RU2518324C2 - Homogenous preparations of il-28 and il-29 - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly to genetic engineering, and may be used in preparing drugs for hepatitis C. What has been offered is using a pegylated methionine-containing form of N-terminal truncated IL-29 variant with a substituted cysteine residue in a position corresponding to position 171 of an amino acid sequence of a wild-type mature protein expressed in a heterologous system in the form of high homogenous preparations when preparing a drug for hepatitis C.

EFFECT: invention may be used in preparing the drugs for hepatitis C.

3 cl, 37 tbl, 45 ex

Description

BACKGROUND

Cytokines play an important role in the regulation of blood formation and immune responses, and can also influence the development of lymphocytes. The family of human class II cytokines includes subtypes of interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), IL-10, IL-19 (US patent No. 5985614), MDA- 7 (Jiang et al., Oncogene 11, 2477-2486 (1995)), IL-20 (Jiang et al., Oncogene 11, 2477-2486 (1995)), IL-22 (Xie et al., J. Biol Chem. 275, 31335-31339 (2000)) and AK-155 (Knappe et al., J. Virol. 74, 3881-3887 (2000)). Most cytokines bind and transmit signals through cytokine receptors of class I or class II. Members of the class II human cytokine receptor family include interferon-αR1 (IFN-αR1), interferon-γ-R2 (IFN-γ-R2), interferon-γ R1 (IFN-γ R1), interferon-γR2 (IFN-γR2), IL-10R (Liu et al., J. Immunol. 152, 1821-1829 (1994)), CRF2-4 (Lutfalla et al., Genomics 16, 366-373 (1993)), IL-20Rβ (Blumberg et al ., Cell 104, 9-19 (2001) (also known as zcytor7 (US patent No. 5945511) and CFR2-8 (Kotenko et al., Oncogene 19, 2557-2565 (2000)), IL-20Rβ (Blumberg et al ., ibid., (2001)) (also known as DIRS1 (PCT WO 99/46379)), IL-22RA1 (receptor-α1 for IL-22, a message was sent to HUGO for examination) (also known as IL-22R ( Xie et al., J. Biol. Chem. 275, 31335-31339 (2000)), zcytor11 (US Pat. No. 5,965,704) and CRF2-9 (Kotenko et al., Oncogene 19, 2557-2565 (2000)) tissue factor.

Class II cytokine receptors are typically dimers formed by two separate receptor chains, subunits of the α and β receptor (Stahl et al., Cell 74, 587-590 (1993)). In the general case, α subunits are the main proteins that bind to cytokines, and β subunits are required for the formation of high affinity sites for binding, as well as for signal transmission. An exception is the IL-20 receptor, in which both subunits are necessary for binding to IL-20 (Blumberg et al., Ibid., 2001).

Class II cytokine receptors are identified by a conservative cytokine binding region of approximately 200 amino acids (D200) located in the extracellular portion of the receptor. Said cytokine binding region includes two type III fibronectin domains (FnIII), each approximately 100 amino acids in size (Bazan JF, Proc. Natl. Acad. Sci. USA 87, 6934-6938 (1990); Thoreau et al., FEBS Lett., 282, 16-31 (1991)). Each FnIII domain contains conserved Cys, Pro, and Trp residues that define the folding structure of seven β-chains in the same way as the constant region of immunoglobulins (Uze et al., J. Interferon Cytokine Res. 15, 3-26 (1995)). The conservative structural elements of the class II cytokine receptor family allow the identification of new members of this family based on the degree of homology of the primary amino acid sequence.

Interleukins are a family of cytokines that mediate immunological responses, including inflammatory responses. The central place in the formation of the immune response belongs to the T-cell, which produces many cytokines and forms the acquired immunity to antigens. The cytokines produced by the T cell are divided into type 1 and type 2 (Kelso, A. Immun. Cell Biol. 76: 300-317 (1998). Type 1 cytokines include IL-2, interferon-gamma (IFN-γ), LT-α and participate in inflammatory responses, in the formation of immunity to viruses, immunity to intracellular parasites and are involved in the transplant rejection reaction. Type 2 cytokines include IL-4, Il-5, IL-6, IL-10 and IL-13 and are involved in humoral responses, in the formation of immunity to helminths, and in allergic responses.Cytokines that are similar to both type 1 and type 2 include IL-3, GM- CSF and TNF-α A number of data suggest that T-cell populations producing cytokines of type 1 and 2 preferably migrate to various types of inflamed tissue.

From a therapeutic point of view of special interest are the interferons (reviews on interferons cm. De Maeyer and De Maeyer-Guignard , "Interferons" in The Cytokine Handbook, 3 ^{rd Edition, Thompson (ed.)} , Pages 491-516 (Academic Press Ltd. 1998 ) and Walsh, Biopharmaceuticals: Biochemistry and Biotechnology, pages 158-188 (John Wiley & Sons 1998)). Interferons exhibit a wide variety of biological activity and can be used to treat certain autoimmune diseases, in particular cancer, as well as to enhance the immune response against infectious agents, including viruses, bacteria, fungi and protozoa. Currently, six forms of interferon have been identified, which are divided into two large groups. The so-called “Type I” interferons include IFN-α, IFN-β, IFN-ω, IFN-δ and interferon-τ. Currently, IFN-γ and one subclass of IFN-α are the only type II interferons.

Type I interferons, which are believed to be derived from the same parent gene, have retained a fairly similar structure and are able to carry out their action through the same receptor on the surface. The human IFN-α / β receptor α-chain contains an extracellular N-terminal region that possesses class II cytokine receptor properties. IFN-γ does not have a significant degree of homology with IFN type I or with an IFN-α subtype of type II, but has a number of biological activities similar to IFN type I.

Practitioners take advantage of the multiplicity of interferon activity to treat a wide range of conditions. For example, one of the forms of IFN-α is approved for use in more than 50 countries for the treatment of painful conditions such as hairy cell leukemia, renal carcinoma, basal cell carcinoma, malignant melanoma, AIDS-associated Kaposi’s sarcoma, multiple myeloma, chronic myelogenous leukemia non-Hodgkin's lymphoma, laryngeal papillomatosis, fungoid mycosis, genital warts, chronic hepatitis B, hepatitis C, chronic hepatitis D, and chronic non-viral hepatitis A, hepatitis non-B / C. The US Food and Drug Administration has approved the use of IFN-β for the treatment of multiple sclerosis, chronic diseases of the nervous system. IFN-γ is used to treat chronic granulomatous diseases, while interferon enhances the patient's immune response in order to destroy the infection caused by bacteria, fungi, pathogenic protozoa. Clinical studies also show that IFN-γ can be used in the treatment of AIDS, leishmaniasis and lepromatous leprosy.

IL-28, IL-28B, and IL-29 constitute a recently discovered new family of proteins whose sequence is homologous to the sequence of type I interferons and which have genomic homology with respect to IL-10. This new family is described in PCT Joint Patent Application WO 02/086087 and Sheppard et al., Nature Immunol. 4: 63-68, 2003); both documents are incorporated herein by reference. In their abilities to induce an antiviral state in IL-28 and IL-29 cells, they functionally resemble type I interferons, however, unlike type I interferons, they do not show antiproliferative activity against certain B-cell lines.

IL-28 and IL-29 are known to have an odd number of cysteines (PCT application WO 02/086087 and Sheppard et al., Supra). The expression of recombinant IL-28 and IL-29 can lead to a heterogeneous mixture of proteins formed by intramolecular disulfide bonds in many conformations. Separation of these forms can be complex and time consuming. Therefore, it is desirable to provide molecules of IL-28 and IL-29, which after expression give one type of formation of intramolecular disulfide bonds, and also develop methods for forming a folded structure and purification of these drugs in order to preserve their homogeneity. Thus, the present invention provides compositions and methods for producing homogeneous preparations of IL-28 and IL-29.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the present description, a number of terms are widely used. In order to facilitate understanding of the invention, the following definitions are given.

Unless otherwise indicated, definitions in the singular, “at least one,” mean one or more than one.

The term “affinity tag” is used herein to refer to a polypeptide segment that can be attached to a second polypeptide, to purify or detect the second peptide, or to create a site for attachment of the second peptide to the substrate. In principle, a peptide or protein for which an antibody or other specific binding agent can be used as an affinity tag. Affinity tags include a polyhistidine tag, protein A (Nilsson et al., EMBO J. 4: 1075, 1985; Nilsson et al., Methods Enzymol. 198: 3, 1991), glutathione S-transferase (Smith and Johnson, Gene 67 : 31, 1988), Glu-Glu affinity tag (Grusenmeyer et al., Proc. Natl. Acad. Sci. USA 82: 7952-4, 1985), substance P, Flag ™ peptide (Hopp et al., Biotechnology 6: 1204-10, 1988), a streptavidin binding peptide or other antigenic epitope or binding domain. See general information in Ford et al., Protein Expression and Purification 2: 95-107, 1991. Affinity tag coding DNAs are available from commercial suppliers (notably Pharmacia Biotech, Piscataway, NJ).

The term “allelic variant” is used herein to mean any of two or more alternative forms of a gene occupying the same chromosomal locus. An allelic variant usually occurs as a result of a mutation and can lead to phenotypic polymophism within a population. A gene mutation can be a silent mutation (no changes in the encoded polypeptide) or can encode polypeptides that have an altered amino acid sequence. The term allelic variant is used in the present description also to refer to a protein encoded by an allelic variant of a gene.

The terms “amino terminal” and “carboxy terminal” are used herein to mean positions in polypeptides. In accordance with the context, these terms are used with reference to a specific sequence or part of a polypeptide to indicate proximity or relative position. For example, some sequence located carboxyterminally relative to the compared sequence in the polypeptide is located proximal to the carboxyl end of the compared sequence, but not necessarily at the carboxyl end of the full length polypeptide.

The term “complement / anti-complement pair” refers to non-identical fragments which under certain conditions form a non-covalently linked stable pair. For example, biotin and avidin (or streptavidin) are prototype members of the complement / anti-complement pair. Other examples of complement / anti-complement pairs are antibody / antigen (or hapten or epitope) pairs, sense / antisense polypeptide pairs, and the like. In the event that subsequent dissociation of the complement / anti-complement pair is desired, the affinity of binding of the complement / anti-complement pair should be <10 ⁹ M ^-1 .

The term “degenerate nucleotide sequence” means a nucleotide sequence that includes one or more degenerate codons (relative to the compared polynucleotide molecule encoding the polypeptide). Degenerate codons contain different nucleotide triplets, but encode the same amino acid residue (for example, GAU and GAC triplets each encode Asp).

The term “expression vector” is used to mean a DNA molecule, linear or cyclic, including a fragment that encodes a polypeptide of interest, operably attached to additional segments that provide for its transcription. Such additional segments include promoter and transcriptional terminator sequences, and may also include one or more replication origin points, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are usually derived from plasmid or viral DNA, or they may contain elements of both.

The term “isolated” as applied to polynucleotides means a polynucleotide that is isolated from its natural gene environment and thus freed from other extraneous or undesirable coding sequences, and exists in a form that is suitable for use in a protein production system obtained by genetic engineering. Such isolated molecules are those that are isolated from the natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free from other genes with which they are usually associated, but may include native 5 ′ and 3 ′ non-translated regions, such as promoters and transcription terminators. The identification of associated sites should be understood by those skilled in the art (see, for example, Dynan and Tijan, Nature 316: 774-78, 1985).

An “isolated” (or “isolated”) polypeptide or protein is a polypeptide or protein that is under conditions different from its natural environment, i.e. not in the blood or tissue of an animal. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, especially other animal polypeptides. The polypeptides are preferably provided in a highly pure form, i.e. with a purity of more than 95%, more preferably with a purity of more than 99%. When used in this context, the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or otherwise glycosylated or functionalized forms.

When using the term “level” in relation to immune cells, such as NK cells, T cells, in particular cytotoxic T cells, B cells and the like, an increased level indicates either an increased number of cells or an increased activity of functions cells.

The term “level” with respect to viral infections refers to a change in the level of viral infection and includes, but is not limited to, a change in the level of CTLs or NK cells (as indicated above), a reduced viral load, an increased titer of an antiviral antibody, decreased serological levels of alanine aminotransferase or improvement, which is determined by histological analysis of the target tissue or target organ. Determining whether these level changes are a significant difference or significant changes can easily be determined by a person skilled in the art.

The term “operably linked” to DNA segments means that the segments are arranged in such a way that they consistently perform their intended function, for example, transcription begins at the promoter and then proceeds through the coding segment to terminate transcription.

The term “ortholog” means a polypeptide or protein derived from one species, which is a functional counterpart of a polypeptide or protein isolated from other species. Differences in orthologs are the result of speciation.

“Paralogs” are distinguishable but structurally related proteins produced by the body. It is believed that paralogs result from duplication of genes. For example, α-globin, β-globin and myoglobin are paralogues with respect to each other.

A “polynucleotide” is a single or double stranded polymer composed of deoxyribonucleotide or ribonucleotide bases that are read from the 5′-end to the 3′-end. Polynucleotides include RNA and DNA and can be isolated from natural sources, synthesized in vitro, or obtained by combining natural or synthetic molecules. The sizes of polynucleotides are expressed in base pairs (abbreviated as “bp”), nucleotides (“n”) or thousands of bases (“so”). If context permits, the last two terms can describe polynucleotides that are single-stranded or double-stranded. If the term is applied to double-stranded molecules, then it is used to denote the full length, in which case it should be understood that it is equivalent to the term “base pair”. It should be apparent to one skilled in the art that the two strands of a double-stranded polynucleotide may slightly vary in length and their ends may have ledges as a result of enzymatic cleavage; thus, not all nucleotides within a double-stranded polynucleotide may be paired.

A “polypeptide” is a polymer composed of amino acid residues that are linked by peptide bonds regardless of whether they are naturally produced or synthetically produced. A polypeptide containing less than about 10 amino acid residues is commonly referred to as a “peptide”.

The term “promoter” is used in the present description according to its value known from the technical field to refer to a part of a gene containing DNA sequences that provide for the binding of RNA polymerase and the initiation of transcription. Promoter sequences are usually, but not always, located at the 5′-end of non-coding regions of genes.

A “protein” is a macromolecule made up of one or more polypeptide chains. A protein may also contain non-peptide components, such as carbohydrate groups. Carbohydrates and other non-peptide substituents can be added to the protein in the cell in which the protein is produced, and vary depending on the type of cell. Proteins are defined herein in terms of amino acid skeleton structures; substituents such as carbohydrate groups are not usually indicated, but they may nevertheless be present.

The term “receptor” means a cell-associated protein that binds to a bioactive molecule (in particular, a ligand) and mediates the effect of the ligand on the cell. Membrane-coupled receptors are characterized by a multi-peptide structure that includes the extracellular ligand binding domain and the intracellular effector domain, which is usually involved in signal transduction. Binding of the ligand to the receptor leads to conformational changes in the receptor that cause an interaction between the effector domain and another molecule or other molecules in the cell. The specified interaction, in turn, leads to a change in metabolism in the cell. Metabolic results associated with receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increased production of cyclic AMPs, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. In general, receptors may be membrane-bound receptors, cytosolic or nuclear receptors; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (in particular, PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL- receptor 6) receptors.

The term “secretory signal sequence” means a DNA sequence that encodes a polypeptide (“secretory polypeptide”), as a component of a larger polypeptide, directs the larger polypeptide along the secretory pathway in the cell in which it is synthesized. The larger polypeptide is usually cleaved to remove the secretory peptide while moving along the secretory pathway.

The term “splicing variant” is used herein to refer to alternative forms of RNA transcribed from a gene. Splicing variation naturally occurs when alternative splicing sites are used inside a transcribed RNA molecule or less often between separately transcribed RNA molecules and can lead to several mRNA molecules transcribed from the same gene. Splicing variants may encode polypeptides having an altered amino acid sequence. The term splicing variant is used in the present description also to refer to a protein encoded by a splicing variant of mRNA transcribed from a gene.

It should be understood that the molecular weights and sizes of polymers determined by qualitative analytical methods (in particular, electrophoresis) are approximate values. In the case when these values are designated as “about” X or “approximately” X, it should be understood that the indicated value of X is given with an accuracy of ± 10%.

“Zcyto20”, “zcyto21”, “zcyto22” are the previously used designations for human IL-28A, human IL-29, and human IL-28B, respectively. The nucleotide and amino acid sequence for IL-28A are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The nucleotide and amino acid sequence for IL-29 are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The nucleotide and amino acid sequence for IL-28B are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. These sequences are fully described in PCT application WO 02/086087, jointly filed by ZymoGenetics, Inc., which is incorporated herein by reference.

“Zcyto24” and “zcyto25” are the previously used designations for mouse IL-28, and they are shown, respectively, in SEQ ID NO: 7, 8, 9, and 10. Polynucleotides and peptides are fully described in PCT application WO 02/086087, together filed by ZymoGenetics, Inc., which is incorporated herein by reference.

“Zcytor19” is the previously used designation for the α-subunit of IL-28, and is given in SEQ ID NO: 11. Polynucleotides and peptides are shown in PCT application WO 02/20569, filed by Schering, Inc., and application WO 02/44209 owned by ZymoGenetics, Inc., which are incorporated herein by reference. “IL-28 receptor" refers to the α-subunit of IL-28 and the CRF2-4 subunit forming a heterodimeric receptor.

The present invention provides polynucleotide molecules, including DNA and RNA molecules encoding mutant IL-28 and IL-29, containing cysteine substitutions that result in expression of a recombinant IL-28 and IL-29 preparation, which is a homogeneous preparation. In accordance with the present invention, a homogeneous preparation of IL-28 and IL-29 is a preparation in which at least 98% of a single intramolecular structure of disulfide bonds in the purified polypeptide is maintained. In other embodiments, implementation of the present invention, the single disulfide conformation in the preparation of the purified polypeptide is 99% homogeneous. In the General case, as described in the present description, these mutants containing substitutions of cysteine, retain some biological activity inherent in IL-28 and IL-29 wild-type. For example, the molecules of the present invention can bind to the IL-28 receptor with a specific specificity. In general, ligand binding to a related receptor is specific when the K _D value is in the range of 100 nM to 100 pM. Specific binding in the range of K _D values from 100 mM to 10 nM corresponds to binding with low affinity. Specific binding in the range of K _D values from 2.5 pM to 100 pM corresponds to binding with high affinity. In another example, the biological activity of mutant IL-28 and IL-29 containing cysteine substitutions occurs when the molecules are able to exhibit a certain level of antiviral activity associated with wild-type IL-28 and IL-29. The determination of the level of antiviral activity is described in detail in the present invention.

When referring to IL-28, this term means both IL-28A and IL-28B. Previously, IL-28A was designated as zcyto20 (SEQ ID NO: 1 and 2), IL-29 was designated as zcyto21 (SEQ ID NO: 3 and 4), and IL-28B was designated as zcyto22 (SEQ ID NO: 5 and 6). (See PCT Application WO 02/086087 and Sheppard et al., Supra). Mouse orthologs for IL-28 were previously designated as zcyto24 (SEQ ID NO: 7 and 8), zcyto25 (SEQ ID NO: 9 and 10).

The wild-type IL-28A gene encodes a 200 amino acid polypeptide as shown in SEQ ID NO: 2. The signal sequence for IL-28A can be predicted as composed of amino acid residues from -25 (Met) to amino acid residue -1 (Ala) from SEQ ID NO: 2. The mature peptide for IL-28A starts at amino acid residue 1 (Val) from SEQ ID NO: 2. The IL-28A helices are predicted as follows: helix A is defined by amino acid residues 31 (Ala) to 45 (Leu); helix B is defined by amino acid residues 58 (Thr) to 65 (Gln); helix C is defined by amino acid residues from 69 (Arg) to 86 (Ala); helix D is defined by amino acid residues 95 (Val) to 114 (Ala); helix E is determined by amino acid residues from 126 (Thr) to 142 (Lys); and the helix F is defined by amino acid residues from 148 (Cys) to 169 (Ala); as shown in SEQ ID NO: 2.

The wild-type IL-29 gene encodes a 200 amino acid polypeptide as shown in SEQ ID NO: 4. The signal sequence for IL-29 can be predicted as composed of amino acids from -19 (Met) to amino acid residue -1 (Ala) from SEQ ID NO: 4, SEQ ID NO: 119, SEQ ID NO: 121. The mature peptide for IL-29 begins at amino acid residue 1 (Gly) of SEQ ID NO: 4. IL-29 is described in PCT application WO02 / 02627. The IL-29 helices are predicted as follows: Helix A is determined by amino acid residues 30 (Ser) to 44 (Leu); helix B is defined by amino acid residues 57 (Asn) to 65 (Val); helix C is defined by amino acid residues 70 (Val) to 85 (Ala); helix D is determined by amino acid residues from 92 (Glu) to 111 (Gln); helix E is determined by amino acid residues 118 (Thr) to 139 (Lys); and the helix F is defined by amino acid residues from 144 (Gly) to 170 (Leu); as shown in SEQ ID NO: 4.

The wild-type IL-28B gene encodes a 200 amino acid polypeptide as shown in SEQ ID NO: 6. The signal sequence for IL-28B can be predicted as composed of amino acids from -21 (Met) to -1 (Ala) from SEQ ID NO: 6. The mature peptide for IL-28B starts at amino acid residue 1 (Val) from SEQ ID NO: 6. The IL-28B helices are predicted as follows: helix A is defined by amino acid residues 31 (Ala) to 45 (Leu); helix B is defined by amino acid residues 58 (Thr) to 65 (Gln); helix C is defined by amino acid residues from 69 (Arg) to 86 (Ala); helix D is defined by amino acid residues 95 (Gly) to 114 (Ala); helix E is determined by amino acid residues from 126 (Thr) to 142 (Lys); and the helix F is defined by amino acid residues from 148 (Cys) to 169 (Ala); as shown in SEQ ID NO: 6.

The present invention provides mutations in wild-type IL-28 and IL-29 sequences, as shown in SEQ ID NO: 1, 2, 3, 4, 5, and 6, which result in the expression of individual forms of IL-28 or IL-29 molecules . Since it is believed that the heterogeneity of the forms is the result of the formation of various combinations of intramolecular disulfide bonds, specific embodiments of the present invention include mutations in cysteine residues in the wild-type IL-28 and IL-29 sequences. Upon expression of IL-28 and IL-29 in E. coli, an N-terminal or amino-terminal methionine is present. For example, SEQ ID NO: 12-17 shows the numbering of nucleotide and amino acid sequences for IL-28A, IL-29, and IL-28B when an N-terminal Met is present. Table 1 shows the possible combinations of cysteine pairs connected by intramolecular disulfide bonds for wild-type IL-28A, IL-28B and IL-29.

