain the Infergen gene with the human interferon beta signal peptide, except that the primers IFNb_Inner and Outer were used instead of the primers IFNa14_Inner and Outer, respectively.

Transient Transfection and Western Analysis

[001056] For transient hyperexpression of Infergen, the Cos-7 cell line was selected. Fugene-6 (Roche Applied Science, Indianapolis, IN) was used as transfection reagent and the manufacturer's instructions were followed. Three days after transfection, the conditioned medium was collected, then filtered through a 0.22 μM cell culture filter system and concentrated using a Centriplus YM-10 filter centrifuge (Millipore, Billerica, MA). The protein concentration was determined. Attached cells were harvested and cell lysate was obtained using conventional methods. In the Western analysis, rabbit polyclonal antibodies obtained against Infergen overexpressed E. coli were used as primary antibodies.

Site-directed mutagenesis

[001057] The location of the characteristic glycosylation sites in the two Infergen fusion proteins is shown in Figure 25. To obtain these mutations, the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used.

results

[001058] Merged constructs were obtained and sequences confirmed. Figure 30 shows the alignment of the amino acid sequence of Infergen (SEQ ID NO: **), human IFN-α14 (SEQ ID NO: **), human IFN-β1 (SEQ ID NO: **), and examples of fusion proteins with signal peptides Human IFN-α14 and IFN-β (SEQ ID NOs: ** and **, respectively). The dominant sequence shown above (SEQ ID NO: **).

[001059] Then, these constructs were transiently transfected into Cos-7 cells and the transfection results were analyzed using a Western blot using rabbit polyclonal antibodies to Infergen. The results are shown in Figure 32.

[001060] Figure 32 depicts a Western blot analysis of transient transfection results. Lanes 1-4 were loaded with a conditioned medium of Cos-7 cells transfected with a plasmid containing the nucleotide sequence encoding: Infergen with signal peptide IFN-α 14 (lane 1); Infergen with IFN-β signal peptide (lane 2); Infergen without signal peptide (lane 3); and (3-galactosidase (lane 4). Lanes 5-8 were loaded with a cell lysate of Cos-7 cells transfected with plasmids containing nucleotide sequences encoding: Infergen with IFN-α 14 signal peptide (lane 5); Inhergen with IFN signal peptide -β (lane 6); Infergen without a signal peptide (lane 7); and (3-galactosidase (lane 8). Lane 9 was loaded with 30 ng of Infergen produced by E. coli obtained from a commercial source.

[001061] The results showed that both fusion proteins were well expressed and mainly secreted by cells into the conditioned medium, while Infergen without a signal peptide was poorly expressed and existed intracellularly. Two fusion genes were selected as templates for glycosylation sites.

[001062] Although the present invention has been described with reference to its specific embodiments, one skilled in the art will appreciate that various changes can be made and equivalent replacements made without departing from the spirit and scope of the claimed invention. In addition, a large number of modifications can be made to adapt a particular situation, material, composition, method, step or steps of the method to the objectives, nature and scope of the present invention. All such modifications are included within the scope of the claims presented here.

Claims (96)

