MX2008000351A - Recombinant interferonî±2 (ifnî±2) mutants - Google Patents

Abstract

The present invention provides IFNα2 mutants and active fragments, analogs, derivatives, and variants thereof that have improved specific agonist or antagonist activity as compared to wild-type IFNα2. The present invention further provides pharmaceutical compositions comprising IFNα2 mutants useful for treating or preventing cancer, autoimmune diseases, infectious diseases or disorders associated with increased expression of IFNα2.

Description

MOTORS OF RECOMBINANT INTERFERON a2 (IFNa2) FIELD OF THE INVENTION The present invention relates to recombinant mutants of interferon a2 (IFNa2), fragments, analogs, derivatives, and variants thereof, which have improved agonist or antagonist activity compared to wild-type IFNa2, and pharmaceutical compositions thereof, useful for treat or prevent cancer, autoimmune diseases, infectious diseases and disorders associated with the increased expression of IFN a2.

BACKGROUND OF THE INVENTION Interferons (IFNs) were discovered in 1957 by Isaacs and Lindenmann, and were named for their ability to interfere with viral proliferation. Interferons can also fight bacterial and parasitic infections, inhibit cell division, inhibit spontaneous apoptosis, and promote or prevent cell differentiation. Based on its receptor specificity, two types of interferons are recognized: Type I and type II. Interferons of type I are a family of monomeric proteins that include IFNct and IFNOÜ that are products of leukocytes, IFNP that are produced by fibroblasts, and IFNT that has been described only in ungulate species.

The only known interferon of type II is IFNy dimer, which is produced exclusively by lymphocytes. The alpha interferon family is composed of 13 fully translated genes without introns (which exclude pseudogenes). Each member includes mature proteins of 165 or 166 amino acid residues, with two conserved disulfide bonds: Cysl-Cys98 and Cys29-Cysl38. A high level of sequence homology (80%) is presented between the different alpha interferon subtypes, and homology of about 35% exists between these subtypes and the IFNp. Despite the high homology of the different subtypes, their biological activities, including antiproliferative, antiviral, and immunomodulation differ markedly.

The structure of the different type I interferons has been determined to include murine IFP (UFA, IRMI), human IF (1AU1), human IFN a2 (1RH2, HTF), and ovine IFNT (IB5L). Structurally, interferons are members of the a-helical cytokine family. All type I interferons signal through a common receptor complex composed of IFNAR1 and IFNAR2. The major ligand binding component of type I interferon receptor is IFNAR2, with a binding affinity of -10 n for IFN a2. The structure of the extracellular part of IFNAR2 consists of two similar immunoglobulin domains, with the IFNa2 binding site located in the N-terminal domain and the connecting loop.

The mature IFNARI is a protein of 530 amino acids, with a transmembrane segment composed of 21 residues, and a cytoplasmic domain of 100 residues. The structure of the extracellular part of IFNAR1 is unknown, but from the sequence it can be deduced that it is composed of four similar immunoglobulin domains. The binding of IFN a2 to IFNAR1 is weak, with the affinity measured in an artificial membrane of 1.5-5 μ ?. The bovine IFNARl (BoIFNARl) and the human IFNARl (HuIFNARl), are 68% identical. Secondary domains 2 and 3 of HuIFNARl have been shown to play a critical role in binding to IFN ct2. By exchanging these secondary domains by BoIFNARl homologue, the affinity is substantially increased. The soluble BoIFNARl can bind to human interferons with an affinity of 10 nM, which is 500 times greater than that of HuIFNARl. Murine cells expressing IFNAR2 bind IFN < x2 with an affinity of 8 nM, while cells expressing only IFNARl do not exhibit ligand binding. With the co-expression of IFNAR1 and IFNAR2, a ten fold increase in affinity for IFN a2 was observed. Using in vitro studies on artificial membranes, it was shown that the magnitude of the increase induced by IFNAR1 in the binding affinity of the ternary complex is related to the relative surface concentration of this receptor. The location of the binding site of IFNARI in interferon was mapped in IFN that will be located in the helices of B, C, and D and the DE curl while the IFN a2 Wing mutagenesis has suggested that the IFNAR1 binding site is restricted to helices B and C. A number of studies have suggested that the formation of the ternary complex occurs in a sequential fashion, beginning with the interferon that binds IFNAR2 to form an intermediate complex, and followed by the recruitment of IFNARl. The IFNAR1-IF p-IFNAR2 complex has been shown to have a stoichiometry of 1: 1: 1. The IFNAR1 receptor is an essential component of the interferon receptor complex, with a null IFNAR1 mutation, or the addition of neutralizing Ab against this receptor resulting in a complete lack of antiviral and antiproliferative responses to IFNa and IF. The association of IFNAR1 and IFNAR2 stimulates the activation of the constitutively associated intracellular kinases Jakl and Tyk2, leading to a cascade of tyrosine phosphorylation that results in the dimerization of the transducers of the phosphorylated signal and the activators of transcription (STATs), and transport in the nucleus, where they link to specific DNA sequences and stimulate the transcription of hundreds of sensitive genes. The question remains open on how very similar IFNs induce differential activities in the same cell type. It has been suggested that the biological activities of the different subtypes of IFNa correlate with their respective binding affinities and the type of cells used. The vertebrate type I interferons are recognized by a single shared receptor, composed of two transmembrane proteins (IFNAR1 and IFNAR2), they present their activity through their associated kinases of Jak with the transcription factors Stat as their main objectives ( Brierley, MM &; Fish, E. N. 2002. J. Interferon Cytokine Res. 22, 835-845). Typically recognized by the IgG-like folds of their extracellular domains (hCR domains), IFNAR2 and IFNAR1 are respectively considered as the binding proteins and auxiliary transducing factors-that is, the alpha and beta chains of heteromeric receptors. A difference in dissociation constants of the ligand of the two chains is implicit in the definition. However, both contribute to the creation of high affinity binding sites. The combination of a "common" beta chain with the different recognition chains is a particularity of the heteromeric receptors that respond differentiated to the different ligands. The ability to interact with the different alpha chains establishes the potential connections of the network for the differential expression of the receptor (Kotenko, S. V. &Langer, J. A. 2004. Int. Immunopharmacol 4, 593-608). When, like the IFNAR1, they have the ability to interact with the elements of different signaling access paths, they can establish the connections for differential gene expression (Platanias, LC &Fish, EN 1999. Experimental Hematology 27, 1583-1592) . Human IFNs total 12 different non-allelic alpha, beta, and omega proteins. As expected from a family with marked sequence homology, a shared 3D core structure and a shared receiver, the type I activities of the IFNs overlap. However they can be recognized and even classified by their amino acid differences and numerous examples of relative differences in activity have been observed. The image that emerges is that functional differences appear only in specific physiological contexts. In addition to their local action in granting antiviral protection in almost any cell, they are linked to the development of the antiviral defenses of the second line. It was noted that a possible difference between IFNs could be their potential to bind strongly to IFNAR1 (Roisman et al, 2005. J. Mol. Biol. 353, 271-281). The differential activities of type I NFIs have been the subject of intense research for many years. Particularly it was observed that ??? ß has an additional repertoire of activities on IFNa. The detailed analysis of the differences between these two IFNs has shown that IF P has a higher general activity in the activation of the transcription of the IFN sensitive genes, and is active in the reduced levels of IFN. Linkage studies suggested that affinity for the accessory IFNAR1 subunit is the key difference between IFN a2 and IFN (Jaitin, 2006. Mol Cell Biol. 26, 1888-1897).

IFN oc2 is known to have anti-cancer effects. However, this treatment is not always effective and sometimes results in intolerable side effects related to the dosage and duration of therapy. O 97/12630 describes the treatment of cancer patients with temozolomide together with IFN a2. WO 01/54678 describes treating cancer patients with temozolomide and PEGylated interferon.

Hepatitis C virus (HCV) infection is the most common chronic blood-borne infection in the United States. Although the numbers of new infections have declined, the burden of chronic infection is substantial; the Center for Disease Control estimates 3.9 million (1.8%). of infected people in the United States. Chronic liver disease is the tenth leading cause of death among adults in the United States, and accounts for approximately 25,000 deaths annually, or approximately 1% of all deaths. Studies indicate that 40% of chronic liver disease is related to HCV, resulting in 8,000-10,000 estimated deaths each year. End-stage liver disease associated with HCV is the most frequent frequent indication for liver transplantation among adults. The antiviral therapy of chronic hepatitis C has evolved rapidly in the last decade, with significant improvements seen in the efficacy of the treatment. However, even with combination therapy using PEGylated IFN-a plus ribavirin, 40% to 50% of patients fail therapy, that is, they do not respond or relapse. These patients currently have no effective therapeutic alternative. Particularly, patients who have advanced fibrosis or cirrhosis in liver biopsy are at significant risk of developing complications of advanced liver disease, including ascites, jaundice, varicella hemorrhage, encephalopathy, and progressive liver failure, as well as a risk markedly increased hepatocellular carcinoma. Multiple sclerosis (MS) is a chronic, neurological, autoimmune, demyelinating disease. MS causes blurred vision, unilateral loss of vision (optic neuritis), loss of balance, poor coordination, speech with difficulty, tremors, numbness, extreme fatigue, changes in intellectual function (such as memory and concentration), muscle weakness, paresthesias, and blindness. Many subjects develop chronic progressive disabilities, but long periods of clinical stability can interrupt periods of deterioration. Neurological deficiencies can be permanent or evanescent. The pathology of MS is characterized by an abnormal immune response directed against the central nervous system. Particularly, they are T lymphocytes activated against the myelin lining of the central nervous system causing demyelination. In the demyelination procedure, the myelin is destroyed and replaced by the scars of hardened "sclerotic" tissue known as plaque. These lesions appear in scattered locations throughout the brain, the optic nerve, and the spinal cord. The two types of interferon-beta that are approved in the United States for use in treating MS are interferon-beta and interferon beta Ib. Type I diabetes, also known as autoimmune diabetes or insulin-dependent diabetes mellitus (IDDM), is an autoimmune disease characterized by the selective destruction of pancreatic cells by "T" self-reactive lymphocytes (Bach, 1994, Endocr. 15: 516-542). The pathology of IDDM is very complex involving an interaction between an epigenetic case (possibly a viral infection), the pancreatic islet cells and the immune system in a genetically susceptible host. A number of cytokines, including IFN-a and IFN-α, have been implicated in the pathogenesis of IDDM in humans and in animal models of the disease (Campbell et al., 1991, J. Clin. Invest. 87: 739- 742). It seems that the local expression of IFN-a by pancreatic islet cells in response to potential diabetogenic stimuli such as virus can trigger the insulitic procedure. WO9304699 describes a method for the treatment of insulin-dependent diabetes mellitus comprising administering an IFN-a antagonist.

Based on the increasing level of expression of IFN-a in patients with systemic lupus erythematosus (SLE), IFN-a has also been implicated in the pathogenesis of SLE (Ytterberg and Schnitzer, 1982, Arthritis Rheum, 25: 401-406). O02066649 * describes the anti-IFN-a specific antibodies for the treatment of insulin dependent diabetes mellitus (IDDM) and systemic lupus erythematosus (SLE). The patent application publication E.U.A. No. 20040230040 describes variants of the cysteine a of interferon-2. The patent application publication E.U.A. No. 20040002474 describes the homologs of interferon α having antiproliferative activity in a test based on the human line of Daudi cells.

The patent E.U.A. No. 4,588,585 describes mutated IFN-p-lb in which Cysl7 is changed to Serl7 via a transition from T to A at the first bases of codon 17, which prevents the incorrect formation of the disulfide bond. WO2005016371 describes a pharmaceutical composition comprising an improved recombinant human IFN-p-lb variant with a larger specific activity. Available treatments for cancer, infectious diseases, multiple sclerosis, and autoimmune disorders associated with increased expression of IFN a2 are expensive, effective only in a certain percentage of patients and adverse side effects are not uncommon. There remains a medical need not covered for adequate therapeutic methods that are safe, reliable, effective, and cost-effective.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides novel IFNa2 mutants that have important therapeutic utility. Variants of the invention have improved specificity as agonists or antagonists compared to wild-type IFN a2. The present invention describes for the first time the finding that a recombinant · IFN a2 mutant mimics the binding properties of IFNp (SEQ ID NO: 3) and shows the key characteristics of IFNp differential activity. This includes increased antiproliferative activity in vitro and in vivo, and the specific sub-regulation of the IFNAR2 receptor.

According to a first aspect, the present invention provides a recombinant polypeptide of interferon a2 (IFN a2), an active fragment, an analogue, a derivative and variant thereof, wherein the polypeptide comprises a mutation selected from at least one amino acid substitution within amino acid residues 57-89, and at least one amino acid substitution of residues 159-165 of the amino acid at the C-terminal, as well as combinations thereof, wherein the polypeptide has agonist activity or specific antagonist activity, with respect to wild type aN IFN (SEQ ID NO: 2) .

According to some embodiments, the polypeptide comprises at least one mutation selected from the group consisting of H57A (SEQ ID NO: 24), E58A (SEQ ID NO: 25), Q61A (SEQ ID NO: 26), H57Y (SEQ ID NO: 27), E58N (SEQ ID NO: 28), Q61S (SEQ ID NO: 29), and combinations thereof, wherein the polypeptide has enhanced specific agonist or antagonist activity, relative to wild type aN IFN (SEQ ID NO: 29). NO: 2). According to one embodiment, the present invention provides a triple mutant H57A, E58A, Q61A (SEQ ID NO: 5) having enhanced specific activity with respect to wild-type a2 IFN (SEQ ID NO: 2). According to another embodiment, the present invention provides a quadruple mutant N65A, L80A, Y85A, Y89A (SEQ ID NO: 6) having antagonist activity with respect to wild type aN IFN.

According to a further embodiment, the present invention provides an IFNa2 protein comprising a substitution of the C terminal ESLRSKE to KRLKSKE (SEQ ID NO: 7) having enhanced specific activity with respect to the wild-type a2 IFN.