Table 1

The polynucleotide and polypeptide molecules of the present invention have mutations in one or more cysteines present in wild-type IL-28A, IL-29, and IL-28B molecules, and yet they retain biological activity as described herein. Table 2 shows examples of mutations with the replacement of cysteine, in particular, point mutations with the replacement of cysteine (C) with serine (S).

table 2

It has been shown that all members of the family in question bind to the same class II cytokine receptor, IL-28R. The α-subunit of IL-28 was previously designated as the zcytor19 receptor. Without going into theory, we can assume that these molecules are manifested in all signals transmitted through the IL-28 receptor along the same path. The IL-28 receptor is described in co-filed PCT patent application WO 02/44209. which is incorporated herein by reference; Sheppard et al., Supra; Kotenko et al., Nature Immunol. 4: 69-77, 2003; and PCT WO 03/040345. IL-28R is a member of class II cytokine receptors, which are characterized by the presence in their extracellular domains of one or more cytokine receptor modules (CRM). Other class II cytokine receptors include zcytor11 (US Pat. No. 5,965,704 co-owned), CRF2-4 (access number Z17227 in the GenBank database), IL-10R (access numbers U00672 and NM_001558 in the GenBank database), DIRS1, zcytor7 (US patent No. 5945511 co-ownership) and tissue factor. The IL-28 receptor, like all known class II receptors, with the exception of the alpha chain of the receptor for interferon alpha / beta, contains only one class II CRM in its extracellular domain.

Cytokines with bundles of four helices are also grouped by the length of their constituent helices. The dyno-chain cytokines typically consist of helices containing 24-30 residues, and they include IL-6, ciliary neurotrophic factor (CNTF), leukemia inhibition factor (LIF) and human growth hormone (hGH). Short-chain cytokines typically consist of helices containing 18-21 residues, and they include IL2, IL-4, and GM-CSF. Studies using CNTF and IL-6 showed that the helix of CNTF can replace the equivalent helix in IL-6, while the chimera acquires the ability to bind CNTF. Thus, it turns out that the functional domains of four-helix cytokines are determined by the degree of structural homology, regardless of sequence identity, and can maintain functional integrity in the chimera (Kallen et al., J. Biol. Chem. 274: 11859-11867, 1999). Thus, mutant IL-28 and IL-29 polypeptides containing cysteine substitutions may be useful for preparing chimeric fusion molecules, in particular with other interferons, in order to determine and modulate the specificity of receptor binding. Of particular interest are fusion proteins that combine the helical and loop regions of interferons and cytokines, such as TNF-α, IL-10 and human growth hormone.

The present invention provides polynucleotide molecules, including DNA and RNA molecules, which encode, for example, mutant IL-28 or IL-29 polypeptides containing cysteine substitutions. For example, the present invention provides degenerate nucleotide sequences encoding the IL-28A C48S, Met IL-28A C49S, IL-28A C50S, Met IL-28A C51S, IL-29 C171S and Met IL-29 C172S polypeptides described herein. For specialists it should be clear that due to the degeneracy of the genetic code in these polynucleotide molecules, significant variations in sequences are possible. SEQ ID NOs: 30, 31, 32, 33, 34, and 35 are degenerate DNA sequences that encompass all DNA molecules encoding IL-28A C48S, Met IL-28A C49S, IL-28A C50S, Met IL-28A C51S, IL -29 C171S and Met IL-29 C172S, respectively. It should be understood by those skilled in the art that degenerate sequences of SEQ ID NO: 30, 31, 32, 33, 34, and 35 also denote all RNA sequences encoding SEQ ID NO: 30, 31, 32, 33, 34, and 35, by substituting T on U and, thus, are also considered in the present description.

The IL-28A polypeptides of the present invention also include mutations in the second cysteine, C2, mature polypeptide. For example, C2 from the N-terminus or amine-end in the polypeptide with SEQ ID NO: 2 is cysteine at amino acid position 48 or position 49 (with an additional N-terminal Met) if the polypeptide is expressed in E. coli (see, for example, SEQ ID NO: 13). The specified second cysteine (of which there are seven, as in IL-28B), or C2 in IL-28A, can be mutated, for example, by substitution with serine, alanine, threonine, valine or asparagine. Mutant IL-28A C2 molecules of the present invention include, for example, polynucleotide molecules shown in SEQ ID NOs: 20 and 22, including DNA and RNA molecules that encode C2 mutant polypeptides of IL-28A, as shown in SEQ ID NO : 21 and 23, respectively. SEQ ID NOs: 36 and 37 are additional IL-28A C2 polypeptides of the present invention.

In addition to IL-28A C2 mutants, the present invention also includes IL-28A polypeptides containing mutations at the position of the third cysteine, C3, mature polypeptide. For example, C3 from the N-terminus or amine-end in the polypeptide with SEQ ID NO: 2 is cysteine at position 50 or position 51 (with an additional N-terminal Met) if the polypeptide is expressed in E. coli (see, for example, SEQ ID NO: 13). Mutant IL-28A C3 molecules of the present invention include, for example, polynucleotide molecules shown in SEQ ID NOs: 24 and 26, including DNA and RNA molecules that encode mutant IL-28A C3 polypeptides as shown in SEQ ID NO: 25 and 27, respectively. SEQ ID NOs: 38 and 39 are additional IL-28A C3 polypeptides of the present invention.

The IL-28A polypeptides of the present invention include, for example, SEQ ID NO: 2, 13, 19, 21, 23, and 25, which are encoded by the polynucleotide molecules of IL-28A shown in SEQ ID NO: 1, 12, 18, 20, 22 and 24, respectively. Additional IL-28A polypeptides of the present invention include, for example, SEQ ID NO: 36, 37, 38, and 39.

The IL-28B polypeptides of the present invention also include mutations in the second cysteine, C2, mature polypeptide. For example, C2 from the N-terminus or amine-end in the polypeptide with SEQ ID NO: 6 is cysteine at amino acid position 48 or position 49 (with an additional N-terminal Met) if the polypeptide is expressed in E. coli (see, for example, SEQ ID NO: 17). The specified second cysteine (of which there are seven, as in IL-28A), or C2 in IL-28B, can be mutated, for example, by substitution with serine, alanine, threonine, valine or asparagine. The mutant IL-28B C2 molecules of the present invention include, for example, the polynucleotide molecules shown in SEQ ID NOs: 122 and 124, including DNA and RNA molecules that encode mutant IL-28B C2 polypeptides, as shown in SEQ ID NO: 123 and 125, respectively. Additional mutant IL-28B C2 molecules of the present invention include polynucleotide molecules shown in SEQ ID NOs: 130 and 132, including DNA and RNA molecules that encode mutant IL-28B C2 polypeptides shown in SEQ ID NOs: 131 and 133 , respectively (PCT publication WO 03/066002 (Kotenko et al.)).

In addition to IL-28B C2 mutants, the present invention also includes IL-28B polypeptides, including mutations at the position of the third cysteine, C3, mature polypeptide. For example, C3 from the N-terminus or amine-end in the polypeptide with SEQ ID NO: 6 is cysteine at position 50 or position 51 (with an additional N-terminal Met) if the polypeptide is expressed in E. coli (see, for example, SEQ ID NO: 17). Mutant IL-28B C3 molecules of the present invention include, for example, polynucleotide molecules shown in SEQ ID NOs: 126 and 128, including DNA and RNA molecules that encode mutant IL-28B C3 polypeptides as shown in SEQ ID NO: 127 and 129, respectively. Additional mutant IL-28B C3 molecules of the present invention include polynucleotide molecules shown in SEQ ID NOs: 134 and 136, including DNA and RNA molecules that encode mutant IL-28B C3 polypeptides shown in SEQ ID NOs: 135 and 137 , respectively (PCT publication WO 03/066002 (Kotenko et al.)).

The IL-28B polypeptides of the present invention include, for example, SEQ ID NO: 6, 17, 123, 125, 127, 129, 131, 133, 135 and 137, which are encoded by the polynucleotide molecules of IL-28B shown in SEQ ID NO: 5 , 16, 122, 124, 126, 128, 130, 132, 134, and 136, respectively.

The IL-29 polypeptides of the present invention also include, for example, mutations in fifth cysteine, C5, mature polypeptide. For example, C5 from the N-terminus of a polypeptide with SEQ ID NO: 4 is cysteine at position 171 or position 172 (with an additional N-terminal Met) if the polypeptide is expressed in E. coli (see, for example, SEQ ID NO: 15) . Said fifth cysteine, or C5 from IL-29, can be mutated, for example, by substitution with serine, alanine, threonine, valine or asparagine. These mutant IL-29 C5 polypeptides have the structure of C1 (Cys15 from SEQ ID NO: 4) / C3 (Cys112 from SEQ ID NO: 4) and C2 (Cys49 from SEQ ID NO: 4) / C4 (Cys145 from SEQ ID) disulfide bonds NO: 4). Additional mutant IL-29 C5 molecules of the present invention include polynucleotide molecules shown in SEQ ID NO: 26, 28, 82, 84, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, and 160, including DNA and RNA molecules that encode mutant IL-29 C5 polypeptides shown in SEQ ID NO: 27, 29, 83, 85, 139, 141, 143, 145, 147, 149, 151, 153, 155 , 157, 159 and 161, respectively. Additional mutant IL-29 C5 molecules of the present invention include polynucleotide molecules shown in SEQ ID NOs: 86, 88, 94 and 96, including DNA and RNA molecules that encode mutant IL-29 C5 polypeptides shown in SEQ ID NO : 87, 89, 95 and 97, respectively (PCT publication WO 03/066002 (Kotenko et al.)). Additional mutant IL-29 C5 molecules of the present invention include polynucleotide molecules shown in SEQ ID NOs: 102, 104, 110, and 112, including DNA and RNA molecules that encode mutant IL-29 C5 polypeptides shown in SEQ ID NO : 103, 105, 111 and 113, respectively (PCT publication WO 02/092762 (Baum et al.)).

In addition to IL-29 C5 mutants, the present invention also includes IL-29 polypeptides containing mutations at the position of the first cysteine, C1, mature polypeptide. For example, C1 from the N-terminus of a polypeptide with SEQ ID NO: 4 is cysteine at position 15 or position 16 (with an additional N-terminal Met) if the polypeptide is expressed in E. coli (see, for example, SEQ ID NO: 15) . These mutant IL-29 C1 polypeptides should therefore have a predicted disulfide bond structure of C2 (Cys49 from SEQ ID NO: 4) / C4 (Cys145 from SEQ ID NO: 4) and C3 (Cys112 from SEQ ID NO: 4) / C5 (Cys171 of SEQ ID NO: 4). Additional mutant IL-29 C1 molecules of the present invention include polynucleotide molecules as shown in SEQ ID NOs: 74, 76, 78 and 80, including DNA and RNA molecules that encode mutant IL-29 C1 polypeptides shown in SEQ ID NO: 75, 77, 79, and 81, respectively. Additional mutant IL-29 C1 molecules of the present invention include polynucleotide molecules shown in SEQ ID NOs: 90, 92, 98 and 100, including DNA and RNA molecules that encode mutant IL-29 C1 polypeptides shown in SEQ ID NO : 91, 93, 99, and 101, respectively (PCT publication WO 03/066002 (Kotenko et al.)). Additional mutant IL-29 C1 molecules of the present invention include polynucleotide molecules shown in SEQ ID NOs: 106, 108, 114 and 116, including DNA and RNA molecules that encode mutant IL-29 C1 polypeptides shown in SEQ ID NO : 107, 109, 115 and 117, respectively (PCT publication WO 02/092762 (Baum et al.)).

The IL-29 polypeptides of the present invention, for example, SEQ ID NO: 4, 15, 27, 29, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161, which are encoded by the polynucleotide molecules of IL-29 shown in SEQ ID NO: 3, 14, 26, 28, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158 and 160 may additionally include the signal sequence shown in SEQ ID NO: 119, or the signal sequence shown in SEQ ID NO: 121. In addition, the present invention includes the IL-29 polypeptides shown in SEQ ID NO: 40 and 41. A polynucleotide molecule encoding a signal sequence polypeptide of SEQ ID NO: 119 is shown in SEQ ID NO: 118. A polynucleotide molecule encoding a signal sequence polypeptide of SEQ ID NO: 120 is shown in SEQ ID NO: 121.

In accordance with one aspect, the present invention provides an isolated polypeptide comprising a sequence that is at least 90% or 95% identical to the amino acid residue sequences selected from the group consisting of SEQ ID NO: 2, 4, 6, 8 , 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91 , 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145 , 147, 149, 151, 153, 155, 157, 159 and 161. The polypeptide may optionally include at least 15, at least 30, at least 45, or at least 30 followed by flax amino acids in the amino acid sequences shown in SEQ ID NO: 2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161. In another embodiment of the present invention, the isolated polypeptide is composed of amino acid residues selected from the group comprising SEQ ID NO: 2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161. Polypep the tweed may contain conservative amino acid substitutions, compared with amino acid sequences selected from the group including SEQ ID NO: 2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161.

In accordance with another aspect, the present invention provides a fusion protein comprising a polypeptide that comprises a sequence of amino acid residues selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 13, 15, 17, 19, 21 , 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 , 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 159 and 161; and a polyalkyl oxide moiety. The polyalkyl oxide moiety may optionally be polyethylene glycol, such as mPEG / propionic aldehyde of 20 kDa or mPEG / propionic aldehyde of 30 kDa. Polyethylene glycol may be linear or branched. Polyethylene glycol can be covalently attached to the N-terminus or C-terminus of the polypeptide.

In accordance with another aspect, the present invention provides a fusion protein comprising a first polypeptide and a second polypeptide connected by a peptide bond, wherein the first polypeptide contains an amino acid residue sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161; and a second polypeptide. The second polypeptide may optionally be a fragment of an antibody. An antibody fragment may optionally be F (ab '), F (ab), Fab', Fab, Fv, scFv, and / or a minimal recognizable unit. The second polypeptide may optionally be human albumin. The second polypeptide may optionally be a polypeptide selected from the group consisting of affinity tags, toxins, radionuclides, enzymes and fluorophores.

Table 3 presents the single-letter codes that are used in SEQ ID NO: 30, 31, 32, 33, 34, and 35 to indicate degenerate nucleotide positions. “Permissions” means nucleotides indicated by a code letter. “Complement” means the code for the complementary nucleotide (s). For example, the code letter Y denotes either C or T, and its complement R denotes A or G, while A is complementary to T, and G is complementary to C.

Table 3

Degenerate codons that are used in SEQ ID NO: 30, 31, 32, 33, 34 and 35, covering all possible codons for a given amino acid, are shown in Table 4.

Table 4

It should be clear to one skilled in the art that some uncertainty is introduced when establishing a degenerate codon, which represents all possible codons encoding each amino acid. For example, a degenerate codon for serine (WSN) may, in some circumstances, encode arginine (ARG), and a degenerate codon for arginine (MGN) may, in some circumstances, encode serine (AGY). A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by degenerate sequences can encode variant amino acid sequences, however, one skilled in the art can easily identify variant sequences by comparison with an amino acid sequence, for example, SEQ ID NO: 19, 21, 23, 25, 27, 29 , 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111 , 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161. Variant sequences may be easily tested on functionally as described herein.

It should be understood by one skilled in the art that various species may exhibit “preference for codon use”. For general information see Grantham et al., Nuc. Acids Res. 8: 1893-912, 1980; Haas, et al., Curr. Biol. 6: 315-24, 1996; Wain-Hobson, et al., Gene 13: 355-64, 1981; Grosjean and Fiers, Gene 18: 199-209, 1982; Holm, Nuc. Acids Res. 14: 3075-87, 1986; Ikemura, J. Mol. Biol. 158: 573-97, 1982. As used herein, the term “preference for codon” or “preferred codons” is a term used in the art and refers to codons for translating a protein that are most commonly used in cells of a particular type , and thereby one or more representatives of possible codons encoding each amino acid are preferred (see Table 4). For example, the amino acid threonine (Thr) can be encoded by ACA, ACC, ACG, or ACT, however, in mammalian cells, the most commonly used codon is ACC; in other species, for example, insect cells, yeast, viruses, or bacteria, other codons for Thr may be preferred. Preferred codons for a particular species can be introduced into the polynucleotides of the present invention using various methods known in the art. The introduction of sequences with preferred codons into recombinant DNA can, for example, increase protein production due to the fact that protein translation becomes more efficient in a particular cell type or species. Therefore, the sequence with a degenerate codon given in SEQ ID NO: 30, 31, 32, 33, 34 and 35, serves as a matrix for optimizing the expression of polynucleotides in various types of cells and species that are commonly used in this field and which are given in the present description. Sequences containing preferred codons can be tested and optimized for expression in various forms and tested for their functionality, as described in the present description.

In accordance with another aspect, the present invention provides an isolated polynucleotide selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158 and 160.

In accordance with another aspect, the present invention provides an isolated polynucleotide that is capable of hybridizing with a sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158 and 160, or its complement under hybridization conditions under the influence of 50% formamide, 5xSSC (1xSSC: 0.15 M sodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt solution (100x Denhardt solution: 2% (w / v) ph cola 400, 2% (w / v) polyvinylpyrrolidone, 2% (w / v) bovine serum albumin, 10% dextran sulfate and 20 mg / ml denatured fragmented salmon sperm DNA at a temperature of from about 42 ° C to about 70 ° C, while an isolated polynucleotide encodes a polypeptide having antiviral activity. The encoded polypeptide does not necessarily have antiviral activity against hepatitis B and / or hepatitis C. An isolated polynucleotide may optionally encode at least a portion of a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 13, 15 , 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97 , 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151 , 153, 155, 157, 159 and 161. An isolated polynucleotide can encode a polypeptide represented by the sequences of SEQ ID NO: 2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29 , 36, 37, 38, 39, 40, 41, 75, 77, 7 9, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161.

In accordance with another aspect, the present invention provides an isolated polynucleotide encoding a polypeptide, wherein the encoded polypeptide is selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23 , 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 , 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161.

In accordance with another aspect, the present invention provides an isolated polynucleotide encoding a polypeptide, wherein the sequence of the encoded polypeptide is at least 90% or 95% identical to the sequence selected from the group consisting of SEQ ID NO: 2, 4, 6 , 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89 , 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143 , 145, 147, 149, 151, 153, 155, 157, 159 and 161, and wherein the encoded polypeptide has antiviral activity. The encoded polypeptide does not necessarily have antiviral activity against hepatitis B and / or hepatitis C.

As indicated previously, isolated polynucleotides of the present invention include DNA and RNA. Methods for producing DNA and RNA are known in the art. In general, RNA is isolated from a tissue or cell that produces a large amount of mutant RNA IL-28 or IL-29 containing cysteine substitutions. Similar tissues and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77: 5201, 1980) or by screening conditioned medium from various types of cells for activity against target cells or tissues. Once the activity of RNA-producing cells or tissues has been established, complete RNA can be obtained by extraction with guanidine isothiocyanate, followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18: 52-94, 1979). Poly (A) ⁺ RNA is obtained from complete RNA according to the method developed by Aviv and Leder (Proc. Natl. Acad. Sci. USA 69: 1408-12, 1972). Complementary DNA (cDNA) is obtained from Poly (A) ⁺ RNA by known methods. Alternatively, genomic DNA can be isolated. Polynucleotides encoding mutant IL-28 or IL-29 polypeptides containing cysteine substitutions are then identified and isolated, for example, by hybridization or PCR.

Full-sized clones encoding mutant IL-28 or IL-29 containing cysteine substitutions can be obtained by conventional cloning methods. Clones of complementary DNA (cDNA) are preferred, although for a number of applications (in particular, for expression in transgenic animals) it may be preferable to use a genomic clone or modify a cDNA clone to include at least one genomic intron. Methods for obtaining cDNA or genomic clones are well known and can easily be carried out by specialists in this field of technology; they include using the sequence or part thereof disclosed in the present invention to prepare a probe or primer from a library. Expression libraries can be probed with antibodies to IL-28 receptor fragments or other specific binding partners.

For specialists, it is obvious that the sequence shown, for example, in SEQ ID NO: 1, 3, 5, respectively, is a mutation of single alleles in the human IL-28 and IL-29 bands and that variations in alleles and alternative splicing are possible. For example, an IL-29 variant has been identified where the amino acid residue 169 (Asn) shown in SEQ ID NO: 4 is an Arg residue, as described in WO 02/086087. Similar allelic variants are included in the present invention. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from various individuals according to standard techniques. Allelic variants of the DNA sequence shown in SEQ ID NO: 1, 3 and 5, including allelic variants containing silent mutations, and allelic variants in which mutations lead to changes in the amino acid sequence, are included, in addition to cysteine mutations, in the scope of the present invention , as well as proteins that are allelic variants of the sequences SEQ ID NO: 2, 4, and 6. cDNA generated from alternatively spliced mRNAs that retain the properties of mutant IL-28 or IL-29 polypeptides containing substitutions cysteine, are within the scope of the present invention, as well as the polypeptides encoded by said cDNA and mRNA molecules. Allelic variants and splicing variants of these sequences can be cloned by probing cDNA or genomic libraries from various individuals or tissues according to standard techniques known in the art, and mutations in polynucleotides encoding cysteine or cysteine residues can be introduced as described herein.

According to methods of implementing the present invention, an isolated variant or nucleic acid molecules encoding mutant IL-28 and IL-29 that contain cysteine substitutions can hybridize under stringent conditions with nucleic acid molecules having the nucleotide sequence of SEQ ID NO: 18, 20, 22 , 24, 26, 28, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116 , 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158 and 160, or with nucleic acid molecules having a nucleotide sequence complementary to SEQ ID NO: 18, 20, 22, 24, 26, 28, 74, 76, 7 8, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158 and 160. In general, stringent conditions are chosen so that the temperature is approximately 5 ° C lower than the temperature melting point (T _m ) for a particular sequence at a given ionic strength and pH. T _m represents the temperature (at a given ionic strength and pH) at which 50% of the target sequence hybridizes into a probe with an ideal pairwise arrangement of the bases.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA, and DNA-RNA, can hybridize if the nucleotide sequences have some degree of complementarity. In hybrids, base pair mismatch in the double helix is acceptable, but the degree of mismatch affects the stability of the hybrid. T _m hybrid with erroneously paired bases decreases by 1 ° C for every 1-1.5% of erroneously paired bases. Varying the stringency of hybridization conditions allows you to control the degree of mismatch that occurs in the hybrid. The degree of severity increases as the hybridization temperature increases and the ionic strength used in the hybridization buffer decreases.

The person skilled in the art easily adapts these conditions when used for a particular polynucleotide hybrid. The value of T _m for a particular target sequence is the temperature (under given conditions) at which 50% of the target sequence hybridizes into a perfectly paired probe sequence. Conditions that affect T _m include the size and content of base pairs in the polynucleotide probe, the ionic strength of the solution in which hybridization is performed, and the presence of destabilizing agents in the solution in which hybridization is performed. A large number of equations are known in the art for calculating T _m specifically for DNA, RNA, and DNA-RNA hybrids and sequences of polynucleotide probes of various lengths (see, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989); Ausubel et al, (eds.), Current Protocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26: 227 (1990)). Sequencing software such as OLIGO 6.0 (LSR; Long Lake, Minnesota) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto, California), as well as Internet sites, are available tools for analyzing a specific sequence and calculating a value T _m based on user-defined criteria. Similar programs can also analyze the sequence under given conditions and identify suitable probe sequences. Typically, the hybridization of a longer polynucleotide sequence containing> 50 base pairs is carried out at a temperature of about 20-25 ° C lower than the calculated value of T _m . For smaller probes containing <50 base pairs, hybridization is usually carried out at a temperature approximately 5-10 ° C lower than the calculated value of T _m . These conditions provide the maximum speed of hybridization of DNA-DNA and DNA-RNA hybrids.