1. A hyperglycosylated variant of parental interferon type 1, wherein the hyperglycosylated variant of parental interferon type 1 has been modified to include at least three additional glycosylated sites, wherein at least one of the glycosylation sites is represented by an amino acid substitute selected from the group consisting of [D99N], [S99N], [R99N], [D105N], [E134N], [E134T], [G134N], [G134T] and [F136T], while the positions of the amino acid substitute are numbered in accordance with the maintenance of the parental interferon type 1 in the aftermath telnostyah shown in Figure 24, using the numbering adopted in this figure. 2. The hyperglycosylated variant of type 1 interferon according to claim 1, wherein the added glycosylation site is located at the site of the parent interferon type 1 at a position homologous to the position of the glycosylation site in the second type 1 interferon. 3. The hyperglycosylated variant of type 1 interferon according to claim 1, wherein the parent body interferon type 1 has been given a regulatory form suitable for human therapy. 4. The hyperglycosylated variant of type 1 interferon according to claim 2, wherein the second type of interferon is selected from the group consisting of interferon α2b (IFN-α2b), interferon α 14 (IFN-α14), interferon β (IFN-β) and interferon ω (IFN -ω). 5. The hyperglycosylated variant of type 1 interferon according to claim 1, wherein the parent type 1 interferon is interferon α (IFN-α). 6. The hyperglycosylated variant of type 1 interferon according to claim 1, wherein the parent type 1 interferon is interferon β (IFN-β). 7. The hyperglycosylated variant of type 1 interferon according to claim 1, where the parent type 1 interferon is interferon - τ (IFN-τ). 8. The hyperglycosylated variant of type 1 interferon according to claim 1, where the parent type 1 interferon is interferon - ω (IFN-ω). 9. The hyperglycosylated variant of interferon type 1 according to claim 1, where the parent interferon type 1 is a consensus interferon type 1. 10. The hyperglycosylated variant of interferon type 1 according to claim 1, where the parent interferon type 1 is a hybrid interferon type 1. 11. The hyperglycosylated variant of type 1 interferon according to claim 5, wherein at least one of the amino acid substitutes is selected from the group consisting of [D99N], [D105N], [E134N], [E134T], [G134N] and [G134T] . 12. The hyperglycosylated variant of interferon type 1 according to claim 5, where interferon α is interferon alfacon-1. 13. The hyperglycosylated variant of interferon type 1 according to claim 12, wherein the hyperglycosylated variant of interferon type 1 is the parent interferon alfacon-1 containing at least one amino acid substitute selected from the group consisting of [D99N], [D105N], [E134N ], [E134T], [D99N, D105N], [D99N, E134N], [D99N, E134T], [D105N, E134N], [D105N, E134T], [D99N, D105N, E134N], [D99N, D105N, E134T ]. 14. The hyperglycosylated variant of interferon type 1 according to claim 12, wherein the hyperglycosylated variant of interferon is the parent interferon alfacon-1 containing at least one amino acid substitute [D99N]. 15. The hyperglycosylated variant of interferon type 1 according to claim 12, wherein the hyperglycosylated variant of interferon is the parent interferon alfacon-1 containing at least one amino acid substitute [D105N]. 16. The hyperglycosylated variant of interferon type 1 according to claim 12, wherein the hyperglycosylated variant of interferon is the parent interferon alfacon-1 containing at least one amino acid substitute [E134N]. 17. The hyperglycosylated variant of interferon type 1 according to claim 12, wherein the hyperglycosylated variant of interferon is the parent interferon alfacon-1 containing at least one amino acid substitute [E134T]. 18. The hyperglycosylated variant of interferon type 1 according to claim 12, wherein the hyperglycosylated variant of interferon is the parent interferon alfacon-1 containing at least amino acid substitutes [D99N] and [D105N]. 19. The hyperglycosylated variant of interferon type 1 according to claim 12, wherein the hyperglycosylated variant of interferon is the parent interferon alfacon-1 containing at least amino acid substitutes [D99N] and [E134N]. 20. The hyperglycosylated variant of interferon type 1 according to claim 12, wherein the hyperglycosylated variant of interferon is the parent interferon alfacon-1 containing at least amino acid substitutes [D99N] and [E134T]. 21. The hyperglycosylated variant of interferon type 1 according to claim 12, wherein the hyperglycosylated variant of interferon is the parent interferon alfacon-1 containing at least amino acid substitutes [D105N] and [E134N]. 22. The hyperglycosylated variant of interferon type 1 according to claim 12, wherein the hyperglycosylated variant of interferon is the parent interferon alfacon-1 containing at least amino acid substitutes [D105N] and [E134T]. 23. The hyperglycosylated variant of interferon type 1 according to claim 12, wherein the hyperglycosylated variant of interferon is the parent interferon alfacon-1 containing at least amino acid substitutes [D99N], [D105N] and [E134N]. 24. The hyperglycosylated variant of interferon type 1 according to claim 12, wherein the hyperglycosylated variant of interferon is the parent interferon alfacon-1 containing at least amino acid substitutes [D99N], [D105N] and [E134T]. 