The specific mutations of the IFN a2 mutants of the invention are each located in a different position in the protein, and therefore can be combined to produce increasing or additional effects. According to one embodiment, the present invention provides a IFN a2 variant comprising a combination of the triple mutant H57A, E58A, Q61A and the C terminal substitution of ESLRSKE to KRLKSKE (SEQ ID NO: 8), with very high specific activity. According to another embodiment, the present invention provides an IFN a2 variant comprising a combination of the quadruple mutant N65A, L80A, Y85A, Y89A and substitution of the C terminal of ESLRSKE to KRLKSKE (SEQ ID NO: 9), with an affinity of Increasing link to IFNAR2, but with very low biological activity. This variant IFN ct2 is now described to act as an IFN antagonist, which blocks the natural activity of IFNs through its receptors.

According to a further embodiment, the present invention provides a triple imitant H57M, E58D, Q61L (SEQ ID NO: ) having improved specific activity with respect to wild-type IFN 2 (SEQ ID NO: 2).

According to yet another embodiment, the present invention provides a triple mutant H57Y, E58N, Q61S (SEQ ID NO: 11) having improved specific activity with respect to Wild type aN IFN (SEQ ID NO: 2).

According to still another embodiment, the present invention provides an IFN variant a2 comprising a combination of the triple mutant H57M, E58D, Q61L and the C terminal substitution of ESLRSKE to KRLKSKE (SEQ ID NO: 12) having improved specific activity with respect to wild type aN IFN (SEQ ID NO: 2).

According to yet another embodiment, the present invention provides an IFN variant a2 comprising a combination of the triple mutant H57Y, E58N, Q61S and the C terminal substitution of ESLRSKE to KRLKSKE (SEQ ID NO: 13) having improved specific activity with respect to wild type aN IFN (SEQ ID NO: 2).

According to some embodiments, the present invention provides PEGylated IFN a2 mutants with increasing specific activity.

According to another aspect, the present invention provides the DNA molecules that encode the polypeptides of the invention. According to one embodiment, the DNA molecules comprise a sequence selected from SEQ ID NO. : 15-23 SEG ID NO. : 30-35.

According to a further aspect, the present invention provides a vector comprising a DNA molecule of the invention, wherein the vector is capable of expressing a mutant IFN a2 polypeptide in a prokaryotic host cell or eukaryotic host cell. According to another aspect, the present invention provides a host cell comprising a vector of the invention.

According to another aspect, the present invention provides a pharmaceutical composition comprising as active ingredient a recombinant polypeptide of interferon a2 (IFN a2), an active fragment, an analog, a derivative and variant thereof, wherein the polypeptide comprises a mutation selected by at least one amino acid substitution within amino acid residues 57-89, at least one amino acid substitution of residues 159-165 of the amino acid at the C-terminal, as well as combinations thereof, wherein the polypeptide has agonist activity or improved specific antagonist, with respect to wild type aN IFN (SEQ ID NO: 2), which further comprises a pharmaceutically acceptable vehicle. According to some embodiments, the pharmaceutical composition comprises the recombinant polypeptide of interferon a2 (IFN a2), which has any of the fragments, analogs, derivatives and variants thereof of SEQ ID NO. : 5-13 and SEC ID NO. : 24-29, fragments, analogs, variants and derivatives thereof. According to one embodiment, the pharmaceutical composition comprises the recombinant polypeptide of interferon a2 (IFN a2) having any fragments, analogs, derivatives and variants thereof of SEQ ID NOs: 5, 7, 8, 10, 11, 12 and 13, wherein the polypeptide has improved specific agonist activity. , According to another embodiment, the pharmaceutical composition comprises the recombinant polypeptide of interferon a2 (IFN ct2) having any of SEQ ID NOs: 6 and 9, fragments, analogs, derivatives and variants thereof, wherein the polypeptide has antagonistic activity specific enhanced. According to a further aspect, the present invention provides a method for treating or preventing a disorder or disease associated with modulation of IFN which comprises administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of the invention, wherein the disorder or disease is selected from the group consisting of cancer, autoimmune disease and infectious disease. According to one embodiment, the autoimmune disease is multiple sclerosis (MS). According to a preferred embodiment, the MS 'is selected from the group consisting of recurrent relapse MS, secondary progressive MS, primary progressive MS, and progressive relapse MS.

According to some embodiments, mutants of IFNa2 having any of SEQ ID NO. : 5, 7, 8, 10, 11, 12 and 13 are useful for treating or preventing MS. According to one embodiment said cancer is selected from the group consisting of hairy cell leukemia, Kaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia, non-Hodgkins lymphoma and melanoma. According to another embodiment, the present invention provides a method of inhibiting the growth of cancer cells, comprising exposing the cancer cells to a therapeutically effective amount of an IFNa2 mutant having any of SEQ ID NO. : 5, 7, 8, 10, 11, 12 and 13, fragments, analogs, and derivatives thereof.

According to one modality, the infectious disease is infection by hepatitis virus. According to some modalities, hepatitis is selected from the group consisting of hepatitis A, hepatitis B and hepatitis C.

According to another embodiment, IFNa2 mutants having any of SEQ ID NO: 5, 7, 8, 10, 11, 12 and 13 are useful for treating or preventing infection by infectious hepatitis virus. According to another embodiment, the present invention describes methods of treatment or prophylaxis of disorders associated with increased expression of IFNa2 including but not limited to insulin dependent diabetes mellitus (IDD) and systemic lupus erythematosus (SLE) comprising administering to a subject in need thereof an IFNcc2 mutant having SEQ ID NO. : 6, and 9, fragments, analogs, and derivatives thereof.

According to yet another aspect, the present invention provides the use of IFNa2 mutants of the invention for the preparation of a medicament for the treatment or prevention of disorders or diseases associated with the modulation of IFN.

These and other embodiments of the present invention will be apparent in conjunction with the figures, description and claims that follow.

BRIEF DESCRIPTION OF THE FIGURES FIGURES 1A-1C show the immobilization of the extracellular domain of the type I interferon receptor (IFNAR1-EC) by affinity capture and binding of IFN a2. FIG. 1A shows the binding curve to capture IFNAR1 with immobilized DB2 mAb, followed by crosslinking with AA3 mAb. FIG. IB shows analysis of the affinity of the steady state. The binding of IFN oc2 E58A (thick gray) and E96A (thin black) at different concentrations between 0.25 and 4 μ (see figures in figure), to IFNAR1 immobilized on the surface. FIG. 1C shows the dissociation reaction of IFN (x2, IFN and the triple mutant, H57A, E58A, Q61 A (SEQ ID NO: 5) of IFNAR1-EC immobilized on the surface followed in real time.The dissociation rate is related to directly to binding affinity, with the data summarized in Table 1 and 3. FIGURE 2 shows the mutant analysis of IFNAR1 and IFNAR2 that bind to IFN a2 Interferons of type I are aligned in relation to IFN a2. The residues that are underlined are those in which the mutant for Ala did not change the binding to any receptor.The signs and above symbolize whether the mutation caused an increase or decrease in IFN a2 binding affinity over the mutation (see Table 1) The above number symbolizes whether the change is due to the link to IFNAR1 (1) or IFNAR2 (2). The residues of the C-terminal in IFN a8 are in bold to mark the changes that were made in SEC ID 7. The residuals in box are those referring to SEC ID 5-13, FIGURES 3A-3B show the functional epitope for binding IFNAR1 to IFNa2. FIG. 3 shows a graph of the change in binding affinity of all mutant proteins analyzed by us to IFNAR1 and IFNAR2. FIG. 3B shows a surface representation of a model of IFNa2 complex with IFNAR2 as previously determined. Residuals that change the link to IFNARl with the mutation are given numbers. Residues that with the mutation increase the binding affinity by >; of 2 times are underlined (57, 58, 61). This image was composed with PyMoI. FIGURES 4A-4C show the concentration dependence of the antiviral and antiproliferative response of interferons in WISH cells. FIG. 4A shows a set of primed data of the antiproliferative response on the administration of a serial dilution of the interferon in a range of 250 nM - 0.48 pM of IFN a2 and 125 nM-0.24 pM of ?? G ß. FIGURES 4B and 4C show the densitometry readings for three independent antiproliferative (4B) and antiviral (4C) experiments that also include the triple mutant H57A, E58A, Q61A (SEQ ID NO: 5). The data of 6 repetitions are shown, including the standard error. The curve was fitted to an equation of the dose response, and represents the best fit for the combined data. FIGURES 5A-5B show the biological activities of the interferon mutarites. FIG. 5A shows the relative antiviral activity (by weight) plotted against the relative antiproliferative activity of 21 unique mutants IFN a2, triple mutant H57A, E58A, Q61A (SEQ ID NO: 5), quadruple mutant N65A, L80A, Y85A, Y89A (SEQ ID NO: 6), (SEQ ID NO: 7), the triple mutant, H57Y, E58N, Q61S (SEQ ID NO: 11) and IFNp. FIG. 5B shows the relative antiviral and antiproliferative activities of the same proteins plotted against their relative binding affinity for IFNARI-EC. All the data are from table 1-4. The linear line represents a theoretical relationship between biological activity and affinity. The points on the line are for mutants where the change in biological activity (antiviral or antiproliferative) is weaker than its change in affinity, and the points below the line are the opposite. FIGURES 6A-6C show the effect of interferon on gene expression observed by stained oligonucleotide microarray experiments. Four different treatments lasting 16 hours with interferon were tested: 0.3 n (1,000 units) IFN a2-wt; 3 n (10,000 units) IFN a2-wt; 0.3 nM HEQ (SEQ ID NO: 5) and 0.15 nM IF (1,000 units). Each condition is represented by treatment with IFN against without treatment in duplicates of microarray with scanning with dye. In addition, four replicates of the microarray experiments consisting of treatment against without treatment of another sample represent treatment without IFN as a control. Fig. 6A shows (for no treatment) the relative expression levels of the 395 genes plotted in ascending order according to the fold change. Fig. 6B shows the expression levels plotted in relation to the expression levels on the addition of 0.15 nM IFNp. The only exception is for the expression level of the untreated control (black dots), which is plotted against a second control set to evaluate the random level of fluctuation. Fig. 6C shows the analysis of the group of genes induced by interferon for the four treatments. The gene expression profiles of IFNP and HEQ (SEQ ID NO: 5) grouped together, with a very short distance between them. The profile of the expression of cells treated with 3 nM groups of IFNa2-wt continues, while the gene expression profile of 0.3 nM IFN a2-wt is further away.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides IFNct2 mutants and active fragments, analogs, derivatives, and variants thereof that have improved agonist or antagonist activity with respect to wild-type IFNa2 (SEQ ID NO: 2). The mutants of the invention provide the improved therapeutic utility which may include at least one advantage selected from improved specificity or selectivity, improved duration of action, improved stability and smaller adverse effects. The present invention further provides pharmaceutical compositions comprising IFNa2 mutants useful for treating or preventing cancer, multiple sclerosis, infectious diseases and disorders associated with increased expression of IFNa2 such as insulin-dependent diabetes mellitus (IDDM) and lupus erythematosus. Systemic (SLE).

Definitions The term interferon (IFN) or interferons (IFNs) refers to the family of secreted proteins, which are cytokines with antiviral, antiprotozoal, immunomodulatory, and cell growth regulating activities. The IFN were originally classified by their sources: leukocytes' IFN-a-? (SEQ ID NO: 1), and IFN-a-2 (SEQ ID NO: 2), IFN-β fibroblasts (SEQ ID NO: 3), and IFN-α immune cells. (SEQ ID NO: 4). The concrete note is made of the IFN alpha, betas, and gamma. Interferon alfa is the main type of interferon produced by white blood cells. The terms "analog", "fragment", "derivative", and "variant" ^ when referring to improved IFNa2 polypeptides of this invention mean analogues, fragments, derivatives, and variants of such improved IFNa2 polypeptides that retain substantially or substantially similar functional activity. the same biological function or activity as the improved IFNa2 polypeptides, as described herein.

An "analogue" includes an improved IFNa2 polypeptide wherein at least one amino acid is substituted by another amino acid to produce an active analog of a polypeptide of the invention having increasing activity, stability or longer half-life with respect to the polypeptides listed in the present. A "fragment" is a portion of an improved IFNa2 polypeptide of the present invention that retains substantially similar functional activity or substantially the same biological function or activity as an enhanced IFNa2 polypeptide, according to the indications of the in vitro assays described herein, as it is described in more detail below. A "derivative" includes all modifications to an improved IFNa2 polypeptide of this invention that substantially preserves the functions described herein and includes the additional function of the structure and the assistant, eg, PEGylated polypeptides, which have a longer half-life. A "variant" includes polypeptides having an amino acid sequence that comprises a combination of the IFNa2 polypeptides of this invention that retain substantially similar functional activity or increased functional activity with respect to original mutant polypeptides IFNoc2. "Substantially similar functional activity" and "substantially the same biological function or activity" each means that the level of biological activity is at about 50% to 100% or more, preferably within 80% to 100% or more, and preferably in about 90% to 100% or more of that biological activity demonstrated by the polypeptide to which it is compared when the biological activity of each polypeptide is determined by the same procedure or assay. The "similarity" between two polypeptides is determined by comparing the amino acid sequence of a polypeptide to the sequence of a second polypeptide. An amino acid of a polypeptide is similar to the corresponding amino acid of a second polypeptide if it is identical or of a conservative amino acid substitution. Conservative substitutions include those described in Dayhoff, M. 0., ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, DC (1978), and in Argos, P. (1989) E BO J 8: 779-785. For example, amino acids belonging to one of the following groups represent conservative changes or substitutions: Ala: - Pro, Gly, Gln, Asn, Ser, Thr: - Cys, Ser, Tyr, Thr; - Val, lie, Leu, Met, ala, Phe; - Lys, Arg, His; - Phe, Tyr, Trp, His; and -Asp, Glu. The "specific activity" as used herein with reference to IFNa2 mutants of the present invention means a biological activity or a function of an IFNa2. The biological activities or functions of IFNa2 are known in the art and include, but are not limited to antiproliferative activity. Such specific activity can be detected and measured using the methods described herein or any method known in the art. "Enhanced specific activity" as used herein means that the specific activity or antagonistic activity of the IFNa2 composition of the present invention is greater than that of a wild type IFNa2 reference composition. The specific activity of an IFNa2 composition of the present invention can be analyzed with respect to the specific activity of the wild-type reference IFNoc2 composition, using a method for detecting and / or measuring a specific activity of IFNa2, for example, as described in the present or as known in the art.