After hybridization, the nucleic acid molecules can, in order to remove non-hybridized nucleic acid molecules, be washed under strictly controlled conditions or under very strictly controlled conditions. Typical stringent washing conditions include washing in a 0.5x - 2x SSC solution with 0.1% sodium dodecyl sulfate (SDS) at 55-65 ° C. Thus, nucleic acid molecules encoding variant or mutant IL-28 or IL-29 polypeptides containing cysteine substitutions hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26, 28, 74 , 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 122, 124, 126, 128 , 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158 and 160, respectively (or its complement), in strict washing conditions, in which strict washing conditions 0.5x - 2x SSC with 0.1% SDS at 55-65 ° C, including 0.5x SSC with 0.1% SDS at 55 ° C or 2x SSC with 0.1% SDS at 65 ° C . One skilled in the art will easily select equivalent conditions for the components, for example, replacing SSC with SSPE in the wash solution.

Typical very stringent washing conditions include washing in a 0.1x - 0.2x SSC solution with 0.1% sodium dodecyl sulfate (SDS) at 50-65 ° C. In other words, nucleic acid molecules encoding a variant or mutant IL-28 or IL-29 polypeptide containing cysteine substitutions hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26, 28, 74 , 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 122, 124, 126, 128 , 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158 and 160 (or its complement), under very strict washing conditions, in which the stringent washing conditions are equivalent 0.1x - 0.2x SSC with 0.1% SDS at 50-65 ° C, including 0.1x SSC with 0.1% SDS at 50 ° C or 0.2x SSC with 0.1% SDS at 65 ° C.

The present invention also provides isolated IL-28 or IL-29 polypeptides in which the sequence is substantially identical to the polypeptides of the present invention, for example SEQ ID NO: 2, 4, 6, 8, 10, 13, 15, 17, 19 , 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101 , 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155 , 157, 159 and 161. The term “significant degree of sequence identity” is used herein to refer to polypeptides whose sequence is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99% or more than 99% identical to the sequences shown in SEQ ID NO: 2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161, or their orthologs. The present invention also includes polypeptides whose amino acid sequence is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more than 99% identical to the polypeptide or its fragment of the present invention. In addition, the present invention includes polynucleotides that encode similar polypeptides. The IL-28 or IL-29 polypeptides of the present invention are preferably recombinant polypeptides. In another aspect, the IL-28 or IL-29 polypeptides of the present invention has at least 15, at least 30, at least 45, or at least 60 consecutive amino acids. For example, the IL-28 or IL-29 polypeptide of the present invention relates to a polypeptide having at least 15, at least 30, at least 45, or at least 60 consecutive amino acids from SEQ ID NO: 2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161. Methods for determining percent identity are described below.

The present invention also contemplates variant nucleic acid molecules that can be identified using two criteria: determining the similarity between the encoded polypeptide and the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 29, 36, 37, 38, 39 , 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123 , 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161, respectively, and / or by hybridization analysis, as described above . Such variants include nucleic acid molecules: (1) which hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26, 28, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158 and 160, respectively (or its complement), in strict washing conditions, where strict washing conditions are 0.5x - 2x SSC with 0.1% SDS at 55-65 ° C; or (2) which encode a polypeptide whose sequence is at least 80%, at least 90%, at least 91%, at least 92%, at least 93% at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more than 99% identical to the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89 , 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143 , 145, 147, 149, 151, 153, 155, 157, 159 and 161. Alternatively, options They can be characterized as nucleic acid molecules: (1) which hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26, 28, 74, 76, 78, 80, 82, 84, 86 , 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140 , 142, 144, 146, 148, 150, 152, 154, 156, 158 and 160, respectively (or its complement), in very strict washing conditions, where the strict washing conditions are 0.1x - 0.2x SSC with 0, 1% SDS at 50-65 ° C; and (2) which encode a polypeptide whose sequence is at least 80%, at least 90%, at least 91%, at least 92%, at least 93% at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more than 99% identical to the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89 , 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143 , 145, 147, 149, 151, 153, 155, 157, 159 and 161, respectively.

The percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio 48: 603 (1986) and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1992). Briefly, the comparison of the primary structure of two amino acid sequences is carried out in such a way as to optimize the alignment weight using a penalty for opening a deletion of 10, a penalty for continuing a deletion of 1, and a Henikoff and Henikoff BLOSUM62 number matrix (ibid. ), as shown in Table 4 (amino acids are indicated by standard single letter codes).

Total identical matches

--------------------------------------------- × 100

[length of longer sequence plus

the number of gaps introduced in a longer

comparison sequence

two sequences]

For specialists, it should be clear that there are many available developed algorithms for comparing the primary structure of two amino acid sequences. The “FASTA” similarity search algorithm developed by Pearson and Lipman is a convenient method for examining the level of identity possessed by the amino acid sequence disclosed herein and the amino acid sequence of the putative IL-28 or IL-29 variant. The FASTA algorithm is described by Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988) and Pearson, Meth. Enzymol. 183: 63 (1990).

Briefly, FASTA primarily characterizes the sequence similarity by identifying areas that are common to the sequences in question (for example, SEQ ID NO: 2) and the test sequence that has either the highest identity density (if the value of the ktup variable is 1) or the largest number pairs of identities (if ktup = 2) without conservative substitutions of amino acids, insertions or deletions. Then, ten regions with the highest density of identities are recorded by comparing the similarities of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are “trimmed” to include only those residues that contribute to the highest alignment weight. If there are several sites with an alignment weight greater than the “cutoff” value (calculated by a certain formula based on the sequence length and ktup value), then the trimmed source regions are examined to determine if the regions can be combined to obtain an approximate gap alignment. Finally, the regions of the two amino acid sequences with the highest alignment weight are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48: 444 (1970); Sellers, SIAM J. Appl. Math. 26: 787 (1974)), which allows for insertion and deletion of amino acids. Preferred parameters for analysis in FASTA format are: ktup = 1, penalty for opening a deletion = 10, penalty for continuing a deletion = 1, and substitution matrix = BLOSUM62. These parameters can be entered into the FASTA program by modifying the numerical matrix file (“SMATRIX”), as explained in Appendix 2 in Pearson, Meth. Enzymol. 183: 63 (1990).

The FASTA format can also be used to determine the sequence identity of nucleic acid molecules using the above ratio. For comparisons of nucleotide sequences, the ktup value may be in the range from one to six, preferably from three to six, and most preferably equal to three, and the values of other parameters are set by default.

Mutant IL-28 or IL-29 polypeptides containing cysteine substitutions or polypeptides with substantially similar sequence identities are characterized in that they contain substitutions, deletions and additions of amino acids. These changes are mostly insignificant, i.e. are conservative amino acid substitutions (see Table 6) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, usually from one to about 30 amino acids; and amino-terminal or carboxy-terminal extension segments, such as amino-terminal methionine residues, small linker peptides containing up to about 20-25 residues, or an affinity tag. Thus, the present invention includes polypeptides containing from about 146 to 207 amino acid residues that form a sequence of at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or more than 99% identical to the corresponding region of the sequence of SEQ ID NO: 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 10 7, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161. A polypeptide comprising an affinity tag may further comprise a proteolytic cleavage site between an IL-28 or IL-29 polypeptide and an affinity tag. Said sites preferably include thrombin cleavage sites and factor Xa cleavage sites.

Table 6
Conservative Amino Acid Substitutions The main ones: arginine lysine histidine Sour: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine

Amino acid residues can be determined to form regions and domains that are critical for maintaining structural integrity. Within these areas, specific residues can be established that are more or less resistant to changes and support the overall tertiary structure of the molecule. Methods for analyzing sequence structure include, but are not limited to, comparing the primary structure of multiple sequences with a high degree of amino acid or nucleotide identity, secondary structural preferences, binary structures, complementary packaging, and latent polar interactions (Barton, Current Opin. Struct. Biol. 5: 372 -376, 1995 and Cordes et al., Current Opin. Struct. Biol. 6: 3-10, 1996). In the general case, when developing modifications in molecules or identifying specific fragments, determining the structure is accompanied by an assessment of the activity of the modified molecules.

Changes in the amino acid sequence in mutant polypeptides of IL-28 or IL-29 containing cysteine substitutions are carried out in such a way as to minimize the destruction of the higher order structure, which is important for the manifestation of biological activity. For example, in the case when a mutant IL-28 or IL-29 polypeptide containing cysteine substitutions has one or more helices, then changes in amino acid residues are carried out in such a way as not to destroy the geometry of the helices and other components of the molecule, where the changes in conformation weaken some critical functions, for example, the binding of molecules to their binding partners. The effect of amino acid sequence changes can be predicted, for example, by computer simulation, as described above, or determined by analysis of the crystal structure (see, for example, Lapthorn et al., Nat. Struct. Biol. 2: 266-268, 1995). Other methods well known in the art compare the folded structure of a variant protein with a standard molecule (e.g., a native protein molecule). For example, you can compare the configuration of cysteine in a variant and a standard molecule. Mass spectrometry and chemical modification using reduction or alkylation provides methods for determining cysteine residues that are associated with the formation of disulfide bonds or do not participate in the formation of such bonds (Bean et al., Anal. Biochem. 201: 216-226, 1992; Gray , Protein Sci. 2: 1732-1748, 1993; and Patterson et al., Anal. Chem. 66: 3727-3732, 1994). It is generally believed that if the modified molecule does not have the same cysteine configuration as the standard molecule, then the folded structure will suffer. Another well-known and recognized way of establishing a folded structure is circular dichroism (CD). Measuring and comparing CD spectra generated by a modified molecule and a standard molecule is a routine procedure (Johnson, Proteins 7: 205-214, 1990). Another well-known method of stacking and structure analysis is crystallography. Known methods for analyzing the folding structure and structural similarity between proteins and polypeptides are nuclear magnetic resonance (NMR, NMR), enzymatic mapping of peptides and mapping of epitopes (Schaanan et al., Science 257: 961-964, 1992).

You can generate the Hopp / Woods hydrophilicity profile for the sequence of the mutant protein IL-28 or IL-29 containing the cysteine substitution, which is shown in SEQ ID NO: 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161 (Hopp et al., Proc. Natl. Acad. Sci USA 78: 3824-3828, 1981; Hopp, J. Immunol. Meth. 88: 1-18, 1986 and Triquier et al., Protein Engineering 11: 153-169, 1998). The establishment of the profile is based on sliding on a window of six residues. Hidden residues G, S and T and open residues H, Y and W are ignored. For specialists it should be clear that when developing modifications of the amino acid sequence of the mutant IL-28 or IL-29 polypeptide containing cysteine substitutions, hydrophobicity or hydrophilicity is taken into account so as not to disturb the overall structure and biological profile. Of particular interest for substitution are hydrophobic residues selected from the group consisting of Val, Leu or Ile, or the group comprising Met, Gly, Ser, Ala, Tyr and Trp.

The identity of essential amino acids can also be established by analyzing sequence similarity between IFN-α and members of the IL-28A, IL-28B, and IL-29 family (as indicated in Tables 1 and 2). Using methods such as the previous analysis in the “FASTA” format, regions with a high degree of similarity are identified in the protein family and used to analyze the amino acid sequence of conserved regions. An alternative approach to identifying a variant polynucleotide that is based on a structure is to detect the ability of a nucleic acid molecule encoding a potential variant gene of IL-28 or IL-29 to hybridize with a nucleic acid molecule, as described above.

Other methods for identifying essential amino acids in the polypeptides of the present invention are techniques known in the art such as site-directed mutagenesis or mutagenic scanning with alanine (Cunningham and Wells, Science 244: 1081 (1989), Bass et al., Proc. Natl. Acad. Sci. USA 88: 4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis and Protein Engineering”, in Proteins: Analysis and Design, Angeletti (ed.), Pages 259-311 (Academic Press, Inc. 1998)) . In the latter method, single alanine mutations are introduced into each residue in the molecule and the resulting mutant molecules containing cysteine substitutions are tested for biological and biochemical activity, as described below, in order to identify amino acid residues that are critical for the activity of the molecule. See also Hilton et al., J. Biol. Chem. 271: 4699 (1996).

The present invention also includes functional fragments of mutant IL-28 or IL-29 polypeptides containing cysteine substitutions, and nucleic acid molecules that encode such functional fragments. A “functional” mutant IL-28 or IL-29 containing cysteine substitutions, or a fragment thereof, as defined herein, is distinguished by its proliferative and differentiating activity, its ability to activate or suppress specialized cell functions, or its ability to specifically bind to an anti-IL-antibody 28 or IL-29 or with an IL-28 receptor (both soluble and immobilized). Specialized activities of mutant IL-28 or IL-29 polypeptides containing cysteine substitutions and their testing are discussed below. As indicated above, the IL-28 or IL-29 polypeptides are characterized by the presence of a folding of six helices. Thus, the present invention further provides fusion proteins, including: (a) polypeptide molecules containing one or more of the above helices; and (b) functional fragments containing one or more of these helices. Another polypeptide part of the fusion protein may be another helical-laid cytokine or interferon, such as IFN-α, or a non-native and / or non-secretory signal peptide that facilitates the secretion of the fusion protein.

Mutant IL-28 or IL-29 polypeptides containing cysteine substitutions of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides, can be obtained by conventional methods using cells into which an expression vector encoding the polypeptide has been introduced. As used herein, the term “cells into which the expression vector has been introduced” includes both cells that have been directly manipulated to introduce exogenous DNA molecules and their progeny that contain the introduced DNA. Suitable host cells are those types of cells that can be transformed or transfected with exogenous DNA and grown in culture medium, and these include bacteria, fungal cells, and cultured higher eukaryotic cells. Techniques for manipulating cloned DNA molecules and techniques for introducing exogenous DNA into various host cells are provided by Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 and Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

In accordance with another aspect, the present invention provides an expression vector that includes the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as described herein; and a transcription terminator.

The present invention also provides an expression vector that contains an isolated and purified DNA molecule, including the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide whose sequence is at least 90% or 95% identical to the polypeptide selected from the group consisting of SEQ ID NO: 19, 21, 23, 25, 27, 29, 36, 37, 38, 39 , 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123 , 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161; and a transcription terminator. A DNA molecule may also include a secretory signal sequence operably linked to a DNA segment. The encoded protein may also contain an affinity tag, as indicated in the present description. The present invention also provides a grown cell containing the above expression vector. The encoded polypeptide may optionally contain at least 15, at least 30, at least 45, or at least 60 consecutive amino acids from the amino acid sequence shown in SEQ ID NO: 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161. The encoded protein may optionally have antiviral activity, in particular against hepatitis B and hepatitis C.

In accordance with another aspect, the present invention provides a cultured cell comprising the expression vector disclosed above.

In accordance with another aspect, the present invention provides a method for producing a protein, which comprises: growing the above cell under conditions in which a DNA segment is expressed; and isolating the protein encoded by the DNA segment.

In general, a DNA sequence encoding a mutant IL-28 or IL-29 polypeptide containing cysteine substitutions operably binds in the expression vector to other genetic elements necessary for its expression, which typically include a promoter and a transcription terminator. A vector usually also contains one or more selectable markers and one or more points of origin of replication, although it should be clear to a person skilled in the art that in some systems, selectable markers can be provided by separate vectors, and replication of exogenous DNA is achieved by integration into the genome of the host cell. The selection of promoters, terminators, selectable markers, vectors and other elements is a common development step and can easily be carried out by a person skilled in the art. Many such elements are described in the literature and are available from commercial sources.

In order to direct mutant IL-28 or IL-29 polypeptides containing cysteine substitutions along the secretory pathway of the host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or presequence) is introduced into the expression vector. The secretory signal sequence may be from a mutant IL-28 or IL-29 containing cysteine substitutions, for example, SEQ ID NO: 119 or 121, or may be obtained from another secreted protein (in particular, t-PA; see US patent No. 5641655) or synthesized de novo. The secretory signal sequence is operably linked to the DNA sequence of mutant IL-28 or IL-29 containing cysteine substitutions, i.e. the two sequences are connected in the correct reading frame and arranged in such a way as to direct the newly synthesized polypeptide along the secretory pathway of the host cell. Secretory signal sequences are usually located at the 5 ′ end of a DNA sequence encoding a polypeptide of interest, although some signal sequences may be located in other places of the DNA sequence of interest (see, in particular, Welch et al., US patent No. 5037743; Holland et al ., US patent No. 5143830).

A variety of suitable recombinant host cells include, but are not limited to, gram-negative prokaryotic host organisms. Suitable E. coli strains include W3110 derived from K12 strains MM294, TG-1, JM-107, BL-21 and UT5600. other suitable strains include BL21 (DE3), BL21 (DE3) pLysS, BL21 (DE3) pLysE, DH1, DH4I, DH5, DH5I, DH5IF ', DH5IMCR, DH10B, DH10B / p3, DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, ER1647, E. coli K12, E. coli K12 RV308, E. coli K12 C600, E. coli HB101, E. coli K12 C600 R.sub.kM .sub.k-, E. coli K12 RR1 (see, e.g., Brown (ed.), Molecular Biology Labfax (Academic Press 1991)). Other gram-negative prokaryotic hosts may include Serratia, Pseudomonas, Caulobacter. Prokaryotic hosts may include gram-positive organisms such as Bacillus, for example B. subtilis and B. thuringienesis, and B. thuringienesis var. israelensis, as well as Streptomyces, e.g. S. lividans, S. ambofaciens, S. fradiae and S. griseofuscus. Suitable strains of Bacillus subtilus include BR151, YB886, MI119, MI120 and B170 (see, for example, Hardy, “Bacillus Cloning Methods”, in DNA Cloning: A Practical Approach, Glover (ed.) (IRL Press 1985)). Standard vectors breeding techniques in prokaryotic hosts are well known in the art (see, e.g., Ausubel et al (eds), Short Protocols in Molecular Biology, 3 ^rd Edition (John Wiley & Sons 1995);... Wu et al , Methods in Gene. Biotechnology (CRC Press, Inc. 1997)). In one embodiment of the present invention, the methods of the invention use mutant IL-28 or IL-29 containing cysteine substitutions that are capable of being expressed in strain W3110 deposited in the American Type Culture Collection (ATCC) under ATCC number # 27325.

If large-scale production of mutant IL-28 or IL-29 containing cysteine substitutions is required using the expression system of the present invention, cyclic fermentation can be used. In general terms, cyclic fermentation consists in the fact that in the first stage, a flask for inoculation is prepared by growing strains of E. coli expressing mutant IL-28 or IL-29 containing cysteine substitutions on a suitable nutrient medium with shaking until the optical density is reached ( OD) in the range of 5 to 20 at 600 nm. A suitable culture medium should contain nitrogen from sources such as ammonium sulfate, ammonium phosphate, ammonium chloride, yeast extract, animal protein hydrolyzate, vegetable protein hydrolyzate, or hydrolyzed casein. Phosphates are supplied from potassium phosphate, ammonium phosphate, phosphoric acid or sodium phosphate. Other components are magnesium chloride or magnesium sulfate, ferrous sulfate or ferrous chloride, and trace amounts of other elements. To improve growth, growth media is supplemented with carbohydrates such as fructose, glucose, galactose, lactose or glycerin. Alternatively, in order to obtain a high yield of a mutant IL-28 or IL-29 protein containing cysteine substitutions, a feed-back cyclic culture method is used. E. coli strains producing mutant IL-28 or IL-29, which contain cysteine substitutions, are grown under conditions similar to those described for the vessel used in the first stage for the preparation of seed in cyclic fermentation.

After fermentation, the cells are separated by centrifugation, resuspended in a homogenizing buffer and homogenized, for example, in an APV-Gaulin homogenizer (Invensys APV, Tonawanda, NY) or in another cell disruption device such as ball mills or ultrasonic disintegrators. Alternatively, the cells are immediately removed from the fermenter and homogenized in an APV-Gaulin homogenizer. The washed preparation from intracellular inclusion bodies is solubilized with a solution of guanidine hydrochloride (5-8 M) or urea (7-8 M) containing a reducing agent, such as beta-mercaptoethanol (10-100 mM) or dithiothreitol (5-50 mM). Solutions can be prepared in Tris, phosphate, HEPES or other suitable buffering agents. Inclusion bodies can also be solubilized with a urea solution (2-4 M) containing sodium lauryl sulfate (0.1-2%). In the process of isolating purified mutant IL-28 or IL-29 containing cysteine substitutions from a transformed E. coli host strain in which mutant IL-28 or IL-29 containing cysteine substitutions accumulates as refractory inclusion bodies, the cells are destroyed and inclusion bodies are separated by centrifugation. Then, inclusion bodies are solubilized and denatured in a 6 M solution of guanidine chloride containing a reducing agent. The reconstituted mutant IL-28 or IL-29, containing cysteine substitutions, is then oxidized during the implementation of the controlled renaturation step. After reconstitution, mutant IL-28 or IL-29, containing cysteine substitutions, can be passed through a filter to clarify and separate insoluble proteins. The solution is then filtered to clarify and separate insoluble proteins. After restoration of the folding of mutant IL-28 or IL-29 containing cysteine substitutions and concentration is carried out, mutant IL-28 or IL-29 proteins containing cysteine substitutions with reduced folded structure are absorbed with diluted buffer on a cation exchange column and purified by the method chromatography with hydrophobic interactions.

According to the present invention, suitable hosts are cultured mammalian cells. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981; Graham and Van der Eb, Virology 52: 456 , 1973), electroporation (Neumann et al., EMBO J. 1: 841-5, 1982), transfection with DEAE-dextran (Ausubel et al., Ibid.), Liposome-mediated transfection (Hawley-Nelson et al., Focus 15: 73, 1993; Ciccarone et al., Focus 15: 80, 1993) and viral vectors (Miller and Rosman, BioTechniques 7: 980-90, 1989; Wang and Finer, Nature Med. 2: 714-6, 1996 ) The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, in Levinson et al., US Pat. No. 4,713,339; Hagen et al. US Pat. No. 4,784,950; Palmiter et al. U.S. Patent No. 4,579,821; and Ringold, US patent No. 4656134. Suitable cultured mammalian cells include COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36: 59-72, 1977) and a Chinese hamster ovary cell line (in particular CHO-K1; ATCC No. CCL 61). Additional cell lines are known in the art and are available from public depositories such as the American Type Culture Collection, Manassas, Virginia. In general, strong transcription promoters, such as those from SV-40 or cytomegaly virus, are preferred. See, for example, US patent No. 4956288. Other suitable promoters include promoters from metallothionein genes (US Pat. Nos. 4,579,821 and 4,601,978) and an adenovirus major late promoter.