25. The hyperglycosylated variant of type 1 interferon according to claim 5, wherein the parent type 1 interferon is interferon α2a (IFN-α2a). 26. The hyperglycosylated variant of interferon type 1 according to claim 25, wherein the hyperglycosylated variant of type 1 interferon is the parent interferon α2a containing at least one amino acid substitute selected from the group consisting of [D105N] and [D99N, D105N]. 27. The hyperglycosylated variant of interferon type 1 according to claim 25, wherein the hyperglycosylated variant of type 1 interferon is the parent interferon α2a containing at least one amino acid substitute [D99N]. 28. The hyperglycosylated variant of interferon type 1 according to claim 25, wherein the hyperglycosylated variant of type 1 interferon is the parent interferon α2a containing at least one amino acid substitute [D105N]. 29. The hyperglycosylated variant of interferon type 1 according to claim 25, wherein the hyperglycosylated variant of type 1 interferon is the parent interferon α2a containing at least amino acid substitutes [D99N] and [D105N]. 30. The hyperglycosylated variant of type 1 interferon according to claim 5, wherein the parent type 1 interferon is interferon α2b (IFN-α2b). 31. The hyperglycosylated variant of interferon type 1 according to claim 30, wherein the hyperglycosylated variant of type 1 interferon is the parent interferon α2b containing at least one amino acid substitute selected from the group consisting of [D99N], [D105N] and [D99N, D105N ]. 32. The hyperglycosylated variant of interferon type 1 according to claim 30, wherein the hyperglycosylated variant of type 1 interferon is the parent interferon α2b containing at least an amino acid substitute [D99N]. 33. The hyperglycosylated variant of interferon type 1 according to claim 30, wherein the hyperglycosylated variant of type 1 interferon is the parent interferon α2b containing at least an amino acid substitute [D105N]. 34. The hyperglycosylated type 1 interferon variant according to claim 30, wherein the hyperglycosylated type 1 interferon variant is the parent interferon α2b containing at least the amino acid substitute [D99N] and [D105N]. 35. The hyperglycosylated variant of type 1 interferon according to claim 5, where the parent interferon type 1 is interferon α14 (IFN-α14). 36. The hyperglycosylated type 1 interferon variant according to clause 35, wherein the hyperglycosylated type 1 interferon variant is the parent interferon α14 containing at least one amino acid substitute selected from the group consisting of [D105N], [E134T] and [D105N, E134T]. 37. The hyperglycosylated variant of interferon type 1 according to claim 35, wherein the hyperglycosylated variant of type 1 interferon is the parent interferon α 14 containing at least an amino acid substitute [D105N]. 38. The hyperglycosylated variant of type 1 interferon according to claim 35, wherein the hyperglycosylated type 1 interferon variant is the parent interferon α14 containing at least an amino acid substitute [E134T]. 39. The hyperglycosylated variant of type 1 interferon according to clause 35, wherein the hyperglycosylated variant of type 1 interferon is the parent interferon α14 containing at least amino acid substitutes [D105N] and [E134T]. 40. The hyperglycosylated variant of type 1 interferon according to claim 5, wherein the parent type 1 interferon is selected from the group consisting of interferon αc, interferon α2c, interferon αd, interferon α5, interferon α6, interferon α4, interferon α4b, interferon α1, interferon αJ, interferon αH, interferon αF and interferon α8. 41. The hyperglycosylated variant of type 1 interferon according to claim 6, wherein at least one amino acid substitute is selected from the group consisting of [S99N], [E134N], [E134T] and [F136T]. 42. The hyperglycosylated variant of interferon type 1 according to claim 6, where the hyperglycosylated variant of interferon type 1 is the parent interferon β containing at least one amino acid substitute selected from the group consisting of [S99N], [E134T], [E134N], [S99N, E134T], [S99N, E134N], [E134T, F136T], [E134N, F136T], [S99N, F134N, F136T] and [S99N, E134T, F136T]. 43. The hyperglycosylated variant of interferon type 1 according to claim 6, where the hyperglycosylated variant of type 1 interferon is the parent interferon β containing at least an amino acid substitute [S99N]. 44. The hyperglycosylated variant of type 1 interferon according to claim 6, wherein the hyperglycosylated variant of type 1 interferon is the parent interferon β containing at least an amino acid substitute [E134T]. 45. The hyperglycosylated variant of interferon type 1 according to claim 6, where the hyperglycosylated variant of type 1 interferon is the parent interferon β containing at least an amino acid substitute [E134N]. 46. The hyperglycosylated variant of interferon type 1 according to claim 6, where the hyperglycosylated variant of type 1 interferon is the parent interferon β containing at least amino acid substitutes [S99N] and [E134T]. 47. The hyperglycosylated variant of interferon type 1 according to claim 6, where the hyperglycosylated variant of type 1 interferon is the parent interferon β containing at least amino acid substitutes [S99N] and [E134N]. 