"IFNa2 composition" refers to an IFNa2 polypeptide, fragment, analog, derivative, or variant thereof, or the composition (e.g., a pharmaceutical composition) comprising an IFNa2 polypeptide, fragment, analog, derivative, or variant thereof. The term "recombinant proteins or polypeptides" refers to proteins or polypeptides produced by recombinant DNA techniques, that is, produced from cells, prokaryotic or eukaryotic, including, for example, microbial or mammalian, transformed by an expression construct. Exogenous recombinant DNA encoding the desired protein or polypeptide. The proteins or polypeptides expressed in most bacterial cultures will typically be free of glycan. The proteins or polypeptides expressed in yeast may have a glycosylation pattern different from that expressed in mammalian cells. An "expression vector" as used herein, refers to a nucleic acid molecule capable of replication and of expressing a gene of interest when transformed, transfected or transduced into a host cell. Expression vectors comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, provide amplification within the host. Selectable markers include, for example, the sequences conferring the markers of antibiotic resistance, which can be used to obtain satisfactory transformants by selection, such as ampicillin, tetracycline and kanamycin resistance sequences, or to provide the nutrients Critics not available from complex media. Suitable expression vectors can be derived from plasmids, for example, from pBR322 or from the different pUC plasmids, which are commercially available. The various expression vectors can be derived from bacteriophage, phagemid, or cosmid expression vectors, which are described in Sections 1.12-1.20 of Sambrook et al., (Molecular Cloning: A Laboratory Manual, 3 ed. , 2001, Cold Spring Harbor Laboratory Press). Isolated plasmids and DNA fragments were excised, adapted, and ligated together in a specific order to generate the desired vectors, as is known in the art (see, for example, Sambrook et al., Ibid). The "native", "natural" or "wild type" (wt) polypeptides or proteins refer to proteins or polypeptides recovered from a source that occurs in nature. The term "native IFN" or "wild type IFN" would include native or wild-type IFN and would include post-translational modifications of IFN, including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, acylation, and cleavage. The term "recombinant expression vector or plasmid" is a replicable DNA vector or the plasmid construct used to either amplify or express the DNA encoding the proteins or polypeptides of the present invention. An expression vector or a plasmid contains DNA control sequences and a coding sequence. DNA control sequences include promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, and enhancers. The recombinant expression systems as defined herein express the proteins or polypeptides of the invention on the induction of the regulatory elements. The term "transformed host cells" refers to cells that have been transformed and transfected with the exogenous DNA. The exogenous DNA may or may not be integrated (ie, covalently linked) to the chromosomal DNA that makes up the genome of the host cell. In prokaryotes and yeast, for example, exogenous DNA can be maintained in an episomal element, such as a plasmid, or stably integrated into chromosomal DNA. With respect to eukaryotic cells, a transformed stable cell is one in which the exogenous DNA has been integrated into the chromosome. This stability is demonstrated by the ability of eukaryotic cell lines or clones to produce through replication a population of daughter cells containing the exogenous DNA.

The term "primer" as used herein, refers to an oligonucleotide, either occurring naturally as in a purified or synthetically produced restriction digestion product, which is capable of acting as the point of initiation of synthesis when is placed under conditions in which synthesis of a primer extension product is induced, which is complementary to a strand of nucleic acid, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and in a adequate temperature and pH. The primer can be either single-stranded or double-stranded and must be long enough to prime the synthesis of the desired extension product in the presence of the inducing agent. The "effective therapeutic amount" refers to that amount of an improved IFNa2 polypeptide of the invention which, when administered to a subject in need thereof, is sufficient to effect the treatment of the condition or disorder. For example for recurrent multiple sclerosis with relapse (MS), cancer, infectious diseases or disorders associated with increased expression of IFNa2 such as insulin dependent diabetes mellitus (IDDM) and systemic lupus erythematosus (SLE). The term "subject" refers to human subjects and non-human subjects.

The term "cancer" means to include all types of cancerous growths or oncogenic procedures, metastatic tissues or transformed malignant cells, tissues, or organs, regardless of the type or the histopathological stage of the invasion. Examples of cancers include but are not limited to solid tumors and leukemias, including: apudoma, coristoma, branquioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker, basal cell, basosquamous, Bro n-Pearce, ductal, Ehrlich's tumor, non-small cell lung, oat cell, papillary cell, bronchiolar, bronchogenic, squamous, and transient cell), histiocytic disorders, leukemia (e.g., B cell, mixed cell, null cell, cell T, chronic T cell, associated with HTLV-II, acute lymphocytic, chronic lymphocytic, mast cell, and myeloid), malignant histiocytosis, Hodgkin's disease, small immunoproliferative, non-Hodgkin's lymphoma, plasmacytoma, reticuloendotheliosis, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, E sarcoma wing, sinovioma, adenofibroma, adenolinforna, carcinosarcoma, chordoma, craniopharyngioma, dysgerminoma, hamartoma, mesenchyme, mesonefroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma, trophoblastic tumor, adeno-carcinoma, adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma , cystadenoma, granulosa cell tumor, ginandroblastoma, hepatoma, hidradenoma, islet cell tumor, Leydig cell tumor, papilloma, Sertoli cell tumor, teak cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, noncromafin, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, sclerotizing angioma, angiomatosis, glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangioma, lymphaniosarcoma, pi nealoma, carcinosarcoma, chondrosarcoma, cystosarcoma, filodes, fibrosarcoma, hemangiosarcoma, leimiosarcoma, leucosarcoma, liposarcoma, lymphaniosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma (for example, Ewing, experimental, Kaposi, and mast cells), neurofibromatosis, and dysplasia cervical and other conditions in which the cells have been immortalized or transformed. "Treating MS" as used herein covers the treatment of disease status in a subject, which characterizes the disease state by symptoms associated with MS, such as weakness, numbness, tremor, loss of vision, pain , paralysis, loss of balance, dysfunction of the bladder and bowel, and cognitive changes (primary symptoms); repeated urinary tract infections, weakness from lack of use, poor postural alignment and trunk control, muscle imbalance, decreased bone density, acute, ineffective breathing, and decubitus ulcer (secondary symptoms); and depression (tertiary symptoms), and includes: (i) inhibiting the condition, that is, stopping its development; or (ii) relieving the condition, that is, causing the condition to regress.The term "hepatitis virus infection" refers to infection with one or more of the hepatitis A, B, C, D, or E virus, with the viral infection of hepatitis carried in the blood of particular interest .

As used herein, the term "liver fibrosis," used alternatively together with "liver fibrosis," refers to the growth of scar tissue in the liver that may occur in the context of a chronic infection of hepatitis.

The term "PEGylated IFNa2 mutants" as used herein means the modified polyethylene glycol conjugates of IFNa2 mutants. The preferred polynitol glycol IFNoc2 mutant conjugates are reversible conjugates that are slowly converted to drugs under physiological conditions, such as are prepared according to the methods described in WO2004089280. Other IFNoc2 mutant conjugates can be prepared by coupling an IFNa2 mutant to a water soluble polymer. A non-limiting list of such polymers includes the various polyalkylene oxide homopolymers such as polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof. As an alternative to polymers based on polyalkylene oxide, effectively non-antigenic materials such as dextran, polyvinylpyrrolidones, polyacrylamides, polyvinyl alcohols, carbohydrate-based polymers and the like may be useful. Such conjugates of the interferon alpha polymer are described in the US patent. No. 4,766,106 and the patent of E.U.A. No. 4,917,888. As defined herein; a "microarray" refers to a plurality of isolated nucleic acid molecules or oligonucleotide probes attached to a support where each of the nucleic acid molecules or the oligonucleotide probes is attached to a support in the only precursor region. selected The term "nucleic acid" is interchangeable with the term "polynucleotide". The term "polynucleotide" refers to a chain of nucleotides. Preferably, the chain has from about 20 to 10,000 nucleotides, preferably from about 150 to 3.55 nucleotides. The term "probe" refers to a polynucleotide sequence capable of hybridizing with a transcript of the gene or complement thereof to form a complex of the polynucleotide / transcript probe of the gene.

The term "gene" includes a region that can be transcribed in the RNA, as the invention contemplates the detection of RNA or the equivalents thereof, for example, cDNA and cRNA. A gene of the invention includes, but is not limited to, genes specific for or involved in a biological process and / or indicative of a biological process, such as apoptosis, differentiation, stress response, aging, proliferation, etc.; genes of the cellular mechanism, for example, cell cycle, signal transduction, metabolism of toxic compounds, and the like; genes associated with disease, for example, genes involved in cancer, multiple sclerosis, infection and the like. For example, the gene may be an oncogene, whose expression within a cell induces that cell to be converted from a normal cell into a tumor cell. Other examples of genes include, but are not limited to, cytokine genes, prion genes, genes that code for molecules that induce angiogenesis, genes that code for adhesion molecules, genes that encode cell surface receptors , genes that encode proteins that are involved in the procedures of invasive and / or metastasis, protease genes as well as the molecules that regulate apoptosis and the cell cycle. According to the present invention, the level of a gene transcript can be determined by measuring the level of transcription of the gene, for example, RNA, using semiquantitative methods such as microarray hybridization or more quantitative methods such as quantitative qPCR. As used in this, the term "regulatory expression and / or activity" generally refers to any method that functions to control or modulate the amount or activity (functionality) of a cellular component. Static regulation maintains expression and / or activity at some given level. Overregulation refers to a relative increase in expression and / or activity. Accordingly, sub-regulation refers to a relative decrease in expression and / or activity. Sub-regulation is synonymous with the inhibition of the given activity of a cellular component.

Characteristics of Enhanced IFNa2 Polypeptides of the Invention Three unique mutations (H57A, E58A, Q61A) (SEQ ID NO: 5) were introduced into IFNoc2 that specifically increase their binding affinity towards the IFNARI receptor by about 30 fold compared to the protein of wild type (SEQ ID: NO 2). The affinity of the mutant IFNa2 protein, as used in the present designated HEQ, is similar to that which is measured for IF P (Table 3), which is higher than for any other subtype of interferon. Evaluating the biological activity of HEQ clearly demonstrates that similar characteristics of IFNP are gained (SEQ ID NO: 3). Thus, HEQ promotes only 2-fold increase in its antiviral potency, but a 25-fold increase in its antiproliferative potency relative to IFNa2-wt (SEQ ID NO: 2) (Table 4). Two additional sets of mutations were introduced in the same positions in IFNa2 as HEQ: H57, E58D, Q61L, designated in the present MDL (SEQ ID NO: 10), and H57Y, E58N, Q61S, designated in the present YNS (SEQ ID NO: 11). The antiviral activity of both is within 3 times that of the wild type (Table 4). However, its antiproliferative activity is higher than that of HEQ of IFNp. For DL, activity is 40 times higher compared to that of weight, and for YNS it is 160 times higher in WISH cells and 70 times higher in MDA231 cells, which is 3-7 times than for IFN (SEQ) ID NO: 3).

The gene transcription profile of HEQ (SEQ ID NO: ) was monitored by microarray experiments of the oligonucleotide. The activation of the HEQ gene is much higher than that of IFNa2 in the same protein concentration, similar to the activation of the gene registered for IFNP (SEQ ID NO: 3). YNS or HEQ could potentially be more effective drugs in the treatment of diseases compared to either IFNa2-wt or ??? ß. The advantage of YNS and HEQ over IFNa2-wt lies in its larger antiproliferative specificity, which could help in reducing side effects and in the treatment of cancer or multiple sclerosis. The advantage of YNS is its higher antiproliferative effect compared with IFN, particularly as measured in human breast cancer cells, MDA231. The present invention further provides a quadruple mutant N65A, L80A, Y85A, Y89A (SEQ ID NO: 6), as used herein designated NLYY, having 1000 times the reduced antiproliferative activity and a reduced antiviral activity of 100 times compared to the wild-type protein but still bind to IFNAR2 in the wild-type affinity (Table 2).

The present invention provides an IFNcc2 protein comprising a substitution of the C-terminus ESLRSKE to KRLKSKE (SEQ ID NO: 7), as used herein is designated cc8-tail. The ad-tail mutant was engineered to have the end of interferon a8 in order to optimize the electrostatic binding energy between IFNa2 and its IFNAR2 receptor. Linkage measurements have shown that the a8-tail mutant has a higher binding affinity of 18 times to IFNAR2 compared to the wild type (Table 2). The antiviral activity of the a8-tail mutant is 3 times higher and the antiproliferative activity is 10 times higher compared to the wild type (Table 2).

The specific mutations of the three IFNa2 mutants of the invention are each located at a different position in the protein, and therefore can be combined to provide additional, or increasing, effects. The present invention provides variants comprising a combination of the triple mutant MEQ (SEQ ID NO: 5); MDL (SEQ ID NO: 10) or YNS (SEQ ID NO: 11), and the replacement of terminal C from ESLRSKE to KRLKSKE (SEQ ID NO: 7). These variants (SEQ ID NO: 8, 12 and 13) may prove to be more effective in treating cancer, specifically as their antiproliferative activity is increasing. On the other hand, its higher binding power can overcome the problem of sub-regulation of the receptor in the cancer cell. Since HEQ is composed of three single point mutations to Ala, and the a8-tail mutant comprises a substitution of the C-terminus of IFNa2 with the C-terminus of the native IFNa8, the possibility of the specific immunogenic response being small. The present invention further provides a variant comprising a combination of the quadruple mutant N65A, L80A, Y85A, Y89A and substitution of the C-terminus from ESLRSKE to KRLKSKE (SEQ ID NO: 9), with an increasing binding affinity to IFNAR2, but with very low biological activity. This variant IFNa2 is an IFN antagonist, which blocks the natural activity of IFN through these receptors..

The improved IFNcc2 compositions of the present invention comprise IFNa2 mutant polypeptides, or fragments, analogs, derivatives, and variants thereof, having at least 2 times, preferably at least 10 times, and preferably at least 100 times, more preferably 1000 times, a specific activity larger than that of a reference wild-type IFNa2 composition. In certain embodiments, the IFNa2 compositions of the present invention have antagonistic activity to wild-type IFNa2.