Drug selection is usually used to select cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants”. Cells that are grown in the presence of a selective agent and capable of transmitting a gene of interest to their offspring are called “stable transfectants”. A preferred selectable marker is a gene encoding antibiotic resistance to neomycin. The selection is carried out in the presence of a neomycin-type drug, such as G-418 or a similar drug. Selection systems can also be used to increase the expression level of a gene of interest, and this process is called “amplification”. Amplification is carried out by growing transfectants in the presence of a low level of selective agent, and then increasing the amount of selective agent, in order to select cells that produce high levels of the introduced gene products. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug-resistant genes (in particular, conferring resistance to hydromycin, conferring multidrug resistance, puromycin acetyltransferase) may be used. Alternatively, markers that introduce an altered phenotype, such as a green fluorescent protein or cell surface proteins such as CD4, CD8, MHC class I, placental alkaline phosphatase, can be used to fractionally separate transfected cells from non-transfected cells, such as FACS-sorting or separation technology using magnetic beads.

Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells, and bird cells. A review of the use of Agrobacterium rhizogenes as a vector is provided in Sinkar et al., J. Biosci. (Bangalore) 11: 47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is described in Guarino et al., US Pat. No. 5,162,222 and WIPO Publication WO 94/06463. Insect cells can be infected with a recombinant baculovirus, usually obtained from Autographa californica nuclear polyhedrosis virus (AcNPV). See King, L.A. and Possee, R. D., The Baculovirus Expression System: A Laboratory Guide, London, Chapman &Hall; O′Reilly, D.R. et al, Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, C.D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995. The second method for producing recombinant baculovirus uses the transposon-based system described by Luckow (Luckow, V.A. et al., J. Virol. 67: 4566-79, 1993). The specified system is sold as a set of reagents Bac-to-Bac (Life Technologies, Rockville. Maryland). This system uses a transfer vector, pFastBac1 ™ (Life Technologies), including a Tn7 transposon to transfer DNA encoding a mutant IL-28 or IL-29 polypeptide that contains cysteine substitutions to the baculovirus genome, maintained in E. coli as a large plasmid dubbed “bacmid.” The pFastBac1 ™ transfer vector uses the AcNPV polyhedrin promoter to activate expression of a gene of interest, in this case mutant IL-28 or IL-29, containing cysteine substitutions. However, pFastBac1 ™ may be substantially modified. The polyhedrin promoter can be removed and replaced with the promoter of the basic baculovirus protein (also known as the Pcor, p6.9 or MP promoter), which was previously expressed in baculovirus infection and has been shown to be convenient for the expression of secreted proteins. See Hill-Perkins, M.S. and Posse, R. D., J. Gen. Virol. 71: 971-6, 1990; Bonning, B.C. et al., J. Gen. Virol. 75: 1551-6, 1994; and Chazenbalk, G. D. and Rapoport, B., J. Biol. Chem. 270: 1543-9, 1995. In such gene constructs of the transfer vector, a long or short version of the main promoter protein can be used. Moreover, transfer vectors can be constructed that replace the secretory signal sequence of native IL-28 or IL-29 with a secretory signal sequence derived from insect proteins. For example, a secretory signal sequence derived from ecdysteroid glucosyl transferase (EGT), bee melittin (Invitrogen, Carlsbad, California) or gp67 baculovirus (PharMingen, San Diego, California) can be used in gene constructs to replace the native IL secretory signal sequence -28 or IL-29. In addition, transfer vectors may include fusion in a frame with DNA encoding an epitope tag at the C- or N-terminus of an expressed mutant IL-28 or IL-29 polypeptide containing cysteine substitutions, for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. USA 82: 7952-4, 1985). Using techniques known in the art, a transfer vector including mutant IL-28 or IL-29, which contains cysteine substitutions, is transformed into E. coli and screened for bacmids that contain an interrupted lacZ gene indicating a recombinant baculovirus. Using known methods, bacmid DNA containing the recombinant baculovirus genome is isolated and used to transfect Spodoptera frugiperda cells, in particular Sf9 cells. A recombinant virus is then produced that expresses a mutant IL-28 or IL-29 containing cysteine substitutions. Stocks of recombinant virus are prepared using methods known in the art.

Recombinant virus is used to infect host cells, typically cells derived from martial worms, Spodoptera frugiperda. For general information see Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, DC, 1994. Another suitable cell line is the High FiveO ™ cell line (Invitrogen) obtained from Trichoplusia ni (US Pat. No. 5,300,435). )

According to the present invention, fungal cells, including yeast cells, can also be used. In this regard, yeast species of interest include Saccharomyces cerevisiae, Pichia pastoris and Pichia methanolica. Methods of transforming S. cerevisiae cells using exogenous DNA and producing recombinant polypeptides from them are disclosed, for example, in Kawasaki, US Patent No. 4,599,311; Kawasaki et al. U.S. Patent No. 4,931,373; Brake, US patent No. 4870008; Welch et al., US patent No. 5037743; and Murray et al., US patent No. 4845075. Transformed cells are selected according to the phenotype determined by the selectable marker, usually by drug resistance or ability to grow in the absence of a specific nutrient (in particular, leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (US patent No. 4931373), which allows you to select transformed cells by growth in glucose-containing nutrient media. Suitable promoters or terminators for use with yeast include promoters and terminators from glycolytic enzyme genes (see, for example, Kawasaki, US Patent No. 4,599,311; Kingsman et al., US Patent No. 4,615,974; and Bitter, US Patent No. 4,971,092) and from genes alcohol dehydrogenase. See also U.S. Patent Nos. 4,990,446, 5,063,154, 5,193,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastorilis, Pichia pastorilis, Pichia pastorilis, are known in the art. Candida maltosa. See, for example, Gleeson et al., J. Gen. Microbiol. 132: 3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279. According to the methods set forth by McKnight et al., US Pat. No. 4,935,349, Aspergillus cells can be used. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., US Pat. No. 5,162,228. Methods of transforming Neurospora disclose Lambowitz, US patent No. 4486533. The use of Pichia methanolica as a host for the production of recombinant proteins is disclosed in US Pat. Nos. 5,955,349, 5,888,768 and 6,001,597, and US Pat.

The polypeptides and proteins of the present invention are preferably purified to a purity of ≥ 80%, more preferably to a purity of ≥ 90%, even more preferably to a purity of ≥ 95%, and most preferably to a pharmaceutical purity of more than 99.9% in relation to impurity macromolecules, in particular, other proteins and nucleic acids, and they do not contain infectious and pyrogenic agents. The purified polypeptide or protein is substantially substantially free of other polypeptides or proteins, in particular polypeptides or proteins of animal origin.

Expressed recombinant mutant proteins of IL-28 or IL-29 containing cysteine substitutions (including chimeric polypeptides or multimeric proteins) are purified using conventional methods used for protein purification, usually using a combination of chromatographic methods. For general information see Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York, 1994. Proteins containing a polyhistidine affinity tag (typically about 6 histidine residues) are purified by affinity chromatography on a polymer containing nickel chelates. See, for example, Houchuli et al., Bio / Technol. 6: 1321-1325, 1988. Proteins containing the glu-glu tag can be purified in the usual manner by immunoaffinity chromatography. See, for example, Grussenmeyer et al., Supra. Maltose containing fusion proteins are purified on an amylose column according to methods known in the art.

Mutant IL-28 or IL-29 polypeptides containing cysteine substitutions can also be obtained by chemical synthesis using methods known in the art, including solid-phase synthesis, partially solid-phase methods, fragment condensation, or classical synthesis in solution. See, for example, Merrifield, J. Am. Chem. Soc. 85: 2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2 ^nd edition), Pierce Chemical Co., Rockford, IL, 1984; Bayer and Rapp, Chem. Pept. Prot. 3: 3, 1986; and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989. In vitro synthesis is particularly convenient for producing small polypeptides.

Using methods known in the art, mutant IL-28 or IL-29 proteins containing cysteine substitutions can be obtained as monomers or multimers; in glycosylated or non-glycosylated form; in pegylated or non-pegylated form; in the form of fusion proteins; or they may or may not include terminal methionine as an amino acid residue. Used in therapy, conjugates of mutant IL-28 or IL-29 containing cysteine substitutions may include fragments of pharmaceutically acceptable water-soluble polymers. The conjugation of interferons with water-soluble polymers has been shown to increase the half-cycle of interferon circulation and reduce the immunogenicity of the polypeptide (see, for example, Nieforth et al., Clin. Pharmacol. Ther. 59: 636 (1996) and Monkarsh et al., Anal. Biochem. 247: 434 (1997)).

Suitable water soluble polymers include polyethylene glycol (PEG), monomethoxy-PEG, mono (C1-C10) alkoxy-PEG, aryloxy-PEG, poly- (N-vinylpyrrolidone) PEG, tresyl-monomethoxy PEG, monomethoxy-PEG / propionic aldehyde, PEG / propionic aldehyde, PEG / bis-succinimidyl carbonate, propylene glycol homopolymers, copolymer of propylene oxide / ethylene oxide, polyoxyethylene polyols (in particular glycerol), monomethoxy-PEG / butyraldehyde, PEG / butyric aldehyde, acetone-methylene-PEG, monomethoxydehyde, monomethoxydehyde PEG PEG / succinimidyl propionate, methoxy PEG / succinimidyl b utanoate, polyvinyl alcohol, dextran, cellulose or other carbohydrate-based polymers. Suitable PEGs can have a molecular weight of from about 600 to about 60,000, including, for example, 5,000 daltons, 12,000 daltons, 20,000 daltons, 30,000 daltons and 40,000 daltons, and can be linear or branched. Conjugates of mutant IL-28 or IL-29 containing cysteine substitutions may contain a mixture of such water-soluble polymers.

One example of a conjugate of mutant IL-28 or IL-29 containing cysteine substitutions includes a fragment of mutant IL-28 or IL-29 containing cysteine substitutions, and a polyalkyl oxide fragment attached to the N-terminus of a fragment of mutant IL-28 or IL-29, containing cysteine replacement. One suitable polyalkyl oxide is PEG. By way of illustration, a mutant IL-28 or IL-29 containing cysteine substitutions can be modified with PEG, and this process is called “pegylation”. The pegylation of a mutant IL-28 or IL-29 containing cysteine substitutions can be carried out using any of the pegylation reactions known in the art (see, for example, EP 0154316, Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9: 249 (1992), Duncan and Spreafico, Clin. Pharmacokinet. 27: 290 (1994), and Francis et al., Int. J. Hematol. 68: 1 (1998)). For example, pegylation can be carried out by an acylation reaction or by an alkylation reaction with a reactive polyethylene glycol molecule. In an alternative approach, cysteine mutant IL-28 or IL-29 conjugates are prepared by condensation of activated PEG, in which the terminal hydroxyl group or amino group in PEG is replaced by an activated linker (see, for example, Karasiewicz et al., US Patent No. 5,382,657) .

PEGylation by acylation requires the reaction of an active ester derivative of PEG with a mutant IL-28 or IL-29 polypeptide containing cysteine substitutions. An example of an activated PEG ester is PEG esterified with N-hydroxysuccinimide. As used herein, the term “acylation” includes the following types of binding of a mutant IL-28 or IL-29 containing cysteine substitutions to a water-soluble polymer: amide, carbamate, urethane, and the like. Methods of pegylated mutant IL-28 or IL-29 containing cysteine substitutions by acylation typically include steps (a) of reacting a mutant IL-28 or IL-29 polypeptide containing cysteine substitutions with PEG (such as a reactive PEG aldehyde derivative ester) under conditions in which one or more PEG groups are attached to a mutant IL-28 or IL-29 containing cysteine substitutions, and (b) isolation of the reaction product (s). In the general case, the optimal conditions for carrying out the acylation reaction are determined based on the known parameters and the desired results. For example, the larger the ratio of PEG: mutant IL-28 or IL-29 containing cysteine substitutions, the greater the percentage of the pegylation product of mutant IL-28 or IL-29 containing cysteine substitutions.

Pegylation by alkylation typically involves reacting a terminal aldehyde derivative of PEG, obtained in particular from propionic aldehyde, butyric aldehyde, acetaldehyde and the like, with mutant IL-28 or IL-29 containing cysteine substitutions in the presence of a reducing agent. PEG groups are preferably attached to the polypeptide via the —CH ₂ —NH ₂ group.

Derivatization by reductive alkylation in order to obtain a mono-pegylated product has the advantage that in this case the difference in reactivity of the various types of primary amino groups available for derivatization can be used. Typically, the reaction is carried out at a pH value that allows you to take advantage of the difference in pKa between the ε-amino groups of the lysine residues and the α-amino group of the N-terminal protein residue. By such selective derivatization, control is made of the addition of a water-soluble polymer containing a reactive group, such as an aldehyde group, to the protein. Conjugation with the polymer is carried out mainly at the N-terminus of the protein without significant modification of other reactive groups, such as amino groups in the lysine side chain.

Reductive alkylation, in order to obtain a substantially homogeneous population of monopolymer molecules of a conjugate of mutant IL-28 or IL-29 containing cysteine substitutions, may include the following steps: (a) reacting a mutant IL-28 or IL-29 polypeptide containing cysteine substitutions with a reactive PEG under reductive alkylation conditions at a pH that allows selective modification of the α-amino group at the amine end of the mutant IL-28 or IL-29 containing cysteine substitutions, and (b) isolation of the product (s) re stocks. The reducing agent used in the reductive alkylation should be stable in aqueous solution and preferably should be able to recover only the Schiff base formed in the early stage of reductive alkylation. Preferred reducing agents include sodium borohydride, sodium cyanoborohydride, dimethylamine borane, trimethylamine borane and a pyridine-borane complex.

In order to obtain a practically homogeneous population of monopolymer conjugates of mutant IL-28 or IL-29 containing cysteine substitutions, the conditions for the reductive alkylation should be such that they allow selective attachment of a fragment of a water-soluble polymer to the N-terminus of mutant IL-28 or IL-29 containing cysteine substitution. Such reaction conditions usually provide a difference in pKa between the amino groups of lysine and the α-amino group at the N-terminus. The pH value also affects the ratio of polymer and protein used. In general, if the pH is low, then a greater excess of the polymer with respect to the protein is desirable, since the lower the reactivity of the N-terminal α-group, the more polymer is required to create optimal conditions. If the pH is higher, then the polymer: mutant IL-28 or IL-29 ratio containing cysteine substitutions should not be so large as more reactive groups are available. Typically, the pH is in the range of 3-9, or 3-6. Another factor to consider is the molecular weight of the water-soluble polymer. In general, the larger the molecular weight of the polymer, the smaller the number of polymer molecules that can attach to the protein. For pegylation reactions, a typical molecular weight is from about 2 kDa to about 100 kDa, from about 5 kDa to about 50 kDa, from about 12 kDa to about 40 kDa, or from about 20 kDa to about 30 kDa. The molar ratio of the water-soluble polymer to the mutant IL-28 or IL-29, containing cysteine substitutions, is usually in the range from 1: 1 to 100: 1. Typically, the molar ratio of the water-soluble polymer to the mutant IL-28 or IL-29 containing cysteine substitutions is from 1: 1 to 20: 1 for polypegylation and from 1: 1 to 5: 1 for mono-pegylation.

General methods for the preparation of conjugates consisting of interferon and fragments of a water-soluble polymer are known in the art. See, for example, Karasiewicz et al., US Pat. No. 5,382,657, Greenwald et al., US Pat. No. 5,738,846, Nieforth et al., Clin. Pharmacol Ther. 59: 636 (1996), Monkarsh et al., Anal. Biochem. 247: 434 (1997). The pegylated derivatives can be separated from the non-pegylated mutant polypeptides of IL-28 or IL-29 containing cysteine substitutions using conventional purification methods such as dialysis, ultrafiltration, ion exchange chromatography, affinity chromatography, size exclusion chromatography, and the like.

Molecules of a mutant IL-28 or IL-29 containing cysteine substitutions of the present invention are able to specifically bind to the IL-28 receptor and / or act as an antiviral agent. An analysis of the binding of mutant IL-28 or IL-29 polypeptides containing cysteine substitutions to the IL-28 receptor can be carried out using conventional methods. Mutant IL-28 or IL-29 containing cysteine substitutions can be iodinated according to the Idobead method (Pierce, Rockford, Illinois) in accordance with the manufacturer's recommendations, and then the obtained ¹²⁵ I-IL-28 or ¹²⁵ I-IL-29 can be used. as described below.

In the first approach, fifty nanograms of ¹²⁵ I-IL-28 or ¹²⁵ I-IL-29 can be mixed with 1000 ng of human IL-28-IgG receptor fusion protein in the presence or absence of possible binding competitors, including unlabeled mutant IL-28, containing cysteine substitutions, mutant IL-29, containing cysteine, IL-28 or IL-29 substitutions. The same binding reactions can be carried out by taking other fusion constructs of human cytokine receptor IgG as a control for determining specificity. After incubation at 4 ° C, protein-G is added to the reaction mixture (Zymed, San Francisco, California) to capture IgG receptor fusion structures and any associated proteins, and the reaction mixture is further incubated for 4 hours at 4 ° C. . Then (protein-G) -sepharose is isolated, washed three times with phosphate-buffered saline and the bound ¹²⁵ I-IL-28 or ¹²⁵ I-IL-29 is measured using a gamma counter (Packard Instruments, Downers Grove, Illinois).

In a second approach, an analysis of the ability of molecules to inhibit the binding of ¹²⁵ I-IL-28 or ¹²⁵ I-IL-29 to receptors immobilized on a plate can be performed. A fragment of the IL-28 receptor, which is an extracellular ligand binding domain, can be adsorbed on the walls of a 96-well plate by overnight incubation with 100 μl per well of a 1 g / ml receptor solution. As a second option, the fusion receptor-human IgG construct can be attached to the walls of a 96-well plate that were coated with an antibody against that part of the fusion protein that is human IgG. After the receptor coated well, it is washed, blocked with SUPERBLOCK (Pierce, Rockford, Illinois) and washed again. Solutions are prepared containing fixed concentrations of ¹²⁵ I-IL-28 or ¹²⁵ I-IL-29 in the presence or absence of increasing concentrations of potential binding competitors, including mutant IL-28 containing cysteine substitutions, mutant IL-29 containing cysteine substitutions, IL -28 or IL-29, and 100 μl of the solution are added to the appropriate wells on the tablet. After incubation for one hour at 4 ° C, the plates are washed and the amount of bound ¹²⁵ I-IL-28 or ¹²⁵ I-IL-29 is determined by counting signals (Topcount, Packard Instruments, Downers Grove, Illinois). The binding specificity of ¹²⁵ I-IL-28 or ¹²⁵ I-IL-29 can be determined using the receptor molecules used in these binding assays, as well as using the molecules used as inhibitors.

In addition to pegylation, in order to extend its half-life, the polypeptide of the present invention can be genetically linked to human albumin. Human albumin is the most common native blood protein in the human circulatory system, which is stored in the circulatory system for more than twenty days. Studies have shown that therapeutically useful proteins that are genetically fused to human albumin have longer half-lives. The fusion protein IL-28-albumin or IL-29-albumin, like pegylation, can provide patients with the possibility of a longer treatment, which will allow the use of more convenient prescription regimens that are as effective or more effective and safe than existing treatment methods ( U.S. Patent No. 6,165,470; Syed et al., Blood, 89 (9): 3243-3253 (1997); Yeh et al., Proc. Natl. Acad. Sci. USA 89: 1904-1908 (1992); and Zeist et al. Horm. Res., 37: 5-13 (1992)).

Similar to the above pegylation and binding to human albumin, the Fc portion of the human IgG molecule can be fused to the polypeptide of the present invention. The resulting fusion protein, due to the Fc fragment, may have an increased half-life in the circulatory system (US patent No. 5750375, US patent No. 5843725, US patent No. 6291646; Barouch et al., Journal of Immunology, 61: 1875-1882 (1998); Barouch et al., Proc. Natl. Acad. Sci. USA, 97 (8): 4192-4197 (April 11, 2000); and Kim et al., Transplant. Proc., 30 (8): 4031-4036 ( Dec. 1998)).

Methods for the determination and diagnosis of viral infections are well known to specialists in this field of technology. The specific method used to assess attenuation of the virus in response to the administration of the molecules of the present invention will depend on the types of virus and whether the infection is an in vitro or in vivo infection. In the case of in vivo infection, the measurement and detection of infection and changes in infection levels may vary depending on the infected subject, type of viral infection, etc. For example, these methods include, but are not limited to, determining changes in the number of CD4 cells, serological testing, determining viral DNA and viral RNA analysis using a routine and quantitative real-time polymerase chain reaction, determining virus-induced antibody levels, immunofluorescence and solid phase immunosorbent assays, cytopathic effects and histology.

Antiviral effects can be direct and indirect. An example of a direct antiviral effect is one in which a mutant IL-28 or IL-29 polypeptide containing cysteine substitutions competes with a viral receptor or coreceptor to block a viral infection. Mutant IL-28 or IL-29 containing cysteine substitutions can be administered parenterally to prevent viral infection or reduce ongoing virus replication or reinfection (Gayowski, T. et al., Transplantation 64: 422-426, 1997). An example of a mediated antiviral effect is one in which a mutant IL-28 or IL-29 containing cysteine substitutions can bind CD4 or another leukocyte receptor and exerts an antiviral effect by modulating the effects of the immune response.

Of particular interest is the use of mutant IL-28 or IL-29 containing cysteine substitutes as an antiviral agent against viral leukemia (HTLV), AIDS (HIV) or gastrointestinal viral infections caused, for example, by rotavirus, calicivirus (in particular Norwalk Agent) and certain strains of pathogenic adenoviruses, hepatitis B and C.

Additional types of viral infections for mutant IL-28 or IL-29 containing cysteine substitutions include, but are not limited to, infections caused by: DNA viruses (in particular herpes viruses such as herpes simplex viruses, Epstein-Barr virus, cytomegalovirus ; poxviruses such as smallpox virus (small poxvirus); hepatadaviruses (in particular hepatitis B virus); papilloma viruses; adenoviruses); RNA viruses (in particular HIV I, II; HTLV I, II; poliomyelitis virus; hepatitis A; coronoviruses such as sudden acute respiratory syndrome (SARS); orthomyxoviruses (in particular influenza viruses); paramyxoviruses (in particular virus measles); rabies virus; hepatitis C virus), flaviviruses, influenza viruses; caliciviruses; rabies viruses, cattle plague virus, arenavirus, etc. In addition, examples of types of virus-induced diseases for which mutant IL-28 or IL-29 containing cysteine substitutions can be used include, but are not limited to, acquired immunodeficiency; severe acute respiratory syndrome (SARS); hepatitis; gastroenteritis; hemorrhagic diseases; enteritis; carditis; encephalitis; paralysis; brochiolite; diseases of the upper and lower respiratory tract; respiratory papillomatosis; arthritis; disseminated disease, meningitis, mononucleosis. In addition, mutant IL-28 or IL-29 containing cysteine substitutions may find various applications in antiviral immunotherapy and in combination with other cytokines, other proteins or small molecules of antiviral agents, and the like.