48. The hyperglycosylated variant of interferon type 1 according to claim 6, where the hyperglycosylated variant of type 1 interferon is the parent interferon β containing at least amino acid substitutes [E134T] and [F136T]. 49. The hyperglycosylated variant of interferon type 1 according to claim 6, where the hyperglycosylated variant of type 1 interferon is the parent interferon β containing at least amino acid substitutes [E134N] and [F136T]. 50. The hyperglycosylated variant of interferon type 1 according to claim 6, where the hyperglycosylated variant of type 1 interferon is the parent interferon β containing at least amino acid substitutes [S99N], [E134N] and [F136T]. 51. The hyperglycosylated variant of interferon type 1 according to claim 6, where the hyperglycosylated variant of type 1 interferon is the parent interferon β containing at least amino acid substitutes [S99N], [E134T] and [F136T]. 52. The hyperglycosylated variant of type 1 interferon according to claim 8, wherein at least one amino acid substitute is selected from the group consisting of [R99N], [G134N] and [G134T]. 53. The hyperglycosylated variant of interferon type 1 of claim 8, where the hyperglycosylated variant of interferon type 1 is the parent interferon ω containing at least one amino acid substitute selected from the group consisting of [R99N], [G134T], [G134N], [R99N, G134N] and [R99N, G134T]. 54. The hyperglycosylated variant of interferon type 1 of claim 8, where the hyperglycosylated variant of interferon type 1 is the parent interferon ω containing at least an amino acid substitute [R99N]. 55. The hyperglycosylated variant of interferon type 1 of claim 8, where the hyperglycosylated variant of interferon type 1 is the parent interferon ω containing at least an amino acid substitute [G134T]. 56. The hyperglycosylated variant of interferon type 1 of claim 8, where the hyperglycosylated variant of interferon type 1 is the parent interferon ω containing at least an amino acid substitute [G134N]. 57. The hyperglycosylated variant of interferon type 1 according to claim 8, where the hyperglycosylated variant of interferon type 1 is the parent interferon ω containing at least amino acid substitutes [R99N] and [G134N]. 58. The hyperglycosylated variant of interferon type 1 according to claim 8, where the hyperglycosylated variant of interferon type 1 is the parent interferon ω containing at least amino acid substitute [R99N] and [G134T]. 59. The hyperglycosylated variant of type 1 interferon according to claim 9, wherein at least one amino acid substitute is selected from the group consisting of [D99N], [D105N], [E134N] and [E134T]. 60. The hyperglycosylated variant of interferon type 1 according to claim 9, where the hyperglycosylated variant of interferon type 1 is the parent consensus interferon containing at least one amino acid substitute selected from the group [D99N], [D105N], [E134N], [E134T ], [D99N, D105N], [D99N, E134N], [D99N, E134T], [D105N, E134N], [D105N, E134T], [D99N, D105N, E134N] and [D99N, D105N, E134T]. 61. The hyperglycosylated variant of interferon type 1 according to claim 9, where the hyperglycosylated variant of interferon type 1 is the parent consensus interferon type 1 containing at least an amino acid substitute [R99N]. 62. The hyperglycosylated variant of interferon type 1 according to claim 9, where the hyperglycosylated variant of interferon type 1 is the parent consensus interferon type 1 containing at least an amino acid substitute [D105N]. 63. The hyperglycosylated variant of interferon type 1 according to claim 9, where the hyperglycosylated variant of interferon type 1 is the parent consensus interferon type 1 containing at least an amino acid substitute [E134N]. 64. The hyperglycosylated variant of interferon type 1 according to claim 9, where the hyperglycosylated variant of interferon type 1 is the parent consensus interferon type 1 containing at least an amino acid substitute [E134T]. 65. The hyperglycosylated variant of interferon type 1 according to claim 9, wherein the hyperglycosylated variant of interferon type 1 is the parent consensus interferon of type 1 containing at least amino acid substitutes [D99N] and [D105N]. 66. The hyperglycosylated variant of interferon type 1 according to claim 9, wherein the hyperglycosylated variant of interferon type 1 is the parent consensus interferon of type 1 containing at least amino acid substitutes [D99N] and [E134N]. 67. The hyperglycosylated variant of interferon type 1 according to claim 9, wherein the hyperglycosylated variant of interferon type 1 is the parent consensus interferon of type 1 containing at least amino acid substitutes [D99N] and [E134T]. 68. The hyperglycosylated variant of interferon type 1 according to claim 9, wherein the hyperglycosylated variant of interferon type 1 is the parent consensus interferon of type 1 containing at least amino acid substitutes [D105N] and [E134N]. 69. The hyperglycosylated variant of interferon type 1 according to claim 9, where the hyperglycosylated variant of interferon type 1 is the parent consensus interferon of type 1 containing at least amino acid substitutes [D105N] and [E134T]. 