Antagonists of the invention An IFNα antagonist is defined to be any substance that is capable of interfering with a biological activity of IFNα in vivo. It is not necessary for the antagonist to completely neutralize the activity of IFNa, but only to do so at a level sufficient to exert a therapeutic activity of IDDM or SLE in vivo. IFNa is known to possess a plurality of biological activities. Antagonists for use herein reduce, inhibit or neutralize any of one or more of these activities. Ordinarily the antagonist interferes with at least one (and preferably all) of the antiviral, antiproliferative or immunomodulatory activity of IFNa.

Antagonists are generally selected from different categories: a soluble form of the interferon alpha receptor, anti-alpha interferon receptor antibodies that block the interferon alpha of the bond or interact correctly with its receptor, antibodies capable of binding and neutralizing interferon alpha itself, and non-interferon polypeptides that compete with interferon alpha for the receptor binding sites but that by themselves do not demonstrate substantial interferon alpha activity. Antagonists of the present invention are mutants of IFNα that antagonize the activity of IFNα and IFN.

Mutagenesis of IFNa2 The effects of altering the amino acids at the specific positions can be tested experimentally by introducing amino acid substitutions and testing the altered IFNa2 polypeptides for biological activity using the assays described herein. Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, site-directed mutagenesis in vitro, using, for example, the QuikChange site-directed mutagenesis kit (Stratagene), etc. Techniques such as alanine scanning mutagenesis are particularly appropriate for this approach. Nucleic Acids The present invention further provides nucleic acid molecules that encode the improved IFNa2 polypeptides of the invention. A nucleic acid molecule can be produced using recombinant DNA technology (eg, amplification of the polymerase chain reaction (PCR), cloning) or chemical synthesis. The nucleic acid sequences include the natural nucleic acid sequences and homologs thereof, which include, but are not limited to, natural allelic variants and modified nucleic acid sequences in which the nucleotides have been inserted, deleted, substituted , and / or inverted such that such modifications do not substantially interfere with the ability of the nucleic acid molecule to encode recombinant IFNa2 polypeptides of the present invention.

A polynucleotide or oligonucleotide sequence can be deduced from the genetic code of a protein, however, the degeneracy of the code must be considered, and the nucleic acid sequences of the invention also include sequences, which degenerate as a result of the genetic code, whose sequences are they can easily determine by those of ordinary skill in the art.

The terms "nucleic acid" and "polynucleotide" as used herein refer to an oligonucleotide, a polynucleotide or a nucleotide and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which can be single- or double-stranded, and represent the sense or antisense strand. The term should also be understood to include, as equivalents, analogues of either RNA or DNA made from nucleotide analogues, and, as applied to that of the described modality. The term "oligonucleotide" refers to a nucleic acid sequence of at least about 6 nucleotides to about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20 to 25 nucleotides, which can be used in PCR amplification or a hybridization assay, or a microarray. As used herein, the oligonucleotide is substantially equivalent to the terms "amplimers," "primers," "oligomers," and "probes," as is commonly defined in the art. As used herein, highly stringent conditions are those that are tolerant of up to about 5% up to about 25% at the divergence of the sequence, preferably up to about 5% to about 15%. Without limitation, examples of highly stringent conditions (-10 ° C below the calculated Tm of the hybrid) use a 0.1 X SSC wash solution (standard saline citrate) and 0.5% SDS in the appropriate Ti (incubation temperature) ) below the calculated Tm of the hybrid. The final severity of the conditions is mainly due to the washing conditions, particularly if the hybridization conditions used are those that allow less stable hybrids to be formed together with stable hybrids. Washing conditions at a higher severity then eliminate the less stable hybrids. A common hybridization condition that can be used with the highly stringent to moderate stringent washing conditions described above is hybridization in a solution of 6 X SSC (or 6 X SSPE), 5 X Denhardt reagent, 0.5% SDS, 100 μg / ml denatured, salmon sperm DNA fragmented to an appropriate Ti. (See generally Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press (1989) for appropriate high stringency conditions).The severity conditions are a function of the temperature used in the hybridization and wash experiments, the molarity of the monovalent cations in the hybridization solution and in the wash solutions and the percentage of formamide in the hybridization solution. In general, the sensitivity for hybridization with a probe is affected by the amount and specific activity of the probe, the amount of the target nucleic acid, the detectability of the marker, the rate of hybridization, and the duration of hybridization. Hybridization rate is maximized at a Ti of about 20 ° C-25 ° C below Tm for AD: DNA hybrids and about 10 ° C-15 ° C below Tm for DNA: RNA hybrids . It is also maximized by an ionic strength of about 1.5M Na +. The speed is directly proportional to the length of the duplex and inversely proportional to the level of non-coincidences. The specificity in hybridization, however, is a function of the difference in stability between the desired hybrids and the "backup" hybrids. Hybrid stability is a function of the length of the duplex, the base composition, the ionic strength, non-matches, and destabilizing agents (if any). The Tm of a perfect hybrid can be estimated for DNA: DNA hybrids using the equation of Meinkoth et al., (Anal Biochem 1984, 138 (2): 267-84).

Expression and Purification of Enhanced IFNct2 Polypeptides There are several ways of expressing and purifying recombinant human IFNα2 in bacteria, particularly in E. coli, to obtain IFNα2 polypeptides that have improved agonist or antagonist activity. Known methods can be used to express cloned genes in bacteria. To obtain high level expression of a cloned eukaryotic gene in a prokaryotic system, it is preferable to construct expression vectors containing a strong promoter for direct transcription of the mRNA. Examples of regulatory regions suitable for this purpose are the promoter and operator region of the E. coli beta-glucosidase gene, the biosynthetic route of E. coli tryptophan, or the promoter to the left of phage A. Inclusion of selection markers in DNA plasmids transformed in E. coli is also useful. Examples of such markers include genes that specify resistance to ampicillin, tetracycline, or chloramphenicol. Post-translational modifications, such as glycosylation, do not occur in the prokaryotic cell expression system of E. coli. In addition, proteins with complex disulfide patterns may be mismatched when expressed in E. coli. With the prokaryotic system, the expressed protein is present either in the cytoplasm of the cell in an insoluble form, in the so-called inclusion bodies, found in the soluble fraction after the cell has been lysed, or is directed in the periplasm by the addition of appropriate secretion signal sequences. If the expressed protein is in insoluble inclusion bodies, solubilization and subsequent refolding of the inclusion bodies is usually required.

Numerous prokaryotic expression vectors are known to those of skill in the art and are commercially available, such as pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), pKK233-2 (Clontech, Palo Alto, CA, USA), and pGEMl (Promega Biotech, Madison, WI, USA). Promoters of general use in recombinant microbial expression systems include the beta-lactamase system (penicillinase) and the lactose promoter (Chang, A.C. et al., 1978, Nature 275: 617-624). The tryptophan (trp) promoter system, and the Tac promoter (Sambrook, J.F. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989). Another system of useful bacterial expression employs the pL promoter of phage lambda and the thermoinducible repressor clts857 (Bernard et al., 1979, gene 5: 59-76). Currently the following protocol was used: The gene coding for IFNa2 was cloned into a two cistron expression vector based on the plasmid of pT7T3-18U, to which a second cistron was added just upstream of the starting codon. The second cistron is the first 9 amino acids of the lpp 5 'gene that includes its SD site flanked by the restriction sites of Xbal and Ndel. The reason for inserting a second cistron was to improve the expression performance of the heterologous protein. In addition, to improve the level of expression, the codons for the first 23 amino acids were changed into: TGT GAT CTG CCG CAG ACT CAC TCT CTG GGT TCT CGT ACT CTG ATG CTG CTG GCT CAG ATG CGT CGT (SEQ ID NO: 14). The proteins are expressed in BL21 cells, overnight in rich medium. IFNa2 is found in inclusion bodies, which dissolve in 8 M urea containing 5 mM DTT. IFNoc2 was refolded by a 20-fold dilution overnight. Protein purification is carried out using standard methodologies. The typical protein yields were 10 mg / L of cell culture. IFNa2 runs as a single band of 18 kDa on SDS-PAGE.

Analogs, Fragments, Derivatives, and Variants of Enhanced IFNct2 Polypeptides An analog, fragment, derivative, or variant of the improved IFNa2 polypeptides of the present invention may be: (i) one in which one or more of the amino acid residues is substituted with a conserved or non-conserved amino acid residue; or (ii) one in which one or more of the amino acid residues include a substitute group; or (iii) one in which the enhanced IFNa2 polypeptide is fused with another compound, such as a compound to increase the half-life of the enhanced IFNa2 polypeptide (eg, polyethylene glycol); or (iv) one in which additional amino acids are fused to the mature polypeptide, improved IFN polypeptide < x2, such as a leader or secretory sequence or a sequence that is employed for the purification of the mature polypeptide, IFNa2 enhanced polypeptide; or (v) one in which the improved IFNa2 polypeptide sequence is fused to a larger polypeptide, i.e. human albumin, an antibody or Fe, for the increasing duration of the effect. Such analogs, fragments, derivatives, and variants are appreciated to be within the scope of those skilled in the art from the teachings attached. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acid side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine) , threonine, tyrosine, cysteine), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta branched side chains (eg, threonine, valine, isoleucine) and side chains aromatics (for example, tyrosine, phenylalanine, tryptophan, histidine). Non-conservative substitutions would not be made for conserved amino acid residues or for amino acid residues residing within a conserved domain of the protein. Fragments or biologically active portions include fragments of the polypeptide suitable for use as a medicament, as a research reagent, and the like. The fragments include peptides comprising amino acid sequences sufficiently similar to or derived from the amino acid sequences of an improved IFNa2 polypeptide of this invention and exhibiting at least one activity of that polypeptide, but including some amino acids that length polypeptides. complete described in this. Typically, the biologically active portions comprise a domain or a portion with at least one activity of the polypeptide. Such biologically active portions may be prepared synthetically or by recombinant techniques and may be evaluated for one or more of the functional activities of a polypeptide of this invention by the means described herein and / or known in the art. Preferred derivatives of the present invention include improved IFNa2 polypeptides that have been fused to another compound, such as a compound to increase the half-life of the polypeptide and / or reduce the potential immunogenicity of the polypeptide (e.g., polyethylene glycol, "PEG") . PEG can be used to impart water solubility, size, slow release rate in the kidney, and reduced immunogenicity to the fusion protein. See, for example, U.S. Patent No. 6,214, 966. In the case of PEGylations, the fusion of the enhanced polypeptide IFNa2 to PEG can be performed by any means known to the skilled person. For example, PEGylation can be performed by first introducing a mutation of the cysteine into the improved IFNa2 polypeptide, followed by the site-specific derivation with PEG-maleimide. The cysteine residue can be added to the C-terminus of the improved IFNcc2 polypeptide. See, for example, Tsutsumi, Y. et al, (2000) Proc. Nati Acad. Sci. USA 97: 8548-8553.

The improved IFNα2 polypeptide variants of this invention include polypeptides having an amino acid sequence sufficiently similar to the amino acid sequence of the original improved IFNα2 polypeptides or combinations thereof. Variants include improved IFNcc2 polypeptides that differ in the amino acid sequence due to mutagenesis. Variants with enhanced activity can be identified by the combinatorial collections of the selection of the mutants, eg, truncation or dot mutants, of improved IFNa2 polypeptides of this invention. In one embodiment, a diversified collection of variants is generated by combinatorial mutagenesis at the level of the nucleic acid and is encoded by a diversified library. A diversified collection of the variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences in such a way that a degenerate set of the potential amino acid sequences of the variant is expressible as individual polypeptides, or, alternatively, as set of larger enhanced IFNa2 polypeptides (e.g., to display phages) containing the set of sequences in this. There are a variety of methods that can be used to produce collections of potential variants of a degenerate oligonucleotide sequence. The chemical synthesis of a degenerate gene sequence can be performed on an automatic DNA synthesizer, and the synthetic gene is then ligated into an appropriate expression vector. The use of a degenerate set of genes allows the provision, in a mixture, of all the sequences that encode the desired set of potential sequences of the variant. Methods for synthesizing degenerate oligonucleotides are known in the art (see, for example, Itakura, K. et al., 1984, Annu., Rev. Biochem. 53: 323-356).

Several techniques are known in the art for selecting gene products from combinatorial collections made by point mutations or truncation, and for selection cDNA collections for gene products having a selected property. Such techniques are adaptable for the rapid selection of libraries generated by the combinatorial mutagenesis of improved IFNa2 polypeptides for the specific activity. The most widely used techniques, which are susceptible to high performance analysis for large selection libraries typically include cloning the library into replicable expression vectors, transforming appropriate cells with the resulting collection of vectors and expressing the combinatorial genes under which detection of a desired activity facilitates the isolation of the vector encoding the gene whose product was detected. Recurrent ensemble mutagenesis (REM), a technique that improves the frequency of functional mutants in collections, can be used in combination with selection assays to identify the desired variants.

Pharmaceutical Compositions of the Invention The invention also provides pharmaceutical compositions that can be administered to a patient to achieve a therapeutic effect. The pharmaceutical compositions of this invention can be prepared for administration by combining an improved IFNa2 polypeptide, having the desired degree of purity in a pharmaceutically effective amount, with pharmaceutically acceptable carriers.

The improved IFNcc2 polypeptides and pharmaceutical compositions of the present invention are useful for parenteral (e.g., intravenous, intramuscular and subcutaneous), topical, oral, inhalable, or local administration. The improved polypeptides of the invention can be used in pharmaceutical compositions, for any suitable method of administration, including but not limited to intravenous, subcutaneous, intramuscular, or intrathecal administration. Thus, the polypeptides described above are preferably combined with an acceptable sterile pharmaceutical carrier, such as five percent dextrose, lactated Ringer's solution, normal saline, sterile water, or any other commercially prepared physiological buffer solution designed for intravenous infusion. It will be understood that the selection of the carrier solution, and the dosage and administration of the composition, vary with the subject and the particular clinical facility, and is governed by standard medical procedures. In accordance with the methods of the present invention, these pharmaceutical compositions can be administered in amounts effective to inhibit or ameliorate the consequences or pathological symptoms associated with MS, cancer, IDDM or SLE.

The administration of improved IFNcc2 polypeptides of the present invention can be by intravenous bolus injection, by constant intravenous infusion, or by a combination of both routes. Alternatively, or in addition, improved IFNa2 polypeptides mixed with appropriate excipients can be taken into the circulation via an intramuscular site. Clearly, the polypeptides are less suitable for oral administration due to the susceptibility to digestion by gastric acids or intestinal enzymes, however in certain embodiments the specific formulations can be used for oral administration.