In clinical settings, diagnostic tests for HCV include serological antibody tests and molecular tests for viral particles. Enzymatic immunoassays are known (Vrielink et al., Transfusion 37: 845-849, 1997), but confirmation using tests such as immunoblotting (Pawlotsky et al., Hepatology 27: 1700-1702, 1998) may be required. Polymerase chain reaction methods are commonly used for qualitative and quantitative assays and are preferred for analysis of viremia and treatment response (Poynard et al., Lancet 352: 1426-1432, 1998; McHutchinson et al., N. Engl. J Med. 339: 1485-1492, 1998). Several commercial tests are available, such as the quantitative RT-PCR method (Amplicor HCV Monitor ™, Roche Molecular Systems, Branchburg, NJ) and the hybridization method using branched DNA probes (deoxyribonucleic acid) (Quantiplex ™ HCV RNA Assay [bDNA], Chiron Corp., Emeryville, California). A non-specific laboratory test for HCV infection, which measures the level of alanine aminotransferase (ALT), is inexpensive and readily available (National Institutes of Health Consensus Development Conference Panel, Hepatology 26 (Suppl. 1): 2S-10S, 1997). A histological evaluation of a liver biopsy is usually considered the most accurate method for determining the development of HCV (Yano et al., Hepatology 26: 1334-1340, 1996). For a review of clinical trials for HCV, see Lauer et al., N. Engl. J. Med. 345: 41-52, 2001.

There are several in vivo models for testing for HBV and HCV that are known in the art. For HCV, the HCV replicon model is a cell-based system for investigating drug efficacy in suppressing HCV replication (Blight et al., Science, 290 (5498): 1972-1974 (Dec. 8, 2000); and Lohmann et al ., Science, 285 (5424): 110-113 (July 2, 1999)). The well-known and recognized in vitro HBV model, which can be used to determine the activity of the tested molecules against HBV, is given by Korba et al., Antiviral Res., 19 (1): 55-70 (1992) and Korba et al., Antiviral Res ., 15 (3): 217-228 (1991).

For example, the effect of mutant IL-28 or IL-29 containing cysteine substitutions on mammals infected with HBV can be studied in a woodchuck model. In short, marmots chronically infected with hepatitis B virus (WHV) develop hepatitis and hepatic cell carcinoma similar to the disease in people chronically infected with HBV. The indicated model was used for preclinical evaluation of antiviral activity. A stable, infected WHV line is obtained, and newborn marmots are injected with serum to obtain animals for studying the effects of certain compounds using this model (for a review see Tannant et al., ILAR J. 42 (2): 89-102, 2001). Chimpanzee can also be used to study the effect on HBV-infected animals of mutant IL-28 or IL-29 containing cysteine substitutions. Using chimpanzees, HBV parameters were determined and studies have shown that the disease in chimpanzees is very similar to the disease in humans (Barker et al., J. Infect. Dis. 132: 451-458, 1975 and Tabor et al., J. Infect. Dis. 147: 531-534, 1983). A chimpanzee model was used to evaluate vaccines (Prince et al., In Vaccines 97, Cold Spring Harbor Laboratory Press, 1997). Therapeutic methods for HBV are usually tested on non-human primates infected with monkey immunodeficiency virus (for a review see Hirsch et al., Adv. Pharmacol. 49: 437-477, 2000 and Nathanson et al., AIDS 13 (suppl. A) : S113-S120, 1999). For a review of the use of non-human primates for the study of HIV, hepatitis, malaria, the respiratory syncytial virus and other diseases, see Sibal et al., ILAR J. 42 (2): 74-84, 2001. A recently developed model of transgenic mice (Guidotti et al., Journal of Virology 69: 6158-6169, 1995) allows high levels of infectious HBV to be reproduced, and has been used as a chemotherapeutic model for HBV infection. Transgenic mice were treated with antiviral drugs, and after treatment, the levels of DNA and HBV RNA in the liver and serum of transgenic mice were determined. After treatment, serum HBV protein levels in transgenic mice can also be determined. This model was used to evaluate the efficacy of lamivudine and IFN-α for lowering the HBV viral titer (Morrey et al., Antiviral Therapy 3: 59-68, 1998).

Moreover, mutant polypeptides and IL-28 or IL-29 proteins containing cysteine substitutions of the present invention can be characterized by their activity, i.e. modulating proliferation, differentiation, migration, adhesion, gene expression or metabolism of the respective cell types. The biological activity of mutant polypeptides and IL-28 or IL-29 proteins containing cysteine substitutions is analyzed using in vitro and in vivo assay methods designed to establish proliferation, differentiation, migration, and adhesion; or analyzed by a change in gene expression or cellular metabolism (in particular, the production of growth factors or other macromolecules). Many suitable methods of analysis are known in the art, and certain types of analysis are provided herein. The most convenient for screening are assays using cultured cells, such as methods for determining the effect of substitutions, deletions or insertions of amino acids.

The activity of mutant proteins of IL-28 or IL-29 containing cysteine substitutions can be determined in vitro using grown cells or in vivo by introducing the molecules of the present invention into suitable animal models. Assays in which proliferation or differentiation of cells are determined are well known in the art. For example, assays that measure proliferation include assays such as neutral red dye chemosensitivity (Cavanaugh et al., Investigational New Drugs 8: 347-354, 1990), inclusion of radiolabeled nucleotides (described, for example, in Raines and Ross, Methods Enzymol. 109: 749-773, 1985; Wahl et al., Mol. Cell Biol. 8: 5016-5025, 1988; and Cook et al., Analytical Biochem. 179: 1-7, 1989), inclusion 5-bromo-2′-deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J. Immunol. Methods 82: 169-179, 1985) and the use of tetrazolium salts (Mosmann, J. Immunol. Methods 65: 55- 63, 1983; Alley et al., Cancer Res. 48: 589-601, 1988; Marshall et al., Growth Reg. 5: 69-84, 1995; and Scudiero et al., Cancer Res. 48: 4827-4833, 1988). Differentiation can be analyzed using suitable progenitor cells, which can be made to differentiate into a more mature phenotype. Assays that measure differentiation include, for example, the determination of cell surface markers associated with phase-dependent expression in tissue, enzymatic activity, functional activity, or morphological changes (Watt, FASEB, 5: 281-284, 1991; Francis, Differentiation 57: 63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all of which are incorporated herein by reference).

The activity of mutant IL-28 or IL-29 proteins containing cysteine substitutions can also be determined using assays based on measuring IL-28-induced or IL-29-induced production of one or more additional growth factors or other macromolecules. It was shown that some members of the protein family, including IL-28 or IL-29, increase in vivo the number of monocytes in the blood circulation. Monocyte activation is important for both innate and acquired immunity. For example, it has been shown that monocytes activate antigen presentation by several mechanisms. Presentation of the antigen promotes the activation and proliferation of T cells, both cytotoxic and helper T cells. The maturation and activation of dendritic cells also contributes to the activation of T cells, as well as both innate and acquired immunity. It has also been shown that activated monocytes and macrophages enhance cytolytic activity. Thus, a mutant IL-28 or IL-29 containing cysteine substitutions is suitable as an anti-infectious agent enhancing the innate, cell-mediated and humoral response. Increased staining of CD14 + monocytes by ICAM was observed, from which it can be concluded that IL-28 or IL-29 play a role in the activation of monocytes. The results showed that members of this family contribute to the development of an antiviral response to the action of viruses, but they are likely to also affect bacteria and parasites.

Monocyte activation assays are performed to (1) evaluate the ability of mutant proteins of IL-28 or IL-29 containing cysteine substitutions, additionally stimulate the activation of monocytes and (2) to study the ability of mutant proteins of IL-28 or IL-29 containing cysteine substitutions, to modulate monocyte-induced or endotoxin-induced activation of monocytes (Fuhlbrigge et al., J. Immunol. 138: 3799-3802, 1987). The levels of IL-1α and TNF-α produced in response to activation were determined by ELISA (Biosource, Inc., Camarillo, CA). Monocyte / macrophage cells, due to CD14 (LPS receptor), are particularly sensitive to the action of endotoxin, and proteins with a moderate level of endotoxin-like activity should activate these cells.

Elevated monocyte levels indicate that mutant IL-28 or IL-29, containing cysteine substitutions, has a direct effect on myeloid progenitor cells in the bone marrow. Enhanced differentiation of myeloid progenitor cells into monocytes is crucial for the restoration of immunocompetence, for example, after chemotherapy. Thus, the introduction of mutant IL-28 or IL-29, containing cysteine substitution, to patients undergoing chemotherapy, contributes to their rehabilitation and the ability to resist infections that are usually associated with chemotherapeutic methods. Thus, the present invention provides methods for increasing the number of monocytes or monocyte precursor cells by culturing either bone marrow or peripheral blood cells together with the molecules of the present invention so that the number of monocytes or monocyte precursor cells increases so as to achieve the indicated effect in vivo or ex vivo. The present invention also provides an in vivo method for administering the molecules of the present invention to a mammal that needs to increase the number of monocytes or monocyte precursor cells. An increase in the number of monocytes or monocyte precursor cells can be determined using methods well known to clinicians, practitioners, and other specialists. Monocyte cells are included in the myeloid line of differentiation of hematopoietic cells, and, therefore, their effect on other cells of this line of differentiation may not seem unusual. For example, when a factor enhances the differentiation or proliferation of one type of cell of the myeloid or lymphoid direction of differentiation, this may affect the production of other cells that share common progenitor cells or stem cells.

Hematopoietic activity of mutant IL-28 or IL-29 proteins containing cysteine substitutions can be determined on various hematopoietic cells in culture. Preferred assays include analysis of colony formation in primary bone marrow culture and analysis of line-specific colony formation in late culture that are known in the art (in particular, Holly et al., WIPO publication WO 95/21920). Bone marrow cells applied to a suitable semi-solid medium (e.g., 50% methyl cellulose containing 15% fetal calf serum, 10% bovine serum albumin and 0.6% PSN antibiotic mixture) are grown in the presence of the test polypeptide and then examined under colony formation with a microscope. Known hemopoietic factors are used as controls. Mitogenic activity of mutant IL-28 or IL-29 polypeptides containing cysteine substitutions in relation to hematopoietic cell lines can be determined as described above.

Cell migration is analyzed by the same procedure as described by Kähler et al. (Arteriosclerosis, Thrombosis, and Vascular Biology 17: 932-939, 1997). A protein is considered chemotactic if it induces cell migration from an area with a low protein concentration to an area with a high protein concentration. A typical analysis is carried out using modified Boyden chambers with polystyrene membranes separating the two chambers (Transwell; Corning Costar Corp.). A test sample diluted with medium containing 1% BSA was placed in a 24-well Transwell lower chamber. After that, the cells are placed on a Transwell insert, which is pretreated with 0.2% gelatin. Cell migration is determined after 4 hours of incubation at 37 ° C. Non-migrated cells are wiped from the upper part of the Transwell membrane, and cells that adhere to the lower surface of the membrane are fixed and stained with 0.1% crystal violet. The stained cells are then extracted with 10% acetic acid and absorbance is measured at 600 nm. After that, the migration is calculated from the standard calibration curve. Cell migration can also be measured by the matrigel method developed by Grant et al. (“Angiogenesis as a component of epithelial-mesenchymal interactions” in Goldberg and Rosen, Epithelial-mesenchymal Interactions in Cancer, Birkhäuser Verlag, 1995, 235-248; Baatout, Anticancer Research 17: 451-456, 1997).

The adhesive activity of cells is analyzed mainly by the method described by LaFleur et al. (J. Biol. Chem. 272: 32798-32803, 1997). Briefly, microtiter plates are coated with a test protein, non-specific sites are blocked with BSA, and cells (such as smooth muscle cells, white blood cells or endothelial cells) are plated at a density of approximately 10 ⁴ -10 ⁵ cells / cell. Wells are incubated at 37 ° C (usually for 60 minutes), then non-adherent cells are removed by gentle washing. The number of adherent cells is estimated by conventional methods (in particular, staining with crystal violet, cell leasing and determining the optical density of the lysate). Control wells are coated with a known adhesive protein, such as fibronectin or vitronectin.

Expression of mutant IL-28 or IL-29 polypeptides containing cysteine substitutions in animals provides a model for further studying the biological effects of overproduction or inhibition of protein activity in vivo. Polynucleotides encoding IL-28 or IL-29 and antisense polynucleotides can be introduced into test animals, such as mice, using viral vectors or naked DNA, or transgenic animals can be obtained.

In one of the in vivo approaches, the viral delivery systems are used in the analysis of the proteins of the present invention. Examples of viruses for this purpose include adenovirus, herpes virus, retroviruses, vaccinia virus and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is currently the most studied gene transfer vector for the delivery of heterologous nucleic acids. For a review, see Becker et al., Meth. Cell Biol. 43: 161-89, 1994; and Douglas and Curiel, Science & Medicine 4: 44-53, 1997. The adenovirus system provides several advantages. Adenovirus can (i) accept relatively large insertions in DNA; (ii) be grown to large titers; (iii) infect a wide range of mammalian cell types; and (iv) be used with many different promoters, including ubiquitous, tissue-specific and regulated promoters. Since adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. See also Wu et al., J. Biol. Chem. 263: 14621-14624, 1988; Wu et al., J. Biol. Chem. 267: 963-967, 1992; and Johnson and Tang, Meth. Cell Biol. 43: 353-365, 1994.

Genetic engineering methods can also produce transgenic mice (Lowell et al., Nature 366: 740-742, 1993) that are capable of expressing a mutant IL-28 or IL-29 gene containing cysteine substitutions, or mice showing a complete lack of mutant function the IL-28 or IL-29 gene containing cysteine substitutions called “knockout mice” (Snouwaert et al., Science 257: 1083, 1992). These mice can be used to study in vivo the mutant gene IL-28 or IL-29 containing cysteine substitutions and the protein encoded by it. Preferred promoters for transgenic expression include promoters from the metallothionein and albumin genes.

Most cytokines, as well as other proteins produced by activated lymphocytes, play an important biological role in cell differentiation, activation, recruitment and homeostasis of cells throughout the body. Mutant IL-28 or IL-29 containing cysteine substitutions and inhibitors of their activity are expected to find wide therapeutic application. Specified therapeutic uses include the treatment of diseases that require immune regulation, including autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, severe pseudoparalytic myasthenia gravis, systemic lupus erythematosus, and diabetes. IL-28 or IL-29 may be important for the regulation of inflammation and therefore may be suitable for the treatment of rheumatoid arthritis, asthma and sepsis. IL-28 or IL-29 may play a role in mediating oncogenesis, and therefore a mutant IL-28 or IL-29 antagonist containing cysteine substitutions may be useful in treating cancer. IL-28 or IL-29 may be suitable for modulating the immune system, and therefore antagonists of mutant IL-28 or IL-29 containing cysteine substitutions can be used to reduce transplant rejection, for graft versus host disease, and to enhance immunity to infectious diseases, for the treatment of patients with damage to the immune system (in particular HIV ⁺ patients) or for the improvement of vaccines.

It was shown that members of the protein family of the present invention have an antiviral effect that resembles the antiviral effect of interferon-α. The use of interferons is approved in the United States for the treatment of autoimmune diseases, genital warts, chronic hepatitis C, bladder carcinoma, cervical carcinoma, laryngeal papillomatosis, fungal mycosis, chronic hepatitis B, Kaposi’s sarcoma, human immunodeficiency virus, human malignant disease and hairy ”cells and multiple sclerosis. In addition, a mutant IL-28 or IL-29 containing cysteine substitutions can be used to treat forms of arteriosclerosis, such as atherosclerosis, by inhibiting cell proliferation. Thus, the present invention encompasses the use of mutant IL-28 or IL-29 proteins containing cysteine substitutions, mutant IL-28 or IL-29 polypeptides containing cysteine substitutions, and peptides having IL-28 or IL-29 activity for the treatment of these conditions, as well as for the treatment of retinopathy. The present invention also encompasses the use of mutant IL-28 or IL-29 proteins containing cysteine substitutions, mutant IL-28 or IL-29 polypeptides containing cysteine substitutions, and peptides having IL-28 or IL-29 activity for the treatment of lymphoproliferative diseases including B cell lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, acute myelocytosis, chronic myelocytosis.

It was also shown that interferons induce the expression of antigens by cultured cells (see, for example, Auth et al., Hepatology 18: 546 (1993), Guadagni et al., Int. J. Biol. Markers 9: 53 (1994), Girolomoni et al., Eur. J. Immunol. 25: 2163 (1995), and Maciejewski et al., Blood 85: 3183 (1995). This activity enhances the ability to identify new tumor-associated antigens in vitro. Moreover, the ability of interferons to increase the level of expression of human tumor antigens indicates that interferons can be used to adjuvant regulation of immunotherapy or enhance immunoscintigra aphii using antitumor antigenic antibodies (Guadagni et al., Cancer Immunol. Immunother. 26: 222 (1988); Guadagni et al., Int. J. Biol. Markers 9: 53 (1994)). Thus, the present invention includes use of mutant IL-28 or IL-29 proteins containing cysteine substitutions, mutant IL-28 or IL-29 polypeptides containing cysteine substitutions, and peptides having IL-28 or IL-29 activity as adjuvants for immunotherapy or to improve immunoscintigraphy using antitumor antigenic antibodies.

The activity and effect of a mutant IL-28 or IL-29 containing cysteine substitutions on tumor development and metastasis can be determined in vivo. To study the effect of polypeptides, compounds, and other treatments on tumor development, several syngeneic mouse models have been developed. In these models, tumor cells after several transfers in the culture medium are implanted into mice of the same line as the tumor donor. In recipient mice, cells develop into tumors that have similar characteristics, and in some models, metastases also develop. Suitable tumor models for the authors' studies include, among others, Lewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No. CRL-6323). Both represent commonly used tumor lines that are syngenic to C57BL6 mice that are easy to grow and easy to manipulate in vitro. Tumors obtained by implantation of any of these cell lines can metastasize to the lungs of C57BL6 mice. A Lewis lung carcinoma model has recently been used in mice to identify an angiogenesis inhibitor (O'Reilly MS, et al., Cell 79: 315-328, 1994). C57BL6 / J mice are treated with an experimental agent, either by daily injection of a recombinant protein, agonist or antagonist, or by a single injection of recombinant adenovirus. Three days after treatment, 10 ⁵ to 10 ⁶ cells are implanted in the skin on the back. Alternatively, the cells themselves can be infected with a recombinant adenovirus prior to implantation, for example, one that expresses a mutant IL-28 or IL-29 containing cysteine substitutions so that the protein is more likely to be synthesized at the location of the tumor or intracellularly rather than systemically. In mice, distinguishable tumors appear within 5 days. Tumors are allowed to grow for up to 3 weeks, and during this time in the treated control group they can reach sizes of 1500-1800 mm ³ . Throughout the experiment, tumor size and body weight are carefully monitored. Once the animals are killed, the tumors are removed along with the lungs and liver and weighed. It was found that lung mass correlates with metastatic tumor burden. As an additional measurement, the surface of the lung affected by metastases is calculated. The excised tumor, lungs and liver are prepared for histological examination, immunohistochemistry and in situ hybridization using methods known from the technical field and described in this description. In this way, it is possible to study the effect of the expression of the polypeptide of interest, in particular, mutant IL-28 or IL-29 containing cysteine substitutions, on the tumor's ability to restore the vasculature and metastasize. In addition, in addition to using adenoviruses, implanted cells can be temporarily transfected with mutant IL-28 or IL-29 containing cysteine substitutions. The use of stable transfectants of mutant IL-28 or IL-29 containing cysteine substitutions, as well as the use of inducible promoters to activate the expression of mutant IL-28 or IL-29 containing cysteine substitutions, are known in the art and can be used in this system for analysis of activation metastasis. Moreover, a purified conditioned medium, including mutant IL-28 or IL-29, which contains cysteine substitutions, can be directly introduced by injection into a mouse model and, thus, used in this system. For general information see O′Reilly MS, et al., Cell 79: 315-328, 1994; and Rusciano D, et al., Murine Models of Liver Metastasis. Invasion Metastasis 14: 349-361, 1995.

Mutant IL-28 or IL-29 containing cysteine substitutions can also be used to treat myocarditis, a disorder that occurs when the heart becomes involved in the inflammatory process. It is believed that the result of infection of a virus, bacterium, fungus, or parasite is lymphocyte infiltration or myocytolysis (see, for example, Brodison et al., J. Infection 37: 99 (1998)). For the treatment of infections associated with myocarditis, mutant IL-28 or IL-29 containing cysteine substitutions can be administered intravenously or subcutaneously. Mutant IL-28 or IL-29 containing cysteine substitutions can also be administered intravenously as an immunoregulatory cytokine in the treatment of autoimmune myocarditis. Dosages of interferon can be extrapolated using an autoimmune myocarditis model in A / J mice (Donermeyer et al., J. Exp. Med. 182: 1291 (1995)).

Recent reports have highlighted the role of type I interferons in preventing virus-induced diabetes by inducing strong antiviral status in pancreatic beta cells in the early stages of viral infection (Flodstroem et al., Nature Immunology 3: 373-382 (2002)). This prevents the loss of beta cells due to virus-induced cell death and the accompanying autoimmune reaction. Mutant IL-28 or IL-29, containing cysteine substitutions, also induces an antiviral state in cells that express IL-28 receptor. The IL-28 receptor is highly expressed in pancreatic tissues, and therefore mutant IL-28 or IL-29, containing cysteine substitutions, can play a role in preventing virus-induced diabetes and beta cell death. In addition, the role of type I interferon in preventing virus-induced diabetes can be extended to other virus-induced autoimmune diseases, and therefore mutant IL-28 or IL-29 containing cysteine substitutions can play a role in preventing other diseases, such as muscle sclerosis, lupus and virus-induced autoimmune tissue diseases that express the IL-28 receptor.

Mutant IL-28 or IL-29 polypeptides containing cysteine substitutions can be administered individually or in combination with other vasculogenic or angiogenic agents, including VEGF. When using a mutant IL-28 or IL-29 containing cysteine substitutions, in combination with an additional agent, both compounds can be administered simultaneously or separately, depending on the particular condition being treated.

Mutant IL-28 or IL-29 containing cysteine substitutions are useful in the treatment of oncogenesis, and therefore could be used to treat cancer. IL-28 can inhibit B-cell tumor lines, and thus it may be therapeutically useful to treat patients with mutant IL-28 or IL-29 containing cysteine substitutions in order to transfer the B cells of the tumor cells to a less proliferative state . The ligand can be administered in combination with other agents that have already found practical use, including conventional chemotherapeutic agents, as well as immunomodulators, such as interferon-alpha. It has been shown that alpha / beta interferons are effective in treating certain types of leukemia and animal models of the disease, and the inhibitory effect of interferon alpha and mutant IL-28 or IL-29 containing cysteine substitutions on growth can complement the effect of tumor-induced cell lines B -cells.