70. The hyperglycosylated variant of interferon type 1 according to claim 9, wherein the hyperglycosylated variant of interferon type 1 is the parent consensus interferon of type 1 containing at least amino acid substitutes [D99N], [D105N] and [E134N]. 71. The hyperglycosylated variant of interferon type 1 according to claim 9, wherein the hyperglycosylated variant of interferon type 1 is the parent consensus interferon of type 1 containing at least amino acid substitutes [D99N], [D105N] and [E134T]. 72. The hyperglycosylated variant of type 1 interferon according to claim 10, wherein at least one amino acid substitute is selected from the group consisting of [D99N], [D105N], [E134N] and [E134T]. 73. The hyperglycosylated variant of interferon type 1 of claim 10, where the hyperglycosylated variant of interferon type 1 is the parent hybrid interferon containing at least one amino acid substitute selected from the group consisting of [D99N], [D105N], [E134N], [E134T], [D99N, D105N], [D99N, E134N], [D99N, E134T], [D105N, E134N], [D105N, E134T], [D99N, D105N, E134N] and [D99N, D105N, E134T]. 74. The hyperglycosylated variant of interferon type 1 of claim 10, where the hyperglycosylated variant of interferon type 1 is the parent hybrid interferon type 1 containing at least an amino acid substitute [D99N]. 75. The hyperglycosylated variant of interferon type 1 of claim 10, where the hyperglycosylated variant of interferon type 1 is the parent hybrid interferon type 1 containing at least an amino acid substitute [D105N]. 76. The hyperglycosylated variant of interferon type 1 of claim 10, where the hyperglycosylated variant of interferon type 1 is the parent hybrid interferon type 1 containing at least an amino acid substitute [E134N]. 77. The hyperglycosylated variant of interferon type 1 of claim 10, where the hyperglycosylated version of interferon type 1 is the parent hybrid interferon type 1 containing at least an amino acid substitute [E134T]. 78. The hyperglycosylated variant of interferon type 1 of claim 10, where the hyperglycosylated variant of interferon type 1 is the parent hybrid interferon type 1 containing at least amino acid substitutes [D99N] and [D105N]. 79. The hyperglycosylated variant of interferon type 1 according to claim 10, where the hyperglycosylated variant of interferon type 1 is the parent hybrid interferon type 1 containing at least amino acid substitutes [D99N] and [E134N]. 80. The hyperglycosylated variant of interferon type 1 according to claim 10, where the hyperglycosylated variant of interferon type 1 is the parent hybrid interferon type 1 containing at least amino acid substitutes [D99N] and [E134T]. 81. The hyperglycosylated variant of interferon type 1 according to claim 10, where the hyperglycosylated variant of interferon type 1 is the parent hybrid interferon type 1 containing at least amino acid substitutes [D105N] and [E134N]. 82. The hyperglycosylated variant of interferon type 1 according to claim 10, where the hyperglycosylated variant of interferon type 1 is the parent hybrid interferon type 1 containing at least amino acid substitutes [D99N] and [E134T]. 83. The hyperglycosylated variant of interferon type 1 according to claim 10, where the hyperglycosylated variant of interferon type 1 is the parent hybrid interferon type 1 containing at least amino acid substitutes [D99N], [D105N] and [E134N]. 84. The hyperglycosylated variant of interferon type 1 according to claim 10, where the hyperglycosylated variant of interferon type 1 is the parent hybrid interferon type 1 containing at least amino acid substitutes [D99N], [D105N] and [E134T]. 85. A pharmaceutical composition comprising a hyperglycosylated variant of type 1 interferon according to one of claims 1 to 12, 13-84, 93-96 and a pharmaceutically acceptable excipient. 86. The pharmaceutical composition according p, where the pharmaceutically acceptable excipient is an excipient suitable for oral use. 87. The use of a therapeutically effective amount of a hyperglycosylated variant of interferon type 1 according to one of claims 1 to 12, 13-84, 93-96 for the manufacture of a medicament for the treatment of a disease used to treat interferon type 1. 88. The application of claim 87, wherein the disease is a fibrotic disease. 89. The application of claim 87, wherein the disease is a proliferative disease. 90. The application of claim 89, wherein the proliferative disease is cancer. 91. The application of claim 87, wherein the disease is a viral infection. 92. The use of claim 91, wherein the viral infection is hepatitis C. 93. The hyperglycosylated variant of interferon type 1 according to claim 1, subsequently containing an amino acid substitute [S101T]. 94. The hyperglycosylated variant of type 1 interferon according to claim 1, subsequently containing an amino acid substitute [E107S]. 95. The hyperglycosylated variant of interferon type 1 according to claim 6, in which the hyperglycosylated variant of interferon type 1 is the parent interferon β containing at least an amino acid substitute [F136T]. 96. A hyperglycosylated variant of type 1 interferon according to claim 40, wherein at least one amino acid substitute is selected from the group consisting of [D99N], [D105N], [E105N], [G134N], [G134T], [E134M ] and [E134T].

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