In preparing the compositions in oral liquid dosage forms (e.g., suspensions, elixirs and solutions), typical pharmaceutical media, such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like, may be employed. Similarly, when preparing oral solid dosage forms (e.g. powders, tablets and capsules), carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like are employed. Because of their ease of administration, tablets and capsules represent a desirable oral dosage form for the pharmaceutical compositions of the present invention. Polypeptides are less suitable for topical administration, however specific formulations can be used for local treatment.

For topical administration, the improved IFNa2 polypeptides of the present invention can be formulated using soft, moisturizing bases, such as ointments or creams. Examples of suitable ointment bases are petrolatum, petrolatum plus volatile silicone emulsions, lanolin and water in oil emulsions.

For administration by inhalation, the IFNa2 polypeptides for use in accordance with the present invention are conveniently administered in the form of an aerosol spray presentation of a pressurized pack or a nebulizer with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to supply a measured quantity. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mixture of the peptide and suitable powder bases such as lactose or starch.

The improved IFNa2 polypeptides and pharmaceutical compositions of the present invention are particularly useful for intravenous administration. The compositions for administration commonly comprise a solution of the improved IFNa2 polypeptide dissolved in a pharmaceutically acceptable carrier, preferably in an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. The compositions can be sterilized by well-known, conventional sterilization techniques. A typical pharmaceutical composition for intravenous administration can be readily determined by one of ordinary skill in the art. The amounts administered are clearly protein-specific and depend on their potency and pharmacokinetic profile. Current methods for preparing compositions that are administered parenterally are known or are apparent to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 18th ed., Ack Publishing Company, Easton, PA, 1990 .

Compositions containing improved IFNa2 polypeptides of the invention or a cocktail thereof (ie, with other proteins) can be administered in therapeutic treatments. In therapeutic applications, the compositions are administered to a patient suffering from MS, cancer, and disorders associated with increased expression of INF2a2.

IFNa2 in sufficient quantity to cure or at least partially arrest the disorder. An adequate amount to achieve this is defined as "effective therapeutic dose". The effective amounts for this use depend on the severity of the disease and the general health status of the patient.

Simple or multiple administrations of the compositions can be administered depending on the dosage and the frequency that is required and tolerated by the patient. In any case, the composition should provide a sufficient amount of improved IFNa2 polypeptides of this invention to effectively treat the patient. Generally, depending on the intended mode of administration, the acceptable pharmaceutical compositions contain about 1% to about 99% by weight of an improved IFNa2 polypeptide of the invention, and 99% to 1% by weight of a suitable pharmaceutical excipient or carrier . Preferably, the composition will be from about 5% to 75% by weight of an improved IFNa2 polypeptide of the invention, with the remainder being suitable excipients or pharmaceutical carriers.

The improved IFNa2 polypeptides of the present invention, or their pharmaceutically acceptable compositions, are administered in a therapeutically effective amount, which varies depending on a variety of factors including the specific activity of the particular improved IFNa2 polypeptide employed; the metabolic stability and the length of action of the improved IFNa2 polypeptide; age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the combination of the drug; the severity-specific disease status; and the therapy that the host experiences. Generally, a therapeutically effective daily dose is from about 0.1 g to about 1000 μg / 1 ^ of body weight per administration of an improved IFNa2 polypeptide of the invention, preferably, from about 0.5 μg to about 100 μg / kg of body weight per administration. For example, for administration to a 70 kg person, the dosage range would be from about 10 μg to about 100 g by administration of an improved IFNα polypeptide of the invention depending on the treatment regimen. For example, if the improved IFNa2 polypeptide of the invention or the formulation containing the polypeptide is administered from one to several times a day, then a lower dose would be necessary if a formulation were administered weekly, or monthly or less frequently.

It is anticipated that the IFNa2 polypeptides of. The present invention is administered in appropriate doses. The doses are generally adjusted to or below the maximum tolerated dose (MTD). Signs indicative of interferon toxicity are noted in terms of hematological toxicity, anemia, thrombocytopenia, leukopenia: in terms of gastrointestinal toxicity, diarrhea, dyspepsia, dysphagia, N / V, pain. abdominal; regarding increases in liver toxicity in bilirubin, alkaline phosphatase and LFTs; in the kidney and bladder, microscopic hematuria, pyuria, azotemia, proteinuria, acute renal failure, nephrotic syndrome, glucosuria, albuminuria; in terms of pulmonary, orthopaedic, dyspnea, bronchospasm, coughing, pulmonary edema, ARDS; in terms of cardiac syncope of toxicity, MI, SVT, bradycardia, tachycardia, dizziness, hypotension, hypertension. The neurological toxicity are confusion, tremors, numbness, paresthesia, inability to concentrate, drowsiness, hallucinations, encephalopathy, attack, coma, psychomotor retardation, memory dysfunction, dry mouth, sweating, personality disorder, agitation, neuropathy, depression, anxiety, aphasia, retinal infarction with loss of vision, eye pain, hemianopsis, taste change, headache, syncope, insomnia. Dermal toxicity of skin rash, urticaria, epidermal necrosis, maculopapular rash is observed. Metabolic toxicity manifests as hyperglycemia. In addition, coagulation is monitored to increase in PT / PTT. Also the presence of pharyngitis, alopecia, fatigue, malaise, anorexia, weight loss, fever, chills, myalgia, arthralgia, cyanosis are potential toxic responses to interferon.

Therapeutic Indications Multiple sclerosis (MS) The two forms of IFN, INFpla and IFNplb, are approved for the treatment of MS that relapses. IFN-ß is also used to treat genital warts. The improved IFNa2 polypeptides of this invention are useful in the above diseases, disorders, or conditions, and are also useful for the treatment of other forms of MS, including secondary progressive MS, primary progressive MS, and progressive relapse MS. By "useful" it means that improved IFNoc2 polypeptides are useful for the treatment of diseases, for example, either to prevent the disease, or to prevent the progress of the disease to a more serious state, or to ameliorate or reduce a symptom or consequences of the disease, such as MS. Cancer Despite numerous advances in cancer treatment, well-known lifestyle changes that can greatly reduce the risk of cancer, and the early warning signs that some cancers provide, numerous patients still develop cancer for which conventional therapies are not available which offer any reasonable hope of cure or significant relief. IFNa2 is known to have anti-cancer effects.

A person suffering from advanced cancer may exhibit one or more of the following signs or symptoms: presence of cancerous tumor, fatigue, pain, diminished state of tumor load behavior, and the well-known symptoms associated with each specific cancer .

To practice the invention, IFNa2 mutants are administered to the patient exhibiting one of more of the aforementioned signs or symptoms in sufficient amounts to eliminate or at least alleviate one or more of the signs or symptoms. Infection of Hepatitis Virus (HCV) Current therapies to treat HCV infection suffer from certain drawbacks. Dosage regimens involve daily injections (QD), every other day (QOD), or three times weekly (TIW) of IFN-cc during extended treatment periods from one or more of the following drawbacks: (1) regimens of dosage are uncomfortable to the patient and, in some cases, result in reduced patient compliance; (2) dosing regimens are often associated with adverse effects, causing additional discomfort to the patient, and, in some cases, resulting in reduced patient compliance; (3) dosing regimes result in "peaks" (Cmax) and "troughs" (Cmin) in the concentration of IFN-a serum, and, during periods of "trough", the virus can replicate, and / or infect additional cells, and / or mutate; (4) In many cases, the log reduction in viral concentration during the early viral response is insufficient to effect the sustained viral response that ultimately results in the release of the virus. The present invention could have a significant impact on the improvement of the therapeutic potential of infection of the treated hepatitis virus.

Insulin dependent diabetes mellitus (IDDM) The etiology of IDDM is a matter of great discussion. It seems to be multifactorial, including genetic predisposition and environmental influences. Strong associations are identified between IDDM and the specific HLAs are encoded by the major histocompatibility complex region located at the short end of chromosome 6. The higher high risk alleles are DR3 and DR4. Environmental influences through importance include viruses, and various lines of evidence support this view. Certain diabetogenic or associated viruses (mumps, measles, rubella, M encephalomyocarditis, coxsackievirus B, and reovirus) have been epidemiologically associated with the development of IDDM or diabetes of the cause of the boat when they are inoculated into rodents. Diabetogenic viruses can directly infect B cells in the culture. In addition, coxsackievirus B4 has been isolated from the pancreas of a child with the new onset IDDM who died of severe ketoacidosis, and this virus produced diabetes in experimental animals along with viral antigens in the B cells of the infected animals. Interferon alpha B cells that secrete IDDM patients, thus the antagonistic IFNa2 polypeptides of this invention could have a significant impact on the improvement of the therapeutic potential of IDDM.

Lupus erythematosus (LE) LE is an autoimmune disease that causes inflammation and damage to body tissues and parts, including the joints, kidneys, heart, lungs, brain, blood vessels, and skin. The most common symptoms of LE include swollen or painful joints (arthritis), fever, prolonged or extreme fatigue, skin rashes and kidney problems. Although the cause of LE is still unknown, LE is believed to be caused by a combination of genetic, environmental, and possibly hormonal factors. LE may be characterized by periods of illness or exacerbation and periods of well-being or remission. Currently, the objectives of effective LE treatment are to prevent widening, minimize organ damage and complications, and maintain normal bodily functions. Medications commonly prescribed for LE include nonsteroidal anti-inflammatory drugs (NSAIDs), acetaminophen, corticosteroids, antimalarials and immunomodulators. Due to the limited success of currently available medications and their potentially serious side effects, it is important to provide an alternative effective treatment for LE. The composition of the invention can be effective in minimizing and / or eliminating different symptoms in patients with LE. Having now generally described the invention, it should be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the present invention.