In accordance with another aspect, the present invention provides a pharmaceutical composition comprising an isolated peptide that is selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25 , 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 , 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161 and a pharmaceutically acceptable carrier.

For pharmaceutical use, mutant IL-28 or IL-29 proteins containing cysteine substitutions are prepared by conventional methods in the form of a composition for topical application or parenteral, in particular, intravenous or subcutaneous administration. In general, pharmaceutical formulations include a mutant IL-28 or IL-29 polypeptide containing cysteine substitutions in combination with a pharmaceutically acceptable carrier such as saline, saline buffered saline, 5% dextrose in water, and the like. The preparations may also include one or more adjuvants, preservatives, solubilizers, buffering additives, albumin to prevent loss of protein on the walls of ampoules, etc. Methods of formulation are well known in the art and include, for example, in Remington:.. The Science and Practice of Pharmacy, Gennaro, ed, Mack Publishing Co., Easton, PA, 19 ^th ed, 1995. The mutant IL-28 or IL -29, containing cysteine substitutions, is preferably used with a concentration of about 10 to 100 μg / ml in total, although concentrations in the range of 1 ng / ml to 1000 μg / ml can be used. For topical application, for example, in order to accelerate wound healing, protein is applied in the range of 0.1-10 μg per square centimeter of the wound area, while the exact dose is determined by the doctor based on accepted standards, taking into account the nature and severity of the condition being treated, the patient’s individual characteristics and etc. Dose determination is within the competence of a specialist. Dosage is carried out daily or take breaks in treatment. Intravenous administration is carried out by injection with boluses or infusion, usually within one to several hours. Sustained-release formulations may also be used. In general, a therapeutically effective amount of a mutant IL-28 or IL-29 containing cysteine substitutions is one that is sufficient to cause a clinically significant change in the condition being treated, such as a clinically significant change in viral load or immune function, a clinically significant decrease in morbidity or a clinically significant increase in histological index.

By way of illustration, the pharmaceutical preparations can be presented in the form of a kit containing a container containing the IL-28 or IL-29 polypeptide of the present invention. Therapeutically valuable polypeptides can be presented in the form of a solution for injection for single or multiple doses or in the form of a sterile powder that is reconstituted before injection. Alternatively, the kit may include a dry powder dispersant, a liquid aerosol generator, or a nebulizer for administering a therapeutic polypeptide. In addition, the kit may include instructions printed on the indications and use of the pharmaceutical composition. Moreover, such information may include an indication that the composition containing the IL-28 or IL-29 polypeptide is contraindicated in patients with known hypersensitivity to the IL-28 or IL-29 polypeptide.

In accordance with another aspect, the present invention provides a method for producing an antibody to a polypeptide, which comprises: vaccinating an animal with a polypeptide selected from the group consisting of SEQ ID NO: 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161, while the polypeptide elicits an immune response in the animal, resulting in to antibody production; and isolation of the antibody from the animal. In accordance with another aspect, the present invention provides an antibody (in particular, a neutralizing antibody) obtained by the above method, wherein the antibody binds to a polypeptide selected from the group consisting of SEQ ID NO: 19, 21, 23, 25, 27 , 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109 , 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161. B in one embodiment of the invention, the antibody disclosed above specifically binds to a polypeptide selected from the group consisting of SEQ ID NO: 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 and 161. In accordance with another aspect, the present invention provides an antibody or antibody fragment that specifically binds to a polypeptide, as described herein. In one embodiment, the antibody is selected from the group consisting of a polyclonal antibody, a murine monoclonal antibody, a humanized antibody derived from a murine monoclonal antibody, a fragment of an antibody, and a human monoclonal antibody. In one embodiment of the present invention, the antibody fragment is an antibody fragment provided herein, wherein said antibody fragment is selected from the group consisting of F (ab ′), F (ab), Fab ′, Fab, Fv, scFv and minimal recognition unit.

In accordance with another aspect, the present invention provides an antigenotypic antibody that specifically binds to an antibody, as described herein.

As used herein, the term “antibodies” includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof, such as F (ab ′) ₂ and Fab fragments, single chain antibodies and the like, including genetically engineered antibodies. Non-human antibodies can be humanized by grafting non-human CDRs into the skeletal and constant regions of a human antibody or by incorporating whole non-human variable regions (with an optional “coating” of their human-like surface due to open residues, thereby producing an antibody with a “veneer” layer) . In some cases, humanized antibodies may retain non-human residues within the variable regions of a human antibody in order to enhance binding properties. By humanizing antibodies, the biological half-life can be increased, and the potential for harmful immune reactions to be reduced. One of skill in the art can generate humanized antibodies containing specific and distinct constant domains (e.g., different Ig subclasses) to facilitate or inhibit various immune functions associated with specific constant regions of an antibody. Antibodies are defined as specifically binding if they bind to a mutant polypeptide or IL-28 or IL-29 protein containing cysteine substitutions with an affinity of at least 10 times that of the control (other than mutant IL-28 or IL- 29, containing cysteine substitution) to a polypeptide or protein. One skilled in the art can readily determine the affinity of a monoclonal antibody (see, for example, Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949).

Methods for producing polyclonal and monoclonal antibodies are well known in the art (see, for example, Hurrell, JGR, Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982; is incorporated into this description by links). The polypeptide immunogen may be a complete molecule or part thereof. If the polypeptide portion is “hapten-like”, then when vaccinating, it is convenient to combine or bind to the macromolecular carrier (such as fissurellia mollusk hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid).

To detect antibodies that specifically bind to mutant IL-28 or IL-29 polypeptides containing cysteine substitutions, a large number of analytical methods known to those skilled in the art can be used. Examples of assays are described in detail in Using Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1999. Some examples of such assays include: combined immunoelectrophoresis, radioimmunoassays, radioimmunoassay, immunoassay solid phase analysis (ELISA), pre- blotting, western blotting, inhibitory and competitive assays and sandwich assays.

For certain applications, including in vitro and in vivo diagnostic uses, it is convenient to use labeled antibodies. Suitable direct labels are radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles, and the like; Biotin-Avidin and other complement / anti-complement pairs can act as indirect labels as intermediates. The antibodies of the present invention can be directly or indirectly associated with drugs, toxins, radionuclides, etc., and the resulting conjugates can be used for in vivo diagnostics or therapeutic applications (for example, to inhibit cell proliferation). For general information, see Ramakrishnan et al., Cancer Res. 56: 1324-1330, 1996.

The present invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1

Mammalian expression plasmids

An expression plasmid containing zcyto20 and zcyto21 is constructed by homologous recombination. Zcyto20 and zcyto21 cDNA fragments were obtained by PCR amplification. As primers for PCR use the following:

zcyto20 / pZMP21: zc40923 and zc43152 SEQ ID NO: 42 and 43, respectively; and zcyto21 / pZMP21: zc40922 and zc43153 SEQ ID NO: 2 and 73, respectively.

The PCR reaction was carried out on a 1% agarose gel and the strip corresponding to the size of the insert was extracted using a QIAquick ™ Gel Extraction kit (Qiagen, Valencia, California).

Plasmid pZMP21, which was cut using BglII, is used for recombination with a fragment of the PCR insert. Plasmid pZMP21 is a mammalian expression vector containing a polygenic expression cluster with the MPSV promoter and multiple restriction sites for insertion of coding sequences; replication initiation site from E. coli; a mammalian selectable expression marker block comprising an SV40 promoter, an enhancer and a replication initiation site, a DHFR gene and an SV40 terminator; and the URA3 and CEN-ARS sequences necessary for selection and replication in S. cerevisiae. It is constructed from pZP9 (deposited in the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, numbered 98668) with yeast genetic elements derived from pRS316 (deposited in the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, numbered 77145), an element of the site of the internal landing site of the ribosome (IRES) from the polio virus and the extracellular domain of CD8 truncated at the C-terminus of the transmembrane domain.

One hundred microliters of competent yeast cells (S. cerevisiae) are independently combined with 10 μl of the DNA insert and 100 ng of the cut pZMP21 vector indicated above, and the mixture is placed in an electroporation cell with an electrode spacing of 0.2 cm. The yeast / DNA mixture is electroporated using a current source (BioRad Laboratories, Hercules, California) with parameters of 0.75 kV (5 kV / cm), ∞ Ohm, 25 μF. Six hundred microliters of a 1.2 M sorbitol solution were added to the cuvette and the yeast was placed as aliquots of 100 μl and 300 μl in two URA-D plates and incubated at 30 ° C. After approximately 72 hours, Ura ⁺ yeast transformants from one cup were resuspended in 1 ml of H ₂ O and carefully centrifuged to obtain a pellet from yeast cells. The cell mass is resuspended in 0.5 ml of lysis buffer solution (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundred microliters of the lysis mixture was placed in an Eppendorf tube containing 250 μl of acid-washed glass beads and 300 μl of a phenol-chloroform mixture, and stirred vigorously for 3 minutes, and then rotated at maximum speed for 5 minutes in an Eppendorf centrifuge. Three hundred microliters of the aqueous phase was transferred to a fresh tube and DNA was precipitated with 600 μl of ethanol (EtOH) and 30 μl of a 3M sodium acetate solution, and then centrifuged at maximum speed for 30 minutes. The DNA pellet was resuspended in 30 μl TE.

Transformation of electrocompetent E. coli host cells (MC1061) is performed using 5 μl of yeast DNA preparation and 50 μl of cells. Cells are electroporated with parameters of 2.0 kV, 25 μF and 400 Ohms. During electroporation, add 1 ml of SOC (2% bactotryptone (Difco, Detroit, Michigan), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ , 20 mM glucose) and cells were aliquoted in 50 μl and 200 μl in two cups of LB AMP (LB culture medium (Lennox), 1.8% Bacto ™ Agar (Difco), 100 mg / L ampicillin).

The inserts of the three clones for each gene construct are sequenced and one clone is selected for each construct containing the correct sequence. Larger isolation of plasmid DNA is carried out using a commercially available kit (QIAGEN Plasmid Mega Kit, Qiagen, Valencia, California) in accordance with the manufacturer's recommendations. The correct constructs are called zcyto20 / pZMP21 and zcyto21 / pZMP21.

Example 2

Expression of mammalian constructs in CHO cells

200 μg of the genetic constructs zcyto20 / pZMP21 and zcyto21 / pZMP21 were digested with 200 PvuI units at 37 ° C for three hours, and then precipitated with isopropyl alcohol and the precipitate was separated by centrifugation in 1.5 ml microtubes. The supernatant was decanted from the precipitate and the precipitate was washed with 1 ml of a 70% ethanol solution and incubated for 5 min at room temperature. The microcentrifuge tube is rotated for 10 min at a speed of 14,000 rpm and the supernatant is separated by suction. The pellet is then resuspended in 750 μl PF-CHO medium under sterile conditions and incubated at 60 ° C for 30 minutes. CHO cells are separated by centrifugation and resuspended in a solution of DNA medium. The DNA / cell mixture was placed in an electroporation cuvette and electroporated under the following conditions: 950 μF, high capacity, and 300 V. The contents were then removed from the cuvette, diluted with 25 ml of PF-CHO medium, and placed in a 125 ml shaker mixer flask. The flask is installed in an incubator on a rocking mixer at 37 ° C, 6% CO ₂ and stirred at a speed of 120 rpm

Example 3

Purification and analysis of zcyto20-CHO protein

A. Purification of zcyto20-CHO Protein

The recombinant zcyto20-CHO protein (IL-28A) is isolated from the pool of DXB11-CHO cell lines. Cultures are grown, and the culture medium is sterilized by filtration through a 0.2 μm filter.

The zcyto20-CHO protein was purified sequentially using a Poros HS 50 column (Applied Biosystems, Framingham, Mass.), Monolithic WCX columns (Isco, Inc., Lincoln, Nebraska), ToyoPearl Butyl 650S columns (TosoH, Montgomeryville, PA) and Superdex columns 75 (Amersham Biosciences, Piscataway, NJ). Before placing on a Poros HS 50 column, the pH of the culture medium from DXB111-CHO was adjusted to 6.0. The column is washed with a solution of 50 mM MES (2-morpholinethanesulfonic acid), 100 mM NaCl with pH 6 and the bound protein is eluted with 10 column volumes (CV) in a linear gradient to 60% 50 mM MES, 2 M NaCl with pH 6. Collected by elution fractions are collected and the presence of zcyto20 protein is confirmed by SDS-PAGE using Coomassie staining. These fractions, which contain zcyto20 protein, are combined, diluted with bidistilled water to an electrical conductivity of approximately 20 mS and placed on a Monolithic WCX column. The column is washed with 93% 50 mM MES, 100 mM NaCl with pH 6 and 7% 50 mM MES, 2 M NaCl with pH 6. The bound protein is eluted with 25 column volumes in a linear gradient from 7% to 50% 50 mM MES, 2 M NaCl pH 6. Eluted fractions were collected and the presence of zcyto20 protein was confirmed by SDS-PAGE using Coomassie staining. Fractions that contain zcyto20 protein are combined, adjusted to 1 M with ammonium sulfate and placed on a ToyoPearl Butyl 650S column. Zcyto20 is eluted in a decreasing gradient of ammonium sulfate and the fractions containing purified zcyto20 are combined and concentrated before injection into the Superdex 75 column. The fractions containing zcyto20 obtained after the gel filtration column are combined, concentrated, filtered through a 0.2 μm filter and frozen at -80 ° C. The concentration of the final purified protein is determined by BCA analysis (Pierce Chemical Co., Rockford, Illinois) and amino acid HPLC analysis.

B. SDS-PAGE and Western blot analysis of zcyto20-CHO protein

The recombinant zcyto20 protein is analyzed by SDS-PAGE (Nupage 4-12% Bis-Tris, Invitrogen, Carlsbad, CA) and Western blotting using rabbit IgG against zcyto21-CEE-BV as the main antibody that cross-interacts with the protein zcyto20-CHO. The gel is subjected to electrophoresis in an Invitrogen Xcell II mini-cell (Carlsbad, California) and transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad Laboratories, Hercules, California) using an Invitrogen Xcell II blotting module according to with the instructions that came with the device. The specified transfer is carried out at 500 mA for 50 min in a buffer solution containing 25 mm Tris base, 200 mm glycine and 20% methanol. The membrane is blocked with a 10% solution of skimmed milk powder in 1x PBS for 10 min, then probed with the main antibody in 1x PBS containing 2.5% skimmed milk powder. The replica is labeled for one hour at room temperature with shaking. To label the second antibody, the replica is washed three times for 10 minutes each with a PBS solution and probed with a goat anti-rabbit IgG-HRP antibody (Pierce Chemical Co., Rockford, Ill.) For one hour. Replica washed with 1X PBS three times for 10 minutes each and developed with a 1: 1 reactant SuperSignal ^® ULTRA (Pierce Chemical Co., Rockford, IL) and the signal recorded with a Lumi-Imager (Boehringer Mannheim GmbH, Germany).

C. Summary of protein purification and analysis

The purified zcyto20 protein under non-reducing conditions predominantly moves from CHO medium to a 4-12% Bis-Tris gel in the form of a doublet with a mass of approximately 20 kDa and to a lesser extent in the form of a triple dimer with a mass of approximately 38 kDa. Under reducing conditions, they are all compressed into one 20 kDa band. Mapping of the peptide by mass spectrometry indicates a mixture of two isomers relative to the disulfide bridge and the presence of an O-linked glycosylation site.

Example 4

Purification and analysis of zcyto21-CHO protein

A. Purification of zcyto21-CHO Protein

Recombinant zcyto21 is obtained from stable DXB11-CHO cell lines. Cultures are grown, and the culture medium is sterilized by filtration through a 0.2 μm filter. Proteins are purified from the conditioned medium, starting with a combination of cationic and anionic exchange chromatography, and then chromatography with hydrophobic interactions and size exclusion chromatography. Before placing on a Poros HS 50 column (Applied Biosystems, Framingham, Mass.), The pH of the DXB111-CHO culture medium was adjusted to 6.0. The column is washed with 1x PBS at pH 6 and the bound protein is eluted with 5x PBS at pH 8.4. Eluted fractions were collected and the presence of zcyto21 protein was confirmed by SDS-PAGE using Coomassie staining. These fractions are then diluted to a conductivity of 13 mS, adjusted to pH 8.4 and passed through a Poros 50 HQ column (Applied Biosystems, Framingham, Mass.). The filtrate containing zcyto21 protein was adjusted with ammonium sulfate to an electrical conductivity of approximately 127 mS and placed on a Toyopearl Phenyl 650S column (TosoH, Montgomeryville, PA). Zcyto21 was eluted in a decreasing gradient of ammonium sulfate and fractions containing purified zcyto21 were combined and concentrated prior to injection into a Superdex 75 column (Amersham Biosciences, Piscataway, NJ). The concentration of the final purified protein is determined by BCA analysis (Pierce Chemical Co., Rockford, Illinois) and amino acid HPLC analysis.

B. SDS-PAGE and Western blot analysis of zcyto20-CHO protein

The recombinant zcyto21 protein was analyzed by SDS-PAGE (Nupage 4-12% Bis-Tris, Invitrogen, Carlsbad, CA) and Western blotting using rabbit IgG against zcyto21-CEE-BV as the main antibody. The gel is subjected to electrophoresis in an Invitrogen Xcell II mini-cell (Carlsbad, California) and transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad Laboratories, Hercules, California) using an Invitrogen Xcell II blotting module according to with the instructions that came with the device. The transfer is carried out at 500 mA for 50 min in a buffer solution containing 25 mm Tris base, 200 mm glycine and 20% methanol. The moving spot is blocked with a 10% solution of skimmed milk powder in 1x PBS for 10 minutes, then probed with the main antibody in 1x PBS containing 2.5% skimmed milk powder. The replica is labeled for one hour at room temperature with shaking. To label the second antibody, the replica is washed three times for 10 minutes each with a PBS solution and probed with a goat anti-rabbit IgG-HRP antibody (Pierce Chemical Co., Rockford, Ill.) For one hour. Replica washed with 1X PBS three times for 10 minutes each and developed with a 1: 1 reactant SuperSignal ^® ULTRA (Pierce Chemical Co., Rockford, IL) and the signal recorded with a Lumi-Imager (Boehringer Mannheim GmbH, Germany).

C. Summary of protein purification and analysis

The purified zcyto21 protein under both reducing and non-reducing conditions moves from CHO medium to a 4-12% Bis-Tris gel in the form of two or more bands with a mass of approximately 20 kDa. Mapping of the peptide by mass spectrometry indicates a mixture of two isomers relative to the disulfide bridge and the presence of an N-linked glycosylation site and several O-linked glycosylation sites.

Example 5

Identification of forms of IL-29

Peaks of fractions from purified IL-29 pools are digested overnight at 37 ° C with trypsin (Roche Applied Science, Indianapolis, Indiana), the purity of which is suitable for sequencing, in a phosphate buffer with a pH of approximately 6.3 so that Minimize rearrangement of disulfide bonds. The product of each cleavage was analyzed by reverse phase HPLC (Agilent, Palo Alto, California) on a chromatograph coupled to a quadrupole time-of-flight hybrid mass spectrometer (Micromass, Milford, Mass.). Spectra are obtained, mass values are converted into charge-to-mass ratios and compared with all theoretically possible peptides and combinations of disulfide-linked peptides that can be obtained by splitting IL-29 with trypsin. Disulfides are determined by comparing the spectra before and after reduction with the assignment of the corresponding masses to the disulfide-linked peptides in IL-29. The substance from fraction # 20 shows the disulfide structure of C15-C112 and C49-C145 with C171 observed as S-glutathionyl cysteine (all refer to SEQ ID NO: 4). The substance from fraction # 51 shows the disulfide structure of C49-C145 and C112-C171 with C15 observed as S-glutathionyl cysteine (all refer to SEQ ID NO: 4).

Example 6

Expression plasmid E. coli

Construction of an Expression Vector, pTAP237

Plasmid pTAP237 was constructed by inserting a PCT-generated linker into the SmaI site of pTAP186 by homologous recombination. The plasmid pTAP186 is obtained from plasmids pRS316 (the “shuttle” vector Saccharomyces cerevisiae) and pMAL-c2, an E. coli expression plasmid derived from pKK223-3 and including the tac promoter and rrnB terminator. Plasmid pTAP186 contains a kanamycin resistance site in which the SmaI site is destroyed and contains NotI and SfiI sites flanking the yeast sequences ARS-CEN6 and URA3, which facilitates their removal from the plasmid by NotI. The PCR generated linker replaces the expression linker sequence in pTAP186 with the synthetic RBS II sequence. It is obtained from 100 pM of each of the oligonucleotides zc29740 and zc29741 shown in SEQ ID NOs: 44 and 45, respectively, and approximately 5 pM of each of the oligonucleotides zc29736 and zc29738 shown in SEQ ID NOs: 46 and 47, respectively. These oligonucleotides are combined by PCR for ten cycles, including 94 ° C for 30 sec, 50 ° C for 30 sec and 72 ° C for 30 sec, followed by annealing at 4 ° C. The resulting PCR products are concentrated by precipitation with two portions of 100% ethanol. The tablet was resuspended in 10 μl of water and used to re-combine with the pTAP186 recipient vector digested with SmaI to obtain a gene construct comprising the synthetic RBS II sequence. Approximately 1 μg of the PCR linker and 100 ng of pTAP186 digested with SmaI are mixed with each other and transformed into competent yeast cells (S. cerevisiae). The yeast is then placed in -URA D plates and left at room temperature for approximately 72 hours. Then Ura + transformants from one cup are resuspended in 1 ml of H ₂ O and centrifuged for a short time to obtain a yeast cell pellet. A tablet of cells is resuspended in 0.5 ml of lysis buffer solution. DNA is isolated and transformed into E. coli MC1061. Clones were screened by colony PCR as described above using 20 pM of each of the oligonucleotides zc29740 and zc29741 shown in SEQ ID NOs: 44 and 45, respectively. Clones that give the correct agarose gel strip size are sequenced. The correct plasmid is called pTAP237.

Example 7

Optimization of codons in mutant IL-29 containing cysteine substitutions

A. Optimization of codons in the generation of wild-type expression construct IL-29

The sequence of the natural human IL-29 gene is poorly expressed in E. coli strain W3110. A study of the codons used in the coding sequence of IL-29 shows that it contains an excess of the least frequently used codons in E. coli, with a CAI of 0.206. The CAI value is a statistical measure of synonymous codon shift and can be used to predict the level of protein production (Sharp et al., Nucleic Acid Res. 15 (3): 1281-95, 1987). Genes encoding highly expressed proteins tend to have high CAIs (> 0.6), while proteins encoded by genes with low CAIs (≤ 0.2) are usually ineffectively expressed. This explains the reason for the poor production of IL-29 in E. coli. In addition, rare codons cluster in the second part of the transcript, which leads to a high likelihood of translation delay, premature translation termination and an error in the combination of amino acids (Kane J.F., Curr. Opin. Biotechnol. 6 (5): 494-500, 1995).