EXAMPLES Methods: (i) Mutagonosis directed to the site. Site-directed mutagenesis was carried out by PCR amplification of the expression of plasmid pT72C with 18-21 nucleotide primers containing the mutated codon using high fidelity polymerases pwo (Boehringer Mannheim) and pfu (Stratagene) as previously described (Piehler, J. &Schreiber, G. 1999, J. Mol. Biol 294, 223-237). After phosphorylation and ligation, the mutated plasmids were transformed into the TG1 cells of E. coli. The sequence of the fully expressed gene containing the mutation was verified by DNA sequence. (ii) Expression and purification of the protein. IFNa2 and IFNAR2-EC were expressed in E. coli and purified by ion exchange and size exclusion chromatography as previously described (Piehler, J. &Schreiber, G. 1999, J. Mol. Biol 289, 57- 67). The concentrations of the protein were determined from the absorption at 280 nM with e2ß? = 18070 cnf XM_1 for IFNa2 and e28? = 26500 crn ^ M "1 for IFNAR2-EC IFNAR1-EC was expressed in the Sf9 insect cells and purified as described (Arduini, RM et al., 1999, Protein Science 8, 1867-1877). purity of the protein was analyzed by SDS-PAGE under non-reducing conditions The concentration of active IFNa2 protein was determined for all mutants by analytical gel filtration with IFNAR2-EC as previously described (Piehler, J. &Schreiber, G. 1999, J. Mol. Biol. 289, 57-67) .For ligand binding studies, IFNa2 and IF p were specifically labeled at the site using maleimide chemistry.The IFNP was directly labeled on its cysteine residue C17 free by 2-fold molar excess incubation of the maleimide (OG488) Oregon Green (Molecular Probes) for 2 h in HBS The IFNa2 was labeled with OG488 after incorporating an additional cysteine residue (S136C mutation), which has been shown so as not to affect the interaction with IFNAR1-EC or IFNAR2-EC (Gavut is et al., 2005, Biophysical Journal 88 (6): 4289-4302). (iii) Growth and refolding of IFNa2-YNS mutates e. The E. coli were grown in a 750 ml flask. The bacterium was centrifuged, resuspended in 30 ml of lysis buffer and sonicated in bacteriology for 30"for five times, followed by centrifugation at 16,000 rpm for 30 ', pelleting (body inclusion) was resuspended in ~ 30ml of the washed solution of triton (0.5% newts, 50 mM Tris8 and 100 mM NaCl). The inclusion bodies were centrifuged at 25,000 g (14,500 rpm in ss34 sorval) and washed for 4-5 times. A final wash was carried out to make triton, using 50mM Tris (8.4) with NaCl of 100m. The purified inclusion bodies were dissolved in 5ml of the 6M guanidine for about 12hr at 4 ° C with gentle agitation. Following the centrifugation at 25000g for 20 ', the supernatant was maintained and the palletizing was discarded. For the refolding, the supernatant was diluted 1:20 in 0.8 M arginine pH 9.3, was stirred slowly while it was done in a vertex and was stirred at 20 ° C for 2 hours. Following centrifugation at 16,000rpm for 20 'and dialysis against Tris 7.4, the protein was purified by ion exchange chromatography to provide: ~ 5 mg / 1 of the pure protein. (iv) Measurements of link affinity. The interaction between the recombinant IFNAR1-EC and IFNa2 was monitored by the reflectometric interference spectroscopy (RIfS) under the conditions through the flow (Schmitt, HM, et al., 1997, Biosens. &Bioelectron., 12, 809-816 ). This method detects the biomolecular interaction at the interfaces as a change in the apparent optical thickness of a thin silica layer. The link to the surface is monitored as a turn in the interference spectrum. A rotation of 1 pm corresponds to approximately 1 pg / mm2 of the protein on the surface. A second method applied for real-time link measurements was by SPR (surface plasmon resonance) using ProteON (Biorad). All measurements were carried out using HBS (20 mM HEPES pH 7.5, 150 mM NaCl and 0.01% triton X100) as a buffered buffer solution. The binding of the IFNARI receptor subunit to the surface of the microplate was made by the two methods. In an anti-IFNAR1-EC mAb DB2 neutralize surface (dextran functionalized with amino AMD 500) was coupled by exposing amino groups by amine coupling chemistry. IFNAR1-EC captured affinity to the surface by crosslinks with a second AA3 mAb. In a second method, the His tagged end of IFNAR1 was ligated directly to the NTA surface of the chip. A detailed description of the method used to measure interferon-IFNAR2-EC interaction is given in (Piehler, J. and Schreiber, G. 2001, Anal. Biochem. 289, 173-186). For the weak link, the determination of the dissociation constant KD was obtained from the equilibrium response plotted against the concentration of the protein and is equipped according to the law of mass action. For close link interactions, constant kinetic ranges were determined and affinity was evaluated from the kapagado / kencendido = KD range as well as directly from the equilibrium response. (v) Antiviral test of protection activity. The antiviral activity of wild-type and mutant IFNa2 was analyzed as the inhibition of the cytopathic effect of vesicular stomatitis virus (VSV) on human WISH cells (Evinger, M. et al., 1981, Arch. Biochem. Biophys. , 319-329). The relative activity of IFNa2 mutants was determined as the concentration necessary for the protection of 50% of the cells relative to the concentration of wild-type IFNa2 required for 50% protection. (vi) Antiproliferative assay. The antiproliferative activity of IFN 2 in the lymphoma cells of Daudi Burkitt was done by assay as described (Piehler, J. et al., 2000, J. Biol. Chem. 275, 40425-40433). The cells were treated for 60 h with IFN. The number of living cells was then determined using a cell staining kit (Biological Industries Co, Israel) based on the colorimetric detection of the cleavage of the tetrazolium salt XTT in formazan. The antiproliferative assay in WISH cells was conducted by adding interferon in different dilutions to the growth medium, and monitoring the density of the cells after 72 hours by staining the purple crystal. (vii) Errors. The error (s) in KD for the IFNa2-IFNAR2 link as measured in RIfS is 20%, and for the relative affinity (p / mut) 30%. For IFN 2-1FNAR1, the error (o) in KD is 35% and for the relative affinity (p / mut) 50%. Using 2s (95% confidence) as the basic confidence level, this should suggest that a minimum of a two-time change can be seen as important. The magnitude of the error (s) for the individual biological activity measurements (if antiviral or antiproliferative) is 35%. Thus, a confidence level 2a between two measurements should suggest that the differences smaller than twice within the experiment error. (viii) Dyed oligonucleotide microarray experiments. Poly-L-lysine-coated glass microarrays containing nearly 19,000 different probes (Compugen human oligonucleotide pool) were purchased from CAG (Center for Applied Genomics, New Jersey). The microarrays were formed into probes with a mixture consisting of cyanine (Cy) 3 or Cy5 labeled cDNA representing the IFN treatment against no treatment (control). WISH cells were treated with different IFNs for 16 hours and their RNA extracted (kit RNeasy Midi, Qiagen), 100 and g of which was subjected to reverse transcription (mutant RT mutant H-M-MLV, Promega) with modified dUTP nucleotide with aminoalyl (Ambion) mixed in the mixture of nucleotides in a ratio of 4: 1 aa-dUTP to «dTTP. The produced cDNA was labeled with the fluorescent probe Cy3 or Cy5 activated with NHS (Amersham), which binds to aa-dUTP. The incorporation was evaluated (NanoDrop spectrophotometer) and the labeled cDNAs were mixed (treatment with one color and control with the other) in equivalent amounts of fluorescent dye (100 pmol each), in 2X final SSC, 0.08% SDS and 6 μ ? of blocking solution (Amersham), in 100 μ? of target volume, denatured at 95 ° C for 3 minutes, cooled and applied between a raised sliding cover (LifterSlip, Erie Scientific Company) and the configuration. Hybridization was done at 55 ° C for 12 hours in the dark. Subsequently, the slide was washed five minutes in 2X SSC, 0.5% SDS at 55 ° C; five minutes in 0.5X SSC at room temperature and finally about five minutes in 0.05X SSC at room temperature. Then it was dried by turning 3 minutes at 1000 rpm, and stored in the dark until swept. (ix) Image of microarray and data analysis. The scanning of the hybridized microarrays was carried out with a DNA microarray sweep (Agilent). Automatic spot detection, backup subtraction and intensity quantification were done with SpotReader software (Niles Scientific). Data normalization, filtering and group analysis were done with GeneSpring software (SiliconGenetics). The analysis is based on the treatment for the control relationship of each point, and includes such manipulations as data filtering by SpotReader output flags, an indication of the quality of the signal, in relation to the backup (noise). Each treatment (condition) is represented by two microarray replicates scanned by two dyes. A starting list of interferon-modulated genes was integrated by genes beyond 1.7 threshold in at least two conditions, tolerating only an "absent" flag (? '), Which is indicative of a "noisy" point, in all the conditions by gene. This criterion was chosen after the genuine envelope or sub-regulation is observed in more than one fair IFN treatment, in order to choose two conditions as a minimum, a low severity threshold is necessary to include modified ISG in relation to lower levels . The list of these genes was exported in an Excel worksheet, and contains the average treatment for the value of the control relation, the replicated values, p-values of test t, which is an estimate of the technical error of microarray with base on the distance between the replicates, and flag codes. The genes are then selected from the eye for importance, by comparing the distance between each pair of replicates and between the conditions, also observing the p-values and the presence of the flag ?? . This tedious analysis was necessary due to the statistical selection that clearly left over / under-regulated out, due to the relatively large noise produced for the lower number of replicates per condition. An average value beyond the threshold 1.7 that has one of its replicates below this threshold, or with a large inter-replicated distance relative to the medium, and without other conditions with similar and important levels, was not considered a bona-fide modulation , but technical noise, and the gene was subsequently excluded from the list. The final list consists of 395 genes, over-regulated or sub-regulated by interferons in 16 hours of treatment. The group analysis was made on this list after importing it back into GeneSpring.

EXAMPLE 1. Characterization of IFNa2-IFNARI-EC interaction using an optical biosensor system The interaction of IFNARI-EC with IFNa2 was investigated by the detection of free solid phase labeled using RIfS. This technology requires the immobilization on the microplate surface of one of the interaction compounds. The previously successfully used method for IFNAR2-EC was used, where the receptor is immobilized using mAbs which are themselves immobilized to the surface by means of amino coupling (Piehler, J. and Schreiber, G. 2001, Anal. Biochem. 289, 173-186). The non-neutralized DB2 mAb anti-IFNAR1 mAb was coupled to AMD 500 surface of amino-functionalized dextran by means of exposed amino groups. IFNARl-EC was captured with affinity to the mAb surface followed by cross-linking with a second AA3 mAb. The procedure as follows in real time RIfS is shown in Fig. 1A. The binding activity of IFNAR1-EC bound to the RIfS surface was determined by injecting the IFNa2 protein at concentrations ranging from 0.12 to 4 μ ?. Due to the lower binding affinity of IFNa2 for kinetic data IFNAR1-EC the association and dissociation may not be determined. Therefore, the binding affinity was determined by plotting the steady-state response against the protein concentration according to the law of mass action. This result in an affinity constant of 1.5 μ? for wild-type IFNa2 (Lamken, P. et al., 2004, J Mol Biol 341, 303-318).

EXAMPLE 2. Mapping of the IFNa.2 liaison site for IFNARl To locate the binding site of IFNAR1 in IFNa2, a Wing sweep of the residues most located in helices B and C was carried out (Fig. 2 and 3). All together, 21 single point mutations were produced, and their binding affinity towards IFNAR2 and IFNAR1 were measured by RIfS or ProteON. As the mutations are located on the opposite surface of the IFNAR2 binding site on IFNa2, the binding affinities of the different mutant proteins towards IFNAR2 were very similar to that of wild-type IFNa2 (Table 1). This is ensured, while validating the proper folding of all mutant proteins, and their active concentrations.

While the binding of IFNa2 with IFNAR2-EC provides a clear kinetic signal, binding to IFNAR1-EC is much weaker and has been analyzed according to the refractive unit (RU) signal of constant state affinity, by varying the concentration of the ligand. In this case, knowledge of the correct and precise concentrations of all IFNa2 proteins is essential. Therefore, all mutant protein concentrations were validated in an analytical gel filtration column for their ability to bind IFNAR2-EC. The results of all link measurements are summarized in Table 1 and 3 and Fig. 3A. Three simple mutations cause an increase in the bond (H57A, E58A, Q61A) while 6 mutations cause a decrease in the bond (F64A, N65A, T69A, L80A, Y85A, Y89A). However, no hot spots for the link were identified, thus, none of the mutations lead to a change of about 5 times in affinity. To corroborate the effects of single mutation, a triple mutant containing the single mutations L80A, Y85A, Y89A (designated herein as LYY) and a quadruple mutant N65A, L80A, Y85A, Y89A (SEQ ID NO: 6) (designated in the present as NLYY) were prepared. The binding of these two mutant proteins was below the threshold of the measurement.

Table 1: Thermodynamic and kinetic constants for mutant IFN interactions < x2 (B and C helix and C-D curl) with IFNAR2-EC and IFNA 1-EC IFNAR2 -EC IFN IFNAR1-EC mutant a.AG ° e K "KD c KD b.AG ° C. KD [nM] wt / mut (kcal / m (kcal / [μ?] Wt / mut (kcal / mo ( kcal ol) mol) 1) ol) . wt 0.011 3 -11.6 0.0 1.6 -7.9 H57A 0.015 4 1.0 -11.4 -0.2 0.69 2.1 -8.4 0.5 E58A 0.011 3 1.3 -11.6 0.0 0.31 4.7 -8.8 0.9 Q61A 0.012 3 1.3 -11.6 0.0 0.59 2.5 -8.5 0.6 Q62A 0.009 3 1.3 -11.6 0.0 1.82 0.8 -7.8 -0.

F64A 0.013 4 1.0 -11.4 -0.2 3.33 0.4 -7.4 -0.

N65A 0.012 3 1.3 -11.6 0.0 5.00 0.3 -7.2 -0.7 S68A 0.010 3 1.3 -11.6 0.0 2.00 0.7 -7.8 -0.

T69A 0.012 3 1.3 -11.6 0.0 3.70 0.4 -7.4 -0.

K70 0.010 3 1.3 -11.6 0.0 2.56 0.6 -7.6 -0.

D71A 1.67 0.9 -7.8 -0.

S73A 0.014 4 1.0 -11.4 -0.2 2.94 0.5 -7.5 -0.

T79A 0.013 4 1.0 -11.4 -0.2 1.45 1.0 -7.9 0.0 L80A 0.019 5 0.8 -11.3 -0.3 8.70 0.2 -6.9 -1.

K83 0.011 3 1.3 -11.6 0.0 1.47 1.0 -7.9 0.0 Y85A 0.019 5 0.8 -11.3 -0.3 4.17 0.4 -7.3 -0.

T86A 0.016 4 1.0 -11.4 -0.2 2.78 0.5 -7.6 -0.4 Y89A 0.017 5 0.8 -11.3 -0.3 5.88 0.3 -7.1 -0.

Q90A 0.013 4 1.0 -11.4 -0.2 1.43 1.0 -7.9 0.0 N93A 0.016 4 1.0 -11.4 -0.2 3.23 0.5 -7.5 -0.

E96A 0.013 4 1.0 -11.4 -0.2 2.86 0.5 -7.5 -0.

V99A 0.010 3 1.3 -11.6 0.0 2.00 0.7 -7.8 -0.

HEQ 0.01 3 1.0 -11.6 0.0 0.083 20 -9.6 1.7 NLYY aS- 0.002 0.15 18 tail -13.3 1.8 D IFN 0.0012 0.1 -13.6 0.066 -9.75 1.8 a AG ° calculated from RTlr \ KD; b AAcP - AG ° mut "AG ° wt; c KD is determined from the bond in equilibrium at the surface; d Data are from Lamken et al., 2004, J. Mol. Biol. 341, 303-318.

EXAMPLE 3. Antiproliferative activity and anti-viral mutants IFN < x2 The antiviral activity was analyzed as the inhibition of the cytopathic effect of vesicular stomatitis virus (VSV) in WISH cells (human epithelial cell line of amniotic origin). The antiproliferative activity was determined in the WISH cells and in the Daudi cells, such as the 50% inhibition of cell growth (as described in methods). Fig. 4A shows the result of applying a dilution of the series of concentrations of IFN a2 and IFNp for 72 hours in the intensity exhibited by the WISH cells after staining, from which the concentration of interferon that promotes an antiproliferative effect it can be deduced. The wells were explored for densitometry analysis, and the results were plotted against the applied protein concentration (Fig. 4B). The same was done to determine the antiviral potency of interferons in WISH cells (Fig. 4C). The activity point of 50% as well as the rate of change in activity (slope) was deduced from a curve of the IFN response dose, Eq. 1. y = A0 + A / (1 + (c / c50) ') [Ec. 1] where y is the transmittance that is related to the number of cells, A0 is the compensation, A is the amplitude, c is the concentration, C50 is the concentration that gives the activity of 50%, and s is the slope. The average concentrations of IFN a2 and IFN that promote antiviral protection at 50% are 0.85 and 0.43 pM respectively (averaged over 6 experiments). The average concentrations of IFN oc2 and IFNp that promote antiproliferative activity at 50% are 1360 and 29 pM (averaged over 17 and 7 experiments respectively). Interestingly, the IFNoc2 concentration that promotes 50% antiproliferative activity in Daudi cells is only 0.5 pM (Table 2), which is about 1000 times lower than that measured for ISH cells, which highlights the importance of using the same cell line for all experiments. The antiviral and antiproliferative activity of all the interferon mutant proteins was determined in relation to the wild-type a2 IFN (wt / mut) with the results summarized in table 2 and 4. As found with the binding affinity for IFNAR1-EC , the antiviral and antiproliferative activities did not change dramatically with the mutation. The only mutant that causes a significant increase in antiviral activity (> 2 times) is E58A. L80A, Y85A and Y89A cause a significant decrease in antiviral activity (but none greater than 6 times). However, the combined mutants of LYY and NLYY reduced the antiviral potency by 30 and one hundred fold, respectively. This reduction roughly adjusts the additive effects of the only mutations (for LYY, 0.5x0.26x0.20 = 0.029, similar to the result obtained for the triple mutant). The effect on the antiproliferative activity of the same set of mutants is more significant, with E58A and Q61A causing a significant increase in activity, while N65A, T69A, L80A, Y85A and Y89A cause a significant decrease in activity. On the other hand, the magnitude of the decrease in activity as measured in the antiproliferative assay when using ISH cells is significantly greater than that measured for antiviral protection. Multiple mutants of LYY and NLYY reduce activity by 190 and 1100 fold, respectively. In this case, the only mutations overestimate the effect measured for the multiple mutants by about 30 times (for LYY, 0.14x0.035x0.044 = 0.00022 against the experimental measurement value of 0.0057, and for NLYY 0.16x0.14x0. 035x0.044 = 0.000035 against the experimental measurement value of 0.00092). The antiviral and antiproliferative results for all mutants analyzed are plotted in Fig. 5A. For interest, we also add the measurement value for ??? ß. It appears from this graph that the antiproliferative response is much more sensitive to the IFN specific IFN mutant compared to the antiviral response. On the other hand, this trend also fits the data for IFNP, for which the antiviral activity is about 2 times higher than that of IFN a2 (similar to the antiviral activity of the mutant E58A) while its antiproliferative activity is ~ 50 times more high. Comparing the biological activity with the binding affinity measured for IFNAR1 shows that both activities are generally scaled with the binding affinity, but not on the full scale (Fig. 5B).

Table 2: Biological activity of mutants IF < x2 Activity Antiproliferative activity Antiproliferati antiviral (ISH) (DAUDI) IFN (WISH) Mutant Pendien Pendien 50% 50% 50% wt / mut tea wt / mut tea (pM) wt / mufe (nM) (pM) wt / mut wt / mut wt 0.85 1.36 0.5 H57A 0.57 1.5 1.08 0.76 1.8 1.0 1.12 0.56 E58A 0.26 3.3 0.95 0.25 5.4 1.15 0.20 2.45 Q61A 0.45 1.9 1.04 0.49 2.8 1.65 0.54 0.92 Q62A 0.45 1.9 1.05 1.3 0.45 1.12 F64A 0.77 1.1 2.72 0.5 0.68 0.73 N65A 1.06 0.8 0.99 8.00 0.17 1.48 0.58 0.86 S68A 0.57 1.5 0.68 2.0 0.52 0.96 T69A 0.94 0.9 3.40 0.4 0.31 1.61 K70A 0.71 1.2 1.70 0.8 0.33 1.51 D71A 0.61 1.4 1.94 0.7 0.30 1.67 S73A 0.61 1.4 2.27 0.6 0.57 0.88 T79A 0.61 1.4 1.94 0.7 0.42 1.20 L80A 1.70 0.5 9.71 0.14 0.47 1.07 K83A 0.65 1.3 1.36 1.0 0.56 0.89 Y85A 3.27 0.26 1.0 39.0 0.035 1.84 1.52 0.33 T86A 0.77 1.1 1.36 1.0 2.2 0.35 1.41 Y89A 4.25 0.20 1.0 30.0 0.045 3.13 0.16 Q90A 0.71 1.2 0.72 1.9 0.45 1.11 N93A 0.85 1.0 1.94 0.7 0.60 0.84 E96A 1.06 0.8 3.40 0.4 1.25 0.4 V99A 1.13 0.75 1.84 0.74 0.51 0.98 LYY 24.29 0.035 1.32 238 0.0057 4.1 NLYY 92.39 0.0092 1478 0.00092 3.2 HEQ 0.41 2.07 0.05 24.18 ot8-tail 0.3 3 0.13 10 0.43 2 0.72 0.03 42.86 0.63 LYY is the triple mutant L80A, Y85A, Y89A. NLYY (SEQ ID NO: 6) is the quadruple mutant including LYY and N65A. The activity point at 5 was obtained by adjusting the data in Equation 1. a The slope is from Eq. 1.

EXAMPLE 4. Ternary complex stability is improved by HEQ compared to IFN oc2 Since three mutations for Ala were found to increase the binding by 2 to 4 times, these three IFN a2 mutations (H57A, E58A, Q61A) were combined into a single IFN a2 mutant protein, as used in the present, designated HEQ (SEQ ID NO: 5). The binding of IFNs to the extracellular domain of the type I interferon receptor (IFNARI-EC), and to the ternary complex that included IFNAR2-EC and IFNARI-EC was studied by internal reflection fluorescence internal spectroscopy (TIRFS). IFNAR2-EC and IFNARI-EC had lateral extensions through their C-terminal histidine tags on lipid bilayers supported in solids. The kinetics of the dissociation of IFN < x2-wt, HEQ, and IFN of IFNARl-EC as observed by TIRFS was compared in Fig. 1C. A 20-fold increase in the lifetime of the complex was observed for HEQ compared to IFN a2, and the similar kinetics of dissociation for IF P (Table 3). In contrast, the very similar rate constants of the association were obtained for the three species. From these data, a dissociation constant of the equilibrium KD = 83 nM was determined for the HEQ / IFNARl-EC complex, KD = 33 nM was determined for the YNS / IFNARI-EC complex, KD = 1.6 μ? for the complex IFNa2-wt / IFNAR1-EC and KD = 66 nM for the IFN / IFNARI-EC complex. The binding affinity of HEQ to IFNAR2-EC is similar to that measured for IFN a2-wt (5 nM), which is about 10 times weaker than that of IFNP (Table 3). Thus, only the affinity of HEQ towards IFNARl is similar to IFNP, but not towards IFNARl.

IFN induces biological activity by the formation of the ternary complex (IFNAR1-IFN-IFNAR2) in 1: 1: 1 stoichiometry. Due to the cooperative binding to the two receptor subunits attached to the surface, the apparent binding affinity of the ligand is higher than the binding to IFNAR2 alone, and depends on the relative and absolute surface concentrations of the receptor (Lamken, P. and others , 2004, J. Mol. Biol 341, 303-318). The great stability of the ternary complex by increasing affinity towards IFNAR1 could be formed in probe by the kinetics of ligand dissociation. Therefore, the dissociation of IFN a2 (SEQ ID NO: 2), HEQ (SEQ ID NO: 5), YNS (SEQ ID NO: 11), a8-tail (SEQ ID NO: 7) and IFNp (SEQ ID NO: 3) were compared at different concentrations of IFNAR1-EC and IFNAR2-EC in the stoichiometric ratio. For IFNP, measurements were made with the mutant I47A in IFNAR2, which binds IFNP at an affinity of 5 nM, similar to that of IFN oc2 towards IFNAR2-wt (Table 3). This compares only the effect of surface concentration of IFNAR1 on ligand dissociation. Although for IFNa2, the kinetics of the dissociation strongly depends on the surface concentration of the receptor, the overlap curves of the dissociation were observed for IFNP and HEQ. This clearly demonstrates the higher stability of the ternary complex formation with IFN and HEQ. Even at the highest concentration of the receptor (which is much higher than the concentration of the cellular receptor at 1000 receptors per cell), a faster dissociation of wild type aN IFN was observed compared to IF P and HEQ at the lower concentrations of the receptor. receiver. However, if concentrations of the receptor surface are increased further, IFN a2-wt eventually dissociates at speeds comparable to IFNP or HEQ. The stability of the ternary complex is a combined function of the surface receptor concentrations and binding affinities towards individual receptors. Assuming that the stability of the ternary complex is related to biological activity, it is not surprising that the differential activation of a more hermetic bond of IFNs can be sequestered in cells with high numbers of the receptor, either natural (similar in Daudi cells) or due to transfection. It also highlights the danger of transfecting cells with cell surface receptors at non-native levels, a generally ignored issue.

Table 3: Speed constants and affinities of the IFN interaction < x2 wild-type, ßβ and IFN a2 mutants with IFNAR1-EC and IFNAR2-EC IFNAR1-EC IFNAR2-EC ka [M "1s" * d Is "1] KD [nM] ta [- X] kd Is" 1] KD [nM] X] a c IFNa2 ~ 2-10b 1500 1 -10 '0.006 0.6 a IFNP 3 · 105 0.020 66 5-107 0.0012 0.024 b HEQ at 3 -105 0.025 83 1 -107 0.006 0.6 Y S 3 -105 0.01 33 1 -107 0.006 0.6 < x8- 7 -107 0.002 0.024 cola a site-specific S136C mutant labeled with Maleimide 488 from Oregon Green; b specific to the site marked with the maleimide 488 of Oregon Green on the free residue C17 c interaction with IFNAR2-EC I47A EXAMPLE 5. Antiviral and antiproliferative activity of IFN a2 mutants: HEQ, MDL, a8-tail and YNS It has been observed that the antiviral potency of IF in WISH cells is only 2 times higher compared to IFNa2, although its antiproliferative activity is about 50 times higher. Figs. 4B-4C show the antiviral and antiproliferative activities of IFN < x2-wt, HEQ (SEQ ID NO: 5) and IFNP, as measured in WISH cells. In these trials, the profile of HEQ activity is comparable to that of IFNp. Despite the large increase in HEQ binding affinity towards IFNAR1, its antiviral potency is only 2 times higher than that of IFN < x2 wild type. Its antiproliferative activity is increasing by ~ 25 times compared to a 42-fold increase in the antiproliferative activity of IFNp (Table 4). The same trend is true also for MDL (SEQ ID NO: 10) and YNS (SEQ ID NO: 11). Although its antiviral activity is approximately that of IFN a2, its antiviral activity is much higher. MDL presents the same level of activity as IFNP, while YNS presents an even higher level of antiproliferative activity. The increasing antiproliferative activities are in line with the increasing affinities of these two IFNs towards IFNAR1. As previously observed, IFN seems to stop the faster growth compared to IFN a2. Consequently, the cell count after applying high concentrations of IFN is smaller for IFNp. This phenomenon can not be compensated using a higher concentration of IFN a2, but it is a qualitative difference (Fig. 4B).

In this aspect, HEQ and YNS behave similar to IFN oc2-wt, and not as IFNp. The slope of the curve of the HEQ titration (which is related to the concentration-dependent beginning) is between that of IFN cc2-wt and IFNp.

Table: Relative biological activities of IFN < x2 of wild type, of the mu an es and of IF P Anti - proliferative activity viral WISH M A231 WISH Relationship EC50 vs. EC50 Ratio EC50 vs. (nM) IFNa (nM) vs. IFNa (pM) IFNa ZFNa 2.0 1 0.7 1 0.6 1 ??? ß 0.03 71 0.07 10 0.3 2.0 YNS 0.013 164 0.01 70 0.12 5.0 MDL 0.085 24 0.6 1.0 HEQ 0.06 35 0.3 2.0 aS- cola 0.2 10 0.2 3.0 EXAMPLE 6. Monitoring gene activation using gene chip technology Gene chips have become a standard technology for obtaining complete information on differential gene activation. In fact, a number of studies have been conducted to investigate the profile of gene expression on the induction of IFN (de Veer, M. J. et al., 2001, J Leukoc, Biol 69, 912-920). The effect of IFNs on gene expression was monitored by microarray experiments of stained oligonucleotides. They treated the WISH cells for 16 hours with 0.3 nM IFN a2; 3 nM (10,000 units) IFN a2; 0.3 nM HEQ and 0.15 nM IFN (1,000 units). The concentrations were chosen to produce > 90% of an antiproliferative response (IFN a2-wt-3 nM, HEQ and IFNβ) or below 10% of the antiproliferative response (IFN a2-wt-0.3 nM) in WISH cells. In each condition the IFN treatment experiment against no treatment was performed in duplicate microarray with dye sweep. In addition, four replicates of the microarray experiments consisting of no treatment versus no treatment were used as additional control. The normalized relationship between the two colors is taken as the level of induction. The criteria chosen to define the list of genes regulated by IFN are described in the section on methods.

The results of the gene chip can be analyzed on a per gene basis, or by looking at general trends of gene induction. Fig. 6 gives an overview of the trends registered for the change in expression levels of 395 genes regulated by IFN, 16 hours after treatment with IFNp (0.15 nM), HEQ and IFN < x2-wt (0.3 nM and 3 nM, respectively). In Fig. 6A (no treatment) the relative expression levels of the 395 genes were plotted in ascending order according to the fold change. The black dots are from the control chip, which contained a mixture of only untreated samples in both color channels. This provides an estimate of the expected random fluctuation. The small and maximum changes in the level of expression recorded for the control chip were 0.65-1.6 times, with the ratio between two channels only 0.75-1.4 times for the top of 95% of the genes. The low level of random fluctuation in gene expression provides a high level of confidence in the quality of the data. Generally, gene expression levels are similar for IFNP (0.15 nM) and HEQ (0.3 nM) (Fig. 6A), while a much lower level of induction was recorded for IFN a2-wt (0.3 nM). The induction of the gene on the addition of 3 nM IFN a2-wt is between the levels recorded for IFN < x2-wt (0.3 nM) and IFNp (0.15 nM) or HEQ (0.3 nM). An alternative way to analyze the differential expression levels is by plotting the rate of change in the activation of the gene between the different treatments (Fig 6B). As IFNp induced the largest change, all other values were calculated relative to the induction of IFNp. The control is the same as in Fig. 6A.

Once again IFNP and HEQ seem to induce the same level of change for all genes (the green line follows the black line that is the control). This means that a single gene does not behave significantly different with induction with IF p against HEQ. This remarkable result shows how both of these IFNs are similar. The largest differential expression is found between IFNp and IFN a2-wt (0.3 nM). Again, the results for IFN a2-wt (3 nM) are among those registered for IFN a2-wt (0 .3 nMs) and HEQ.

The group analysis of the IFN-induced genes for the four treatments was performed to give independent verification of the results presented graphically in Fig. 6 A and 6B. Fig. 6C shows the ples of gene expression of the group of IFNP and HEQ together, with a very short distance between them. The ple of the expression of the cells treated with the IFN a2-wt 3 nM groups follows, although the ple of gene expression of IFN oc2-wt 0. 3 nM is much farther away. The ple of gene expression is in excellent agreement with the differential biological activity observed between INFP and IFN a2, and demonstrates again that HEQ is an IFN a2 with all the characteristics of IFNp.

The gene chips provide detailed knowledge of the expression levels of the individual genes comprising the genome. One of the most interesting aspects of analyzing the gene expression ple of IFN against IFN a2-wt is to investigate differential activation, and the relationship between differential activation and IFN concentration (Der, SD, et al., 1998, Proc. Nat. Acad Sci. USA 95, 15623-15628). Out of the 395 genes induced, 59 over-regulated and 9 sub-regulated genes were selected according to their differential level of activation between the different treatments (Jaitin et al., 2006, Mol Cell Biol 26 (5): 1888-1897, table 3).