It has been shown that the expression level of proteins whose genes contain rare codons can be significantly improved when the level of certain rare tRNA molecules increases in the host (Zdanovsky et al., Applied Enviromental Microb. 66: 3166-3173, 2000; You et al. Biotechniques 27: 950-954, 1999). Plasmid pRARE contains genes encoding tRNA for a number of codons rarely used by E. coli (argU, argW, leuW, proL, ileX and glyT). These genes are controlled by their native promoters (Novy, ibid.). Co-expression with pRARE enhances the production of IL-29 in E. coli and yields approximately 200 mg / L. Based on these data, it can be concluded that the re-synthesis of the gene encoding IL-29, together with the more correct use of codons, provides an improved vector for the expression of large quantities of IL-29.

An optimized codon coding IL-29 sequence is constructed from sixteen overlapping oligonucleotides: zc44566 (SEQ ID NO: 48), zc44565 (SEQ ID NO: 49), zc44564 (SEQ ID NO: 50), zc44563 (SEQ ID NO: 51), zc44562 (SEQ ID NO: 52), zc44561 (SEQ ID NO: 53), zc44560 (SEQ ID NO: 54), zc44559 (SEQ ID NO: 55), zc44558 (SEQ ID NO: 56), zc44567 (SEQ ID NO: 56) : 57). Elongation of these oligonucleotides with a primer followed by PCR amplification gives the full length IL-29 gene with codons optimized for expression in E. coli. The final PCR product is inserted into the pTAP237 expression vector by homologous recombination in the yeast system. The expression genetic construct was extracted from yeast and transformed into competent E. coli MC1061. Resistance of clones to kanamycin is identified by colony PCR. The positive clone is confirmed by sequencing and then transformed into the host producer strain W3110. An IL-29 sequence optimized expression vector is called pSDH184. The resulting gene is very well expressed in E. coli. Expression levels with a new gene construct increase to approximately 250 mg / L.

B. Preparation of the cysteine mutation zcyto21 C172S expression construct with optimized codons

The strategy used to prepare the zcyto21 C172S containing the cysteine mutation is based on the use of the QuickChange Site-Directed Mutagenesis (Stratagene) reagent kit for conducting site-directed mutagenesis. C172S mutation primers are designed in accordance with the manufacturer's recommendations. These primers are designated as ZG44340 (SEQ ID NO: 58) and ZG44341 (SEQ ID NO: 59). PCR for generating zcyto21 C172S containing a cysteine mutation is carried out in accordance with the instructions for the QuickChange Mutagenesis reagent kit. Five identical reactions are carried out in a volume of 50 μl. Each reaction uses 2.5 μl of pSDH175 DNA (does not contain the frame sequence of the yeast vector) as a template. A PCR cocktail was prepared using the following amounts of reagents: 30 μl 10x PCR buffer, 125 ng (27.42 μl) ZG44340, 125 ng (9.18 μl) ZG44341, 6 μl dNTP, 6 μl Pfu Turbo polymerase (Stratagene, La Joyle, CA) and 206.4 μl of water. An aliquot of 47.5 μl of the cocktail is used in each reaction. The PCR conditions are as follows: 1 cycle at 95 ° C for 30 sec, and then 16 cycles at 95 ° C for 30 sec, 55 ° C for 1 min, 68 ° C for 7 min, then 1 cycle at 68 ° C for 7 min and ending with annealing at 4 ° C. All five reaction PCR mixtures are combined in one tube. In accordance with the manufacturer's recommendations, 5 μl of restriction enzyme DpnI was added to the PCR reaction mixture and incubated at 37 ° C for 2 hours. DNA is precipitated by adding a 10% 3 molar solution of sodium acetate and two volumes of 100% ethanol. Precipitation is carried out at -20 ° C for 20 minutes DNA is separated by centrifugation at a speed of 14,000 rpm for 5 minutes and the precipitate is dried rapidly in vacuo. The DNA tablet is resuspended in 20 μl of water. PCR DNA was transformed into E. coli strain DH10B. 5 μl of DNA is mixed with 40 μl of ElectroMAX DH10B cells (Invitrogen). The mixture of cells and DNA is then electroporated in a cuvette with an electrode spacing of 0.1 cm (Bio-Rad) using a Bio-Rad Gene Pulser II ™ and the following parameters: 1.75 kV, 100 Ohm and 25 uF. Electroporated cells grow at 37 ° C for 1 hour. The mixture is placed in a cup on LB medium + 25 μg / ml kanamycin and incubated overnight at 37 ° C. Ten clones were screened for zcyto21 C172S insert. DNA was extracted from all ten clones using the QIAprep ™ Spin Miniprep kit (Qiagen, Valencia, California) and analyzed for insert by restriction enzyme digest with XbaI and PstI. Nine clones that contain the insert are sequenced to confirm the introduction of the zcyto21 C172S mutation. The clone is checked by sequencing and designate it as pSDH188.

Example 8

Expression genetic construct of E. coli IL-29

The IL-29 DNA fragment containing the wild-type sequence was isolated by PCR. The reaction used primers zc41212 (SEQ ID NO: 60) containing 41 base pairs (bp) of the flanking sequence of the vector, and 24 bp corresponding to the amine end of IL-29, and primer zc41041 (SEQ ID NO: 61 ) containing 38 bp, which corresponds to the 3'-end of the vector containing the zcyto21 insert. The PCR conditions are as follows: 25 cycles at 94 ° C for 30 sec, 50 ° C for 30 sec, and 72 ° C for 1 min; followed by annealing at 4 ° C. A small sample (2-4 μl) of the PCR sample, for analysis, was run on a 1% agarose gel with 1X TBE buffer and the expected band corresponding to a fragment of approximately 500 bp was detected. The remaining volume of the reaction mixture, comprising 100 μl, precipitated with 200 μl of absolute ethanol. The resulting pellet was resuspended in 10 μl of water before use to recombine into the pTAP238 recipient vector, cut with SmaI, in order to obtain, as indicated above, the gene construct encoding zcyto21. A clone with the correct sequence is designated as pTAP377. Clone pTAP377 was digested with Not1 / Nco1 (10 μl DNA, 5 ml 3 New England BioLabs buffer mixture, 2 μl Not1, 2 μl Nco1, 31 μl water for 1 hour at 37 ° C) and again ligated with T4 DNA ligase in buffer mixtures (7 μl of the product of the previous cleavage reaction, 2 μl of 5X buffer, 1 μl of T4 DNA ligase). At this stage, the yeast sequence, CEN-ARS, is removed to straighten the vector. pTAP377 DNA for diagnostic purposes was digested with Pvu2 and Pst1 to confirm the absence of yeast sequence. PTAP377 DNA was transformed into E. coli strain W3110 / pRARE, a host strain containing additional copies of the rare E. coli tRNA genes.

Example 9

Expression genetic construct of E. coli IL-28A

A DNA fragment containing the wild-type zcyto20 sequence (as indicated in SEQ ID NO: 1) was isolated by PCR. The reaction used primers zc43431 (SEQ ID NO: 62) containing 41 bp the flanking sequence of the vector, and 24 bp corresponding to the amine end of zcyto20, and primer zc43437 (SEQ ID NO: 63) containing 38 bp, which correspond to the 3′-end of the vector containing the zcyto20 insert. The PCR conditions are as follows: 25 cycles at 94 ° C for 30 sec, 50 ° C for 30 sec, and 72 ° C for 1 min; followed by burning at 4 ° C. A small sample (2-4 μl) of the PCR sample, for analysis, was run on a 1% agarose gel with 1X TBE buffer and the expected band corresponding to a fragment of approximately 500 bp was detected. The remaining volume of the reaction mixture, comprising 100 μl, precipitated with 200 μl of absolute ethanol. The resulting pellet was resuspended in 10 μl of water before use to recombine into the pTAP238 recipient vector, cut with SmaI, to obtain a gene construct encoding zcyto20 as described above. A clone with the correct sequence is designated as pYEL7. It is digested with Not1 / Nco1 (10 μl DNA, 5 μl 3 New England BioLabs buffer mixture, 2 μl Not1, 2 μl Nco1, 31 μl water for 1 hour at 37 ° C) and T4 ligated again with DNA ligase in the buffer mixture (7 μl of the product of the previous cleavage reaction, 2 μl of 5X buffer, 1 μl of T4 DNA ligase). At this stage, the yeast sequence, CEN-ARS, is removed to straighten the vector. The identified pYEL7 DNA is digested with Pvu2 and Pst1 for diagnostic purposes to confirm the absence of a yeast sequence. PYEL7 DNA was transformed into E. coli strain W3110 / pRARE.

Example 10

Expression genetic construct zcyto21 C172S containing cysteine mutation

The strategy used for the preparation of zcyto21 C172S, comprising a mutation of cysteine (SEQ ID NO: 28), based on the use of reagents for carrying out site-directed mutagenesis, ^{QuikChange ® Site-Directed Mutagenesis (Stratagene} , La Dzhoyla, CA). C172S mutation primers are designed according to the manufacturer's recommendations. These primers are designated as ZG44327 and ZG44328 (SEQ ID NO: 64 and 65, respectively). PCR for generating zcyto21 C172S containing a cysteine mutation is carried out in accordance with the instructions for the QuikChange Mutagenesis reagent kit. Five identical reactions are carried out in a volume of 50 μl. Each reaction uses 2.5 μl of pTAP377 DNA (does not contain the frame sequence of the yeast vector) as a template. A PCR cocktail was prepared using the following amounts of reagents: 30 μl 10x PCR buffer, 125 ng (27.42 μl) ZG44327 (SEQ ID NO: 64), 125 ng (9.18 μl) ZG44328 (SEQ ID NO: 65) 6 μl dNTP, 6 μl Pfu Turbo polymerase (Stratagene) and 206.4 μl water. An aliquot of 47.5 μl of the cocktail is used in each reaction. The PCR conditions are as follows: 1 cycle at 95 ° C for 30 sec, and then 16 cycles at 95 ° C for 30 sec, 55 ° C for 1 min, 68 ° C for 7 min, then 1 cycle at 68 ° C for 7 min and ending with a burn at 4 ° C. All five reaction PCR mixtures are combined in one tube. In accordance with the manufacturer's recommendations, 5 μl of restriction enzyme DpnI was added to the PCR reaction mixture and incubated at 37 ° C for 2 hours. DNA is precipitated by the addition of a 10% 3 molar solution of sodium acetate and two volumes of 100% ethanol (Aaper Alcohol, Shelbyville, Kentucky). Precipitation is carried out at -20 ° C for 20 minutes DNA is separated by centrifugation at a speed of 14,000 rpm for 5 minutes and the precipitate is dried rapidly in vacuo. The DNA pellet was resuspended in 20 μl of water. PCR DNA was transformed into E. coli strain DH10B. 5 μl of DNA was mixed with 40 μl of ElectroMAX DH10B cells (Invitrogen, Carlsbad, CA). The mixture of cells and DNA is then electroporated in a cuvette with an electrode spacing of 0.1 cm (Bio-Rad, Hercules, California) using a Bio-Rad Gene Pulser II ™ and the following parameters: 1.75 kV, 100 Ohm and 25 uF. Electroporated cells grow at 37 ° C for 1 hour. The mixture is placed in a cup on LB medium + 25 μg / ml kanamycin and incubated overnight at 37 ° C. Ten clones were screened for the presence of an IL-29 insert. DNA was isolated from all ten clones using the QIAprep ™ Spin Miniprep kit (Qiagen) and assayed for insert by restriction enzyme digest with XbaI (Roche) and PstI (New England Biolabs). Nine clones that contain the insert are sequenced to confirm the introduction of the zcyto21 C172S mutation. The clone is checked by sequencing and designate it as pSDH171. A similar strategy can be used to generate the zcyto21 C15S mutant.

Example 11

Zcyto20 C49S expression genetic construct containing cysteine mutation

The sequence that encodes a zcyto20 C49S containing the cysteine mutation is generated by PCR using overlapping primers (SEQ ID NO: 20). The first 187 bases of the wild-type IL-28A sequence (SEQ ID NO: 1) are generated by PCR amplification using pYEL7 (SEQ ID NO: 67) as the template and oligonucleotide primers zc43431 (SEQ ID NO: 62) and zc45399 (SEQ ID NO: : 66). A second DNA fragment from base 105 to base 531 is generated by PCR amplification using pYEL7 (SEQ ID NO: 67) as template and oligonucleotide primers zc45398 (SEQ ID NO: 68) and zc43437 (SEQ ID NO: 63). Primers zc45399 (SEQ ID NO: 66) and zc45398 (SEQ ID NO: 68) contain a specifically modified sequence that replaces cysteine 49 with serine. The two PCR products are combined and the PCR overlap is amplified using oligonucleotide primers zc43431 (SEQ ID NO: 62) and zc43437 (SEQ ID NO: 63). The final PCR product is inserted into the pTAP238 expression vector by homologous recombination in a yeast system (Raymond et al., Biotechniques. Jan. 26 (1): 134-8, 140-1, 1999). The expression genetic construct is extracted from yeast and transformed into competent E. coli DH10B. Kanamycin-resistant clones were selected by colony PCR. The positive clone is confirmed by sequencing and then transformed into the producing host strain W3110 / pRARE. The expression genetic construct zcyto20 C49S containing the cysteine mutation is called pCHAN9.

Example 12

Zcyto20 expression genetic construct containing cysteine mutation C51S

The sequence that encodes zcyto20 containing the C51S cysteine mutation is generated by PCR using overlapping primers (SEQ ID NO: 24). The first 193 bases of the wild-type IL-28A sequence are generated by PCR amplification using pYEL7 (SEQ ID NO: 67) as template and oligonucleotide primers zc43431 (SEQ ID NO: 62) and zc45397 (SEQ ID NO: 63). A second DNA fragment from base 111 to base 531 is generated by PCR amplification using pYEL7 (SEQ ID NO: 67) as template and oligonucleotide primers zc45396 (SEQ ID NO: 70) and zc43437 (SEQ ID NO: 63). Primers zc45397 (SEQ ID NO: 69) and zc45396 (SEQ ID NO: 70) contain a specifically modified sequence that replaces cysteine 51 with serine. The two PCR products are combined and the PCR overlap is amplified using oligonucleotide primers zc43431 (SEQ ID NO: 62) and zc43437 (SEQ ID NO: 63). The final PCR product is inserted into the pTAP238 expression vector developed by the authors by homologous recombination in a yeast system (Raymond et al., Supra). The expression genetic construct is extracted from yeast and transformed into competent E. coli DH10B. Kanamycin-resistant clones were selected by colony PCR. The positive clone is confirmed by sequencing and then transformed into the producing host strain W3110 / pRARE. The zcyto20 C50S expression genetic construct containing a cysteine mutation is called pCHAN10.

Example 13

Expression of IL-28A, IL-29 and cysteine mutants of E. coli containing cysteine to serine substitutions

In separate experiments, E. coli transformed with each of the expression vectors described in Examples 6-9 is seeded in 100 ml of Superbroth II culture medium (Becton Dickinson, San Diego, CA) with 0.01% Antifoam 289 (Sigma Aldrich, St. Louis, Missouri), 30 μg / ml kanamycin, 35 μg / ml chloramphenicol and grown overnight at 37 ° C. 5 ml of seed is added to 500 ml of the same medium, placed in a 2 L growth flask, and shaken at 250 rpm at 37 ° C until the optical density of OD600 of the culture reaches 4. Then add IPTG to a final concentration of 1 mM and shaking continue for another 2.5 hours. Cells are centrifuged with an acceleration of 4000 × g for 10 min at 4 ° C. The cell pellet was frozen at -80 ° C and stored until further use.

Example 14

Folded restoration and purification of IL-28

A. Preparation of inclusion bodies

Wild-type human IL-29, as described above, is expressed in E. coli strain W3110 as inclusion bodies. The biomass of bacteria obtained by continuous fermentation with water, resuspended in a 50 mm solution of Tris with a pH of 7.3. The suspension is passed three times through an APV-Gaulin homogenizer (Invensys APV, Tonawanda, NY) at a pressure of 8,000 psi. Insoluble materials are separated by centrifugation with an acceleration of 15,000 g for 30 minutes. The tablet is washed successively with 50 mM Tris, 1% (v / v) Triton X100 with a pH of 7.3 and a 4 M urea solution. The inclusion bodies are then dispersed in 50 mM Tris, 6 M guanidine hydrochloride, 5 mM DTT at room temperature for 1 hour. After that, the sample is centrifuged with an acceleration of 15000 g for 1 hour. The supernatant obtained at this stage contains reduced soluble IL-29.

B. Restoration of the folded structure

Solubilized IL-29 is slowly diluted with stirring with a solution of 50 mM Tris, with a pH of 8, 0.75 M arginine, 0.05% PEG3350, 2 mM MgCl ₂ , 2 mm CaCl ₂ , 0.4 mm KCl, 10 mm NaCl, 4 mM reduced glutathione, 0.8 mM oxidized glutathione at room temperature. The final concentration of IL-29 in the folded structure restoring buffer mixture is 0.1 mg / ml. The mixture to restore the folded structure is left overnight at room temperature. Concentrated acetic acid is used to adjust the pH of the suspension to a value of 5. Then the suspension is filtered through a filter with a pore size of 0.2 μm. Analysis of the mixture to restore the folded structure by reverse phase HPLC shows two peaks.

C. Cleaning

The folded structure restoration mixture was diluted (1: 2) in the duct with 50 mM NaOAc at pH 5 and placed on a Pharmacia SP Sepharose Fast Flow cation exchange column (North Peapack, NJ). The column is washed with a solution of 50 mM NaOAc, 400 mM NaCl with a pH of 5 in an amount of 3 column volumes. Bound IL-29 was eluted with a solution of 50 mM NaOAc, 1.4 M NaCl with a pH of 5. Solid (NH ₄ ) ₂ SO _{4 was} added to the pooled pool of fractions obtained by elution in the step using a cation exchange column until the final concentration ( NH ₄ ) ₂ SO ₄ will not be 0.5 M. After this, the substance is placed on a ToyoPearl Phenyl 650S HIC column (Tosoh Biosep, Montgomeryville, PA). Then the column is washed with its three volumes with a solution of 50 mM NaOAc, 1 M (NH ₄ ) ₂ SO ₄ with a pH of 5. A linear gradient of 10 column volumes from 50 mM NaOAc, 1 M (NH ₄ ) ₂ SO ₄ with a pH of 5 to 50 mM NaOAc pH 5 is used to elute the bound zcyto21. The fractions obtained by elution are combined. At this stage, two peaks are observed. The eluted fractions are analyzed by reverse phase HPLC. After the final replacement of the buffer solution with PBS, two products are obtained corresponding to two isomers with respect to the disulfide bond.

Example 15

Folded structure restoration and purification of mutant IL-29 containing cysteine substitutions

As indicated in Example 3, purification of the obtained IL-29 gives two isomers relative to the disulfide bond. To separate these two forms, the HIC FPLC step is carried out. Separation of forms cannot be fully resolved relative to the baseline. In order to obtain substantially purified isomers (> 95%), significant “peak trimming” is necessary. The output at this stage is small and reduces the through yield of the entire process. Final yields are 8% and 9% for forms C15-C112 and C12-C171, respectively. Wild-type IL-29 derived from CHO and baculovirus (BV) systems has similar properties. It was found that the C15-C112 isomer is homologous in structure of the disulfide bond to type I interferons. In addition, when conducting an ISRE analysis (see below), it was found that the C15-C112 form shows 30 times greater biological activity than the C112- S171.

Folded structure restoration and purification of mutein zcyto21 Cys172Ser

Obtaining inclusion bodies, restoring the folded structure and purification of the zcyto21 C172S polypeptide (SEQ ID NO: 29) is carried out similarly to that indicated for wild-type IL-29 (SEQ ID NO: 4). Reverse-phase HPLC of the mixture to restore the folded structure of the mutein shows only one noticeable peak corresponding to wild-type C15-C112 IL-29. Subsequent hydrophobic interaction chromatography shows only one peak. Therefore, there is no need to carry out significant “peak trimming”. The total yield for the whole method is close to 50%. In the ISRE assay of Example 16, the zcyto21 Cys172Ser polypeptide (SEQ ID NO: 29) shows a biological activity equivalent to wild-type C15-C112 IL-29.

Example 16

Antiviral activity: effect in Hela and L929 cells

The primary functional analysis of antiviral activity is carried out using a conditioned medium from temporarily transfected cells of human embryonic kidney (HEK). Full length cDNA for human or mouse IL-28A, IL-28B or IL-29 is cloned into pzp7Z vector by standard methods. Gene constructs of human or murine IL-28A, IL-28B, or IL-29 are transfected into 293 HEK cells. Briefly, for each construct, 700,000 cells per well (on a 6-well plate) were placed 18 hours before transfection on a plate with 2 ml of DMEM + 10% fetal calf serum. For each well, 1.5 μg of human or mouse IL-28A, IL-28B, or IL-29 DNA and 0.5 μg of pIRES2-EGFP DNA (Clontech) are added to 6 μl of Fugene 6 reagent (Roche Biochemicals) in a total volume of 100 μl DMEM. 2 μg of individual pIRES2-EGFP DNA is used as a negative control. Said transfection mixtures after 30 minutes were added to 293 cells previously preplaced onto the plate. After 24 hours, the cell medium was removed and DMEM + 0.1% bovine serum albumin was added. The conditioned medium is collected after 48 hours, filtered through a filter with a pore size of 0.45 μm and used in antiviral and reporter analyzes.

Antiviral assays are performed using human cervical carcinoma cells (HeLa) and mouse fibroblast cells (L929). On the first day, the conditioned medium containing human or mouse IL-28A, IL-28B, or IL-29 is diluted and placed together with 50,000 cells on a 96-well microtiter flat bottom plate. After 24 hours of incubation at 37 ° C, the culture medium is removed and replaced with a medium containing encephalomyocarditis virus with an infection multiplicity index of 0.1. Cells are re-incubated for 24 hours at 37 ° C. The culture wells are then visually evaluated on a 4-point scale for the presence of a cytopathic effect (CPE), which is then expressed as% CPE, as shown in Table 7. As controls, include conditioned medium from cells transfected only with GFP and purified human interferon-a-2a or mouse interferon-alpha.

Table 7
Determination of cytopathic effect Designation Observed Cytopathic Effect (CPE) - CPE is missing +/- Possible CPE (approximately 1% of the monomolecular layer) + CPE is limited to one plaque (approximately 5% of the surface) +1 CPE is limited to three plaques affecting less than 25% of the monolayer one 25% CPE 1-2 37% CPE 2 50% CPE 2-3 62% CPE 3 75% CPE 3-4 87% CPE four 100% CPE

Table 8 shows that the conditioned medium containing human or mouse IL-28A, IL-28B or IL-29, depending on the dose, inhibits viral infection (% CPE) in HeLa cells, while the control conditioned GFP medium is not capable to a significant extent block the manifestation of the cytopathic effect. As shown in Table 9, conditioned medium containing human or murine IL-28A, IL-28B, or IL-29 does not inhibit viral infection in L929 cells. In both experiments, purified interferon showed positive antiviral activity.