The effect on the expression of IFNP and HEQ (SEQ ID NO: ) in most of the genes was much stronger than the effect induced by IFN a2 in the antiviral concentrations of the equivalent activity, ie 0.15 and 0.3 nM, respectively, including numerous genes not influenced by IFN a2 in the concentration of 0.3 nM.

EXAMPLE 7. Tumor prevention model MDA231 To establish subcutaneous (sc) tumors, MDA231 cells grew in vitro to the confluence of 70%, were trypsinized and suspended in PBS at a concentration of 108 cells / ml, and 107 cells were injected sc, on the flank of a mouse hairless. The mice were then randomized and separated into 3 treatment groups of 6 animals per group. The treatment regimen started on day 1 and continued to day 34. The mice were injected s.c. with PBS or a specific treatment twice a week for 34 days. After 34 days of treatment the animals were sacrificed, the tumors were excised and the tumor sizes were determined. The results are summarized in Table 5. The results clearly show that according to tissue culture experiments, YNS is very effective in suppressing the growth of MDA231 breast cancer cells in vivo. After 34 days of treatment with YNS all mice treated with YNS were clear of lumps, while five of six of mice treated with 1 IFN a2 and all six mice treated with PBS had lumps of varying sizes.

Table 5: The results of the MDA231 tumor prevention model Treatment PBS IFNCC2 YNS Days LMSNLMSNLMSN 1 1 0 0 5 4 2 0 0 6 0 5 1 0 4 0 1 0 5 0 3 0 3 0 8 1 2 2 0 1 0 5 0 3 2 1 0 12 1 2 3 0 0 0 5 1 3 1 2 0 15 3 2 3 0 0 0 5 1 1 3 0 2 18 3 0 3 0 0 0 4 2 1 3 0 2 22 3 1 2 0 0 0 4 2 1 0 0 3 26 3 2 1 0 0 1 4 1 0 0 0 6 29 3 2 1 0 0 1 3 2 0 0 0 6 33 3 2 1 0 0 2 3 1 0 0 0 6 34 3 2 1 0 1 2 2 1 0 0 0 6 The mice were injected with the MDA231 cells on day zero, followed by injections of 20 μg / mice of the different treatments at the indicated time. Six mice were treated for each experimental condition. Tumor growth was estimated by the eye for all except day 34. On the last day (34) the mice were sacrificed, and the tumor was removed and measured. L, M, S and N appear for tumor sizes (in parentheses it is the diameter measured on day 34), L - large (> 5 millimeters), M-medium (2.5-5 millimeters), S - small ( < 2.5 mm), N - none. EXAMPLE 8. Experimental autoimmune encephalomyelitis (???) Experimental autoimmune encephalomyelitis (EAE), also known as experimental allergic encephalomyelitis, is an animal model of multiple sclerosis (MS). Various EAE models are known in the art, depending on the induction method, the strain of the animal and the antigen used to induce the disease. EAE is an acute autoimmune disease or chronic, acquired, inflammatory and demyelinating relapse. The different forms of EAE closely resemble the various forms and stages of the MS.

In the present study, EAE is induced by the injection of myelin basic protein (MBP), a known method for the acute phase MS model. In this model the onset of the disease is observed by the appearance of clinical symptoms about 10 days after induction. The disease progresses and increases and clinical peaks of the score around day 15 and spontaneous recovery are observed around day 23 after induction of the disease. Female Lewis rats (average body weight 130-180 g, Harán, Israel) were injected s.c. on the hind paws with 25 μg of the purified myelin basic protein from guinea pigs (MBP, Sigma) emulsified in 0.1 ml of complete Freund's adjuvant (Difco). The animals are kept in a regime of 12 hours of darkness / 12 hours of light, at a constant temperature of 22 ° C, with food and water ad libitum. From the induction that followed on day 8, animals are tracked on daily bases. The results are recorded as clinical score; the score of 0 indicates a normal animal without clinical signs, 0.5 indicates a loss of tonicity in the distant part of the tail, 1 indicates complete paralysis of the tail, 1.5 indicates weakness in a hind leg, 2 indicates weakness in both hind legs, 2.5 indicates in a foreleg, 3 indicates paralysis of all four legs, 4 indicates complete paralysis of the body and moribund state and 5 indicates death. The clinical score of the animals is recorded for ~ 15 days following the onset of the disease until the end of the study 25 days after the induction and the area under curve (AUC) is calculated over this time interval.

Animals exhibiting the symptom of the disease, which could be clinically labeled between 0.5 and 1, are treated with compositions of HEQ (SEQ ID NO: 5), YNS (SEQ ID NO: 11) or vehicle control for three consecutive days from the beginning of the disease (~ on day 9-11 that follows the induction of the disease). Various routes of administration are evaluated, for example, intravenous (in vehicle) or oral by priming (in vehicle) in the volume dose of 5 ml / kg. On the last day of the study (on day 25) euthanasia is aed to animals with sodium pentobarbitone 100 mg / kg i.p.

The results are expressed as mean + SEM and the differences between the treatment groups were analyzed by analysis of variance (ANOVA) followed by Tukey's pst-hoc test. A value of p < 0.05 is considered to be statistically significant and is indicated in the figure by an asterisk on the relevant treatment group.

The validity of the model is established using methylprednisolone as a positive control. When steroids are administered daily for 5 consecutive days i.v. at 30 mg / kg from the day of induction of the disease by injection of MBP, a 34% reduction in AUC was reported. EXAMPLE 9. An animal model of systemic lupus erythematosus (SLE) Female hybrid mice (NZB x NZW) Fi (hereinafter referred to as "NZB / W mice") spontaneously develop a lupus-like disease characterized by the presence of serum autoantibodies to double-stranded DNA (dsDNA). These mice, which eventually experience kidney failure, are often used as a model for directed experimentation for better understanding and treating lupus in humans. Over time, this condition in NZB / W mice progresses to the malfunctioning of the kidney as manifested by the onset of proteinuria.

NZB / W female mice of 32 weeks of age were used in an experiment to determine if NLYY (SEQ ID NO: 6) could delay the development of the disease similar to lupus. The mice are divided into two groups, and each group is treated every third day with NLYY (group of 10 mice) or rat IgG as control (group of 10 mice), administered by intraperitoneal injection. The treatments continue for five weeks, and the mice are evaluated weekly for the presence of proteinuria. The above description of the specific modalities will reveal so completely the general nature of the invention that others can, aing current knowledge, modify and / or adapt easily for different acations to such specific modalities, without undue experimentation and without leaving the generic concept, and, therefore, such adaptations and modifications must and are intended to be included within the meaning and range of equivalents of the modalities set forth. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that numerous alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. It should be understood that the detailed description and the specific examples, while indicating the preferred embodiments of the invention, are given by illustration only, since different changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art. of this detailed description.

Claims (31)

1. A recombinant interferon a2 polypeptide (IFNa2), active fragment, analogue, derivative and variant thereof, the polypeptide comprises a mutation selected from at least one amino acid substitution within amino acid residues 57-89, at least one amino acid substitution of C-terminal amino acid residues 159-165, and combinations thereof, characterized in that the polypeptide has improved specific agonist or antagonist activity, as compared to Wild type IFNa2 (SEQ ID NO: 2).

2. The polypeptide according to claim 1, characterized in that the polypeptide comprises at least one mutation selected from the group consisting of H57A (SEQ ID NO: 24), E58A (SEQ ID NO: 25), Q61A (SEQ ID NO: 26), H57Y (SEQ ID NO: 27), E58N (SEQ ID NO: 28), Q61S (SEQ ID NO: 29), and combinations thereof, wherein the polypeptide has improved specific agonist or antagonist activity, in comparison with wild-type IFNa2 (SEQ ID NO: 2).

3. The polypeptide according to claim 1, characterized in that the polypeptide comprises a triple mutant H57A, E58A, Q61A (SEQ ID NO: 5) having enhanced specific activity, as compared to wild-type IFNa2 (SEQ ID NO: 2) .

4. The polypeptide according to claim 1, characterized in that the polypeptide comprises a quadruple mutant N65A, L80A, Y85A, Y89A (SEQ ID NO: 6) having antagonist activity, as compared to wild-type IFNoc2 (SEQ ID NO: 2) ).

5. The polypeptide according to claim 1, characterized in that the polypeptide comprises a substitution of the C terminal ESLRSKE to KRLKSKE (SEQ ID NO: 7) having improved specific activity, compared to the wild-type IFNa2 (SEQ ID NO: 2) .

6. The polypeptide according to claim 1, characterized in that the polypeptide comprises a combination of the triple mutant H57A, E58A, Q61A and the C-terminal substitution of ESLRSKE to KRLKSKE (SEQ ID NO: 8) having enhanced specific activity, as compared to wild-type IFNa2 (SEQ ID NO: 2).

7. The polypeptide according to claim 1, characterized in that the polypeptide comprises a combination of the quadruple mutant N65A, L80A, Y85A, Y89A and C-terminal substitution of ESLRSKE to KRLKSKE (SEQ ID NO: 9) having antagonistic activity, in comparison with wild-type IFNa2 (SEQ ID NO: 2).

8. The polypeptide according to claim 1, characterized in that the polypeptide comprises a triple mutant H57M, E58D, Q61L (SEQ ID NO: 10) having improved specific activity, compared to wild-type IFNa2 (SEQ ID NO: 2) .

9. The polypeptide according to claim 1, characterized in that the polypeptide comprises a triple mutant H57Y, E58N, Q61S (SEQ ID NO: 11) having improved specific activity, compared to wild-type IFNoc2 (SEQ ID NO: 2) .

10. The polypeptide according to claim 1, characterized in that the polypeptide comprises a combination of the triple mutant H57M, E58D, Q61L and C-terminal substitution of ESLRSKE to KRLKSKE (SEQ ID NO: 12) having enhanced specific activity, as compared to wild-type IFNa2 (SEQ ID NO: 2).

11. The polypeptide according to claim 1, characterized in that the polypeptide comprises a combination of the triple mutant H57Y, E58N, Q61S and the C terminal substitution of ESLRSKE to KRLKSKE (SEQ ID NO: 13) having enhanced specific activity, as compared to wild-type IFNa2 (SEQ ID NO: 2).

12. The polypeptide according to claim 1, characterized in that the polypeptide is also conjugated to PEG.

13. A DNA molecule encoding a polypeptide, characterized in that it comprises a mutation selected from at least one amino acid substitution within amino acid residues 57-89, at least one amino acid substitution of the amino acid residues of C-terminal 159-165, and combinations thereof, wherein the polypeptide has improved specific agonist or antagonist activity, as compared to wild-type IFNa2 (SEQ ID NO: 2).

14. The DNA molecule according to claim 13, characterized in that the DNA molecule comprises a sequence selected from the group consisting of SEQ ID NOS: 15-23 and SEQ ID NOS: 30-35.

15. A vector characterized in that it comprises the DNA molecule according to any of claims 13-14, operatively linked to one or more transcription control elements.

16. A host cell characterized in that it comprises the vector of claim 15.

17. A pharmaceutical composition characterized in that it comprises as an active ingredient a recombinant interferon a2 polypeptide (IFNa2), active fragment, analogue, derivative and variant thereof, the polypeptide comprises a mutation selected from at least one amino acid substitution within the amino acid residues 57-89, and at least one amino acid substitution of C terminal amino acid residues 159-165, and combinations thereof, wherein the polypeptide has improved specific agonist or antagonist activity, as compared to wild-type IFNa2 ( SEQ ID NO: 2), which further comprises a pharmaceutically acceptable carrier.

18. The pharmaceutical composition according to claim 17, characterized in that it comprises the recombinant a2 interferon polypeptide (IFNa2), having any d SEQ ID NOS: 5-13 and SEQ ID NOS: 24-29, fragments, analogues, derivatives and variants of the same.

19. The pharmaceutical composition according to claim 18, characterized in that it comprises the recombinant a2 interferon polypeptide (IFNa2), having any of SEQ ID NOS: 5, 7, 8, 10, 11, 12 and 13, fragments, analogs, derivatives and variants thereof, wherein the polypeptide has improved specific agonist activity.

20. The pharmaceutical composition according to claim 18 characterized in that it comprises the recombinant a2 interferon polypeptide (IFNa2) having any of SEQ ID NOS: 6 and 9, fragments, analogs, derivatives and variants thereof, wherein the polypeptide has antagonistic activity specifies improved.

21. A method of treating or preventing a disorder or disease associated with modulation of interferon (IFN), characterized in that it comprises administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 19, wherein the disorder or disease is selected from the group consisting of cancer, autoimmune disease and infectious disease.

22. The method according to claim 21, characterized in that the cancer is selected from the group consisting of hairy cell leukemia, Kaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia, non-Hodgkins lymphoma and melanoma.

23. The method according to claim 21, characterized in that the autoimmune disease is multiple sclerosis (MS).

24. The method according to claim 23, characterized in that the MS is selected from the group consisting of relapsing relapsing MS, MS, secondary progressive, primary progressive MS, and progressive recurrent MS.

25. The method according to claim 21, characterized in that the infectious disease is infection by hepatitis virus.

26. The method according to claim 25, characterized in that the hepatitis virus is selected from the group consisting of hepatitis A, hepatitis B and hepatitis C.

27. A method for the treatment or prevention of disorders associated with the increased expression of IFNa2, characterized in that it comprises administering to a subject in need thereof an effective amount of the pharmaceutical composition according to claim 20.

28. The method according to claim 27, characterized in that the disorder is insulin dependent diabetes mellitus (IDDM).

29. The method according to claim 27, characterized in that the disorder is systemic lupus erythematosus (SLE).

30. Using a recombinant interferon a2 polypeptide (IFNa2), active fragment, analogue, derivative and variant thereof, the polypeptide comprises a mutation selected from at least one amino acid substitution within amino acid residues 57-89, at least one amino acid substitution of amino acid residues of C-terminal 159-165, and combinations thereof, in, wherein the polypeptide has improved specific agonist or antagonist activity, compared to wild-type IFNa2 (SEQ ID NO: 2) for the preparation of a medicament for the treatment or prevention of disorders or diseases associated with the modulation of IFN.

31. The use of an interferon a2 (IFNa2) mutant according to any of claims 2-12 for the preparation of a medicament for the treatment or prevention of disorders or diseases associated with modulation of IFN.