Table 8
The percentage of the cytopathic effect of human or mouse IL-28A, IL-28B or IL-29 in HeLa cells using conditioned medium (CM, CS) Relative concentration of CS GFP control zcyto20 IL-28A (COP) zcyto21 IL-29 (COP) zcyto22 IL-28B (COP) zcyto24 mouse (cop) zcyto25 mouse (cop) hIFN-a-2a HIFN-a-2a Concentration Not added 87 87 87 87 87 87 87 0 ng / ml 0.008X 87 10 56 0 0 10 fifteen 0.0001 ng / ml 0,0156X 87 2,5 31 0 0 5 8.3 0.001 ng / ml 0,0325X 87 5 10 0 0 5 1.7 0.01 ng / ml 0.0625X 87 2,5 10 0 0 0 0 0.1 ng / ml 0,125X 87 0 5 0 0 0 0 1 ng / ml 0.25X 87 0 0 0 0 0 0 10 ng / ml 0.5X 87 0 0 0 0 0 0 100 ng / ml

Table 9
The percentage of cytopathic effect of human or mouse IL-28A, IL-28B or IL-29 in L929 cells using conditioned medium (KS) Relative concentration of CS GFP control zcyto20 IL-28A (COP) zcyto21 IL-29 (COP) zcyto22 IL-28B (COP) zcyto24 mouse (cop) zcyto25 mouse (cop) hIFN-a-2a HIFN-a-2a Concentration Not added 87 87 87 87 87 87 87 0 ng / ml 0.008X 87 87 87 87 87 87 87 0.0001 ng / ml 0,0156X 87 87 87 87 87 87 87 0.001 ng / ml 0,0325X 87 87 87 87 87 87 87 0.01 ng / ml 0.0625X 87 87 87 87 87 87 58 0.1 ng / ml 0,125X 87 87 87 87 87 87 6.7 1 ng / ml 0.25X 87 87 87 87 87 87 0 10 ng / ml 0.5X 87 87 87 87 87 87 0 100 ng / ml

Example 17

Signal transmission via interferon-induced cascade of responses

The interaction of type 1 interferons with their specific receptor leads to the induction of a number of genes responsible for their antiviral / antiproliferative activity. These include 2′-5′-oligoadenylate synthase (2-5 OAS), double-stranded DNA Pkr kinase (Pkr), phospholipid scramblase, and intercellular adhesion-1 molecule (ICAM-1). Also, induction of genes with still unknown functions is observed, such as the 56 kDa gene product activated by interferon (ISG-56k). In order to determine whether some or all of these genes are induced by treating cells with IL-28A, Daudi B human lymphoid cells are treated for 72 hours with conditioned medium from Sf9 cells infected with baculovirus expressing IL-28A. As a negative control, an air-conditioned medium of Sf9 cells infected with wild-type baculovirus is used. After treatment, cells are harvested and leased to isolate complete RNA. One microgram of total RNA is converted to cDNA using reverse transcriptase and used as a template in PCR, together with examples specific to the human interferon-activated genes given above. Oligonucleotide primers for human glycerol-3-phosphate dehydrogenase (G3PDH) are used as non-interferon-activated control genes. The results show a clear induction of ISG-56k, Pkr, 2-5 OAS and phospholipid stramblase after treatment of cells with IL-28A. No induction was observed for ICAM-1 or non-interferon-activated gene control, G3PDH.

Example 18

Reporter signal transduction analysis

Reporter signal transduction analysis can be used to establish the functional interaction of human or mouse IL-28 or IL-29 with the IL-28 receptor. Human embryonic kidney cells (HEK) are transfected with a reporter plasmid containing an interferon-induced response element (ISRE) that transcribes the luciferase reporter gene in the presence or absence of pZP7 expression vectors containing cDNA for class II cytokine receptors (including DIRS1, IFNαR1, IFNαR2 and IL-28 receptor). Luciferase activity after activation of transfected cells with respect to class II ligands (including IL-28A (SEQ ID NO: 2), IL-29 (SEQ ID NO: 4), IL-28B (SEQ ID NO: 6), zcyto10, huIL10 and huIFNa-2a) reflects the interaction of the ligand with transfected and native cytokine receptors on the cell surface. The results and methods are given below.

Cell transfection

HEK 293 cells are transfected as follows: 700,000 293 cells per well (on a 6-well plate) are placed 18 hours before transfection on a plate with 2 ml of DMEM + 10% fetal calf serum. For each well, 1 μg of pISRE luciferase DNA (Stratagene), 1 μg of cytokine receptor DNA and 1 μg of pIRES2-EGFP DNA (Clontech) are added to 9 μl of Fugene 6 reagent (Roche Biochemicals) in a total volume of 100 μl of DMEM. In the event that cytokine receptor DNA is not used, then 2 μg of pIRES2-EGFP DNA is added. Said transfection mixture was added after 30 minutes to 293 cells previously plated on the plate. After 24 hours, the transfected cells were removed from the plate using a trypsin-EDTA mixture and reapplied in an amount of approximately 25,000 cells per well in a 96 well microtiter plate. Approximately 18 hours before ligand activation, the medium is changed to DMEM + 0.5% fetal calf serum (FBS).

Signal Transduction Analysis

Reporter signal transduction assays are performed as follows: after incubation for 18 hours at 37 ° C in DMEM + 0.5% FBS growth medium, transfected cells are activated by dilutions (in DMEM + 0.5% FBS growth medium) of the following class II ligands; IL-28A, IL-29, IL-28B, zcyto10, huIL10 and huIFNa-2a. After 4 hours of incubation at 37 ° C, the cells are lysed and relative light units (RLU) are measured using a luminometer after adding the luciferase substrate. The results are presented as the multiplicity of RLU induction in experimental samples with respect to the control medium only (RLU of experimental samples / RLU of the control medium = induction ratio). Table 10 shows that IL-28A, IL-29, and IL-28B activate ISRE signal transduction in 293 cells transfected with ISRE-luciferase and give a 15- to 17-fold increase in luciferase activity compared to culture medium alone. The introduction of the alpha-subunit DNA of the IL-28 receptor (SEQ ID NO: 11) into the mixture for transfection with endogenous CRF2-4 (SEQ ID NO: 71) leads to a further 6- to 8-fold increase in ISRE signal transduction by IL -28A, IL-29 and IL-28B, which gives a total activation in the range from 104 to 125 times. None of the other transfected class II cytokine receptor DNAs enhance ISRE signaling. The results indicate that IL-28A, IL-29 and IL-28B functionally interact with the cytokine receptor IL-28. Table 10 also shows that huIFNa-2a is capable of activating signal transduction in 293-transfected ISRE-luciferase cells and leads to a 205-fold increase in luciferase activity compared to culture medium alone. Nevertheless, the addition of IL-28 receptor DNA to transfections leads to an 11-fold decrease in ISRE signal transmission (compared to the action of ISRE luciferase DNA alone), which suggests that overproduction of IL-28 receptor has a negative effect on interferon signal transmission, in contrast to the positive effects that overproduction of IL-28 receptor has on signal transmission of IL-28A, IL-29 and IL-28B.

Table 10
Interferon-stimulated response element (ISRE) of 293 transfected cell signal after activation by class II cytokine (activation ratio) Ligand ISRE luciferase ISRE-luciferase / IL-28R IL-28A (125 ng / ml) fifteen 125 IL-29 (125 ng / ml) 17 108

IL-28B (125 ng / ml) 17 104 huIFNa-2a (100 ng / ml) 205 eighteen Zcyto10 (125 ng / ml) 1.3 one HuIL10 (100 ng / ml) one 0.5

Example 19

Signal Transduction Assays for Cysteine Mutant IL-29

Cell transfection

To obtain 293 HEK cells in which stable overproduction of the IL-28 receptor is possible, 293 cells are transfected as follows: 300,000 293 cells per well (on a 6-well plate) are placed 6 hours before transfection on a plate with 2 ml DMEM + 10% fetal calf serum. For each well, 2 μg of the pZP7 expression vector containing the human IL-28 receptor alpha subunit DNA (SEQ ID NO: 11) is added to 6 μl of Fugene 6 reagent (Roche Biochemicals) in a total volume of 100 μl of DMEM. The specified transfection mixture after 30 minutes is added to 293 cells previously pre-plated on the plate. After 48 hours, the transfected cells are selected using 2 μg / ml puromycin. Puromycin-resistant cells are obtained as a population of cells.

HEK 293 cells excessively producing human IL-28 receptor are transfected as follows: 700,000 293 cells per well (on a 6-well plate) are placed 18 hours before transfection on a plate with 2 ml DMEM + 10% fetal calf serum. For each well, 1 μg KZ157 containing an interferon-induced response element (ISRE), which causes transcription of the luciferase reporter gene, is added to 3 μl of Fugene 6 reagent (Roche Biochemicals) in a total volume of 100 μl of DMEM. The specified mixture for transfection after 30 minutes is added to 293 HEK cells previously applied to the plate. After 48 hours, the transfected cells are removed from the plate using trypsin-EDTA and reapplied with a solution of 500 μg / ml G418 (Geneticin, Life Technologies). Puromycin and G418 resistant cells are isolated as a cell population.

Signal Transduction Analysis

Reporter signal transduction assays are performed as follows: 293 HEK cells excessively producing the human IL-28 receptor and containing KZ157 are treated with trypsin-EDTA and reapplied in an amount of approximately 25,000 cells per well on 96-well plates. After approximately 18 hours of activation, the medium is changed to DMED + 0.5% FBS.

After 18 hours of incubation at 37 ° C in DMED + 0.5% FBS, the transfected cells are activated by dilutions (in DMED + 0.5% FBS) of various forms of zcyto21 derived from E. coli, which includes various types of cysteine binding. After 4 hours of incubation at 37 ° C, the cells are lysed and relative light units (RLU) are measured using a luminometer after adding the luciferase substrate. The results are presented as the multiplicity of RLU induction in experimental samples with respect to the control medium only (RLU of experimental samples / RLU of the control medium = induction ratio).

Table 11 shows that the C1-C3 (C16-C113) form of IL-29 derived from wild-type E. coli is more capable of inducing ISRE signaling than the wild-type C3-C5 (C113-C172) or mixture of Form C1 -C3 wild-type and forms C3-C5 (C16-C113, C113-C172), which all refer to SEQ ID NO: 15.

Table 12 shows that the C1-C3 (C16-C113) form of IL-29 derived from wild-type E. coli and C1-C3 (C16-C113; SEQ ID NO: 15) containing the cysteine mutation (C172S) IL- 29 (SEQ ID NO: 29), obtained from E. coli, are equally capable of inducing ISRE signaling in 293 HEK cells over-producing human IL-28 receptor.

Table 11
ISRE signaling by various forms of IL-29 derived from E. coli (activation ratio) Cytokine Concentration (ng / ml) Form C1-C3 (C16-C113) Form C3-C5 (C113-C172) Mixture C1-C3 and C3-C5 one hundred 36 29th 34 10 38 25 35 one 32 12 24 0.1 10 2 5 0.01 3 one one 0.001 one one one

Table 12
ISRE signaling by various forms of IL-29 derived from E. coli (activation ratio) Cytokine Concentration (ng / ml) Wild-type C1-C3 C1-C3 cysteine mutation C172S 1000 9.9 8.9 one hundred 9.3 8.7 10 9.3 8.1 one 7.8 7 0.1 4.6 3.3 0.01 1.9 1,5 0.001 1.3 0.9

Example 20

Activation of IL-28A, IL-29 and IL-28B under the influence of poly I: C and viral infection

Freshly isolated human peripheral blood mononuclear cells are grown in the presence of polyinosinic acid - polycytidylic acid (poly I: C; 100 μg / ml) (Sigma, St. Louis, Missouri), encephalomyocarditis virus (EMCV) with an MOI of 0.1, or only in a nutrient medium. After 15 hours of incubation, complete RNA is isolated from the cells and treated with RNase-free DNase. 100 ng of total RNA is used as template in a one-step RT-PCR method along with Superscript One-Step RT-PCR, a Platinum Taq reagent kit and gene-specific primers according to the manufacturer's recommendations (Invitrogen).

In untreated cells, the amounts of IL-28A, IL-28B and IL-29, IFN-α and IFN-β RNA are low, up to values below the detection limit. In contrast, the amounts of IL-28A, IL-29, and IL-28B RNA increase both during treatment with poly I: C and under the influence of viral infection, which is also observed for type I interferons. These experiments show that IL-28A, IL-28B and IL-29, like type I interferons, can be activated by double-stranded RNA or viral infection.

Example 21

Signal transmission activity for IL-28, IL-29 compared with IFNα in HepG2 cells

A. Transfection of cells

HepG2 cells are transfected as follows: 700,000 HepG2 cells per well (on a 6-well plate) are placed 18 hours before transfection on a plate with 2 ml DMEM + 10% fetal calf serum. For each well, 1 μg of pISRE luciferase DNA (Stratagene) and 1 μg of pIRES2-EGFP DNA (Clontech) were added to 6 μl of Fugene 6 reagent (Roche Biochemicals) in a total volume of 100 μl of DMEM. The specified transfection mixture after 30 minutes is added to the pre-coated HepG2 cells. After 24 hours, the transfected cells were removed from the plate using a trypsin-EDTA mixture and reapplied in the amount of approximately 25,000 cells per well in 96 well microtiter plates. About 18 hours before ligand activation, the medium is changed to DMEM + 0.5% FBS.

B. Reporter analysis of signal transduction

Reporter analysis of signal transduction is carried out as follows: after incubation for 18 hours at 37 ° C in DMEM + 0.5% FBS growth medium, transfected cells activate 100 ng / ml of ligands IL-28A, IL-29, IL-28B, zcyto24, zcyto25 and huIFN-α2a. After 4 hours of incubation at 37 ° C, the cells are lysed and relative light units (RLU) are measured using a luminometer after adding the luciferase substrate. The results are presented in the form of the multiplicity of RLU activation in experimental samples with respect to the control medium only (RLU of experimental samples / RLU of the control medium = induction ratio). Table 13 shows that IL-28A, IL-29, IL-28B, zcyto24 and zcyto25 activate ISRE signaling in human liver HepG2 cells transfected with ISRE-luciferase.

Table 13
Cytokine-dependent ISRE signal activation multiplicity Cytokine Activation rate IL-28A 5,6 IL-29 four IL-28B 5.8 zcyto24 4.7 zcyto25 3 Huifn-a2a 5.8

Example 22

Antiviral activity of IL-29 compared with IFNα in HepG2 cells

Antiviral assays are adapted for EMCV (American Type Culture Collection # VR-129B, Manassas, Virginia) with human cells (Familletti, P. et al., Methods Enzym. 78: 387-394, 1981). Cells, together with a cytokine, are placed in a dish and incubated for 24 hours before an EMCV challenge test is introduced with a multiplicity of infection ranging from 0.1 to 1. Cells are analyzed for viability using bioassays to capture dyes 24 hours after infection ( Berg, K. et al., Apmis 98: 156-162, 1990). Target cells are treated with MTT and incubated at 37 ° C for 2 hours. The solubilizer solution is added, incubated overnight at 37 ° C and the absorbance is determined at 570 nm. The OD570 value is directly proportional to antiviral activity.

The results show antiviral activity when IL-29 and IFN are tested together with HepG2 cells: IL-29, IFN-β, and IFNα-2a are added at various concentrations to HepG2 cells before EMCV infection and dye capture analysis. The average value and standard deviation of the OD570 value in three cells are plotted. OD570 values are proportional to antiviral activity. For IL-29, the EC50 is 0.60 ng / ml; for IFNα-2a, the EC50 is 0.57 ng / ml; and for IFN-β, the EC50 is 0.46 ng / ml.

Example 23

Expression of IL-28RA mRNA in the liver and lymphocyte subpopulation

In order to further study the mRNA distribution for IL-28RA, semi-quantitative RT-PCR was performed using the SDS 7900HT system (Applied Biosystems, California). One-step RT-PCR was performed using 100 ng of total RNA for each sample and gene-specific primers. For each set of primers, a standard curve is obtained using Bjab RNA and the values obtained for all samples are normalized to HPRT. Normalized data are collected in Tables 14-17. Normalized data for IFNAR2 and CRF2-4 are also given.

Table 14: B and T cells express significant amounts of IL-28RA mRNA. Low values are observed in dendritic cells and in most monocytes.

Table 14 Cage / fabric IL-28RA IFNAR2 CRF2-4 Dendritic cells inactive 0.04 5.9 9.8 Dendritic cells + IFNg 0,07 3.6 4.3 Dendritic cells 0.16 7.85 3.9 CD14 + activated using LPS / IFNg 0.13 12 27 CD14 + monocytes at rest 0.12 eleven 15.4 Human CD14 + Inactive 4.2 TBD TBD Human CD14 + Activated with 1 μg / ml LPS 2,3 TBD TBD Sore Tonsil 3 12,4 9.5 Human B cells + PMA / Iono 4 & 24 hours 3.6 1.3 1.4 Human CD19 + at rest 6.2 TBD TBD

Human CD19 + 4 hour activation using PMA / Iono 10.6 TBD TBD Human CD19 + 24 hour activation by PMA / Iono 3,7 TBD TBD IgD + B cells 6.47 13.15 6.42 IgM + B cells 9.06 15.4 2.18 IgD-B cells 5.66 2.86 6.76 Killer cells + PMA / Iono 0 6.7 2.9 Human CD3 + Inactive 2.1 TBD TBD CD4 + at rest 0.9 8.5 29.1 CD4 +, inactive 18 hours 1,6 8.4 13,2 CD4 ++ Poly I / C 2.2 4,5 5.1 CD4 + + PMA / Iono 0.3 1.8 0.9 CD3 neg., At rest 1,6 7.3 46 CD3 neg., Inactive 18 hours 2,4 13,2 16.8 CD3 neg., + Poly I / C 18 hours 5.7 7 30,2 CD3 neg., + LPS 18 hours 3,1 11.9 28,2 CD8 +, inactive 18 hours 1.8 4.9 13.1 PMA / Iono Activated CD8 + 18 Hours 0.3 0.6 1,1

As can be seen from Table 15, normal liver tissue and liver derived cell lines show significant levels of IL-28RA and CRF2-4 mRNA.

Table 15 Cage / fabric IL-28RA IFNAR2 CRF2-4 Hepg2 1,6 3.56 2.1 HepG2 UGAR 5/10/02 1,1 1,2 2.7 HepG2, CGAT HKES081501C 4.3 2.1 6 HuH7 5/10/02 1,63 16 2 HuH7 Hepatoma - CGAT 4.2 7.2 3,1 Normal liver - CGAT # HXYZ020801K 11.7 3.2 8.4 Liver, NAT - Normal Adjacent Tissues 4,5 4.9 7.7 Liver, NAT - Normal Adjacent Tissues 2.2 6.3 10,4 Hep SMVC, hepatic vein 0 1.4 6.5 Hep SMCA, hepatic artery 0 2.1 7.5 Liver fibroblasts 0 2.9 6.2 Liver carcinoma 3.8 2.9 5.8 Liver adenocarcinoma 8.3 4.2 10.5 SK-Hep-1, liver adenocarcinoma 0.1 1.3 2,5 AsPC-1, human pancreatic adenocarcinoma 0.7 0.8 1.3 Star-shaped cells of the human liver 0,025 4.4 9.7

As can be seen from Table 16, the main airway epithelial cells contain elevated levels of IL-28RA and CRF2-4.

Table 16 Cage / fabric IL-28RA IFNAR2 CRF2-4 U87MG - Glioma 0 0.66 0.99 NHBE Inactive 1.9 1.7 8.8 NHBE + TNF-alpha 2.2 5.7 4.6 NHBE + poly I / C 1.8 There is no data There is no data Small airway epithelial cells 3.9 3.3 27.8 NHLF - Normal Human Pulmonary Fibroblasts 0 There is no data There is no data

As can be seen from Table 17, IL-28RA is present in normal and diseased liver samples, with increased expression in samples infected with hepatitis C and hepatitis B.

Table 17 Cage / fabric IL-28RA CRF2-4 IFNAR2 Liver with dry necrosis 8.87 15.12 1.72 Autoimmune Hepatitis Liver 6.46 8.90 3.07 Congenital giant cell hepatitis 6.29 12.46 6.16 The terminal stage of liver disease 4.79 17.05 10.58 Sudden liver failure 1.90 14.20 7.69 Sudden liver failure 2,52 11.25 8.84 Primary Biliary Cirrhosis 4.64 12.03 3.62 Alcoholic cirrhosis of the liver (Laenneca) 4.17 8.30 4.14 Cryptogenic cirrhosis 4.84 7.13 5.06 Hepatitis C +, accompanied by necrosis 3.64 7.99 6.62 Hepatitis C + 6.32 11.29 7.43 Sudden hepatitis secondary to hepatitis A 8.94 21.63 8.48 Hepatitis C + 7.69 15.88 8.05 Hepatitis B + 1,61 12.79 6.93 Normal liver 8.76 5.42 3.78 Normal liver 1.46 4.13 4.83 Liver, NAT - Normal Adjacent Tissues 3.61 5.43 6.42 Liver, NAT 1.97 10.37 6.31 Human embryo liver 1,07 4.87 3.98 Hepatocellular carcinoma 3,58 3.80 3.22 Liver adenocarcinoma 8.30 10.48 4.17 Hep SMVC, hepatic vein 0.00 6.46 1.45 Hep SMCA, hepatic artery 0.00 7.55 2.10 Liver fibroblasts 0.00 6.20 2.94 Huh7 hepatoma 4.20 3.05 7.24 HepG2 hepatocellular carcinoma 3.40 5.98 2.11 SK-Hep-1, liver adenocarcinoma 0,03 2,53 1.30 HepG2 Inactive 2.06 2.98 2.28

HepG2 + zcyto21 2.28 3.01 2,53 HepG2 + IFNα 2.61 3.05 3.00 Normal female liver - split 1.38 6.45 4,57 Normal liver - split 1.93 4.99 6.25 Normal liver - split 2.41 2,32 2.75 Sick liver - split 2,33 3.00 6.04 Clonetics Primary Hepatocytes 9.13 7.97 13.30

As shown in Tables 18-22, IL-28RA is found in normal B cells, B cells of the lymphoma cell line, T cells, T cells of the lymphoma cell line (Jurkat cells), normal and transformed lymphocytes (B cells and T -cells) and normal human monocytes.

Table 18 HPRT
average IL-28RA
average IL-28RA
normal. IFNAR2 IFNR2
normal. CRF2-4 CRF2-4
normal. CD14 +, 24 hours, inactive # A38 13.1 68.9 5.2 92.3 7.0 199.8 15,2 CD14 +, 24 hours, activated # A38 6.9 7.6 1,1 219.5 31.8 276.6 40.1 CD14 +, 24 hour unactivated # A112 17.5 40.6 2,3 163.8 9,4 239.7 13.7 CD14 +, 24 hour activated # A112 11.8 6.4 0.5 264.6 22.4 266.9 22.6 CD14 +, at rest #X 32,0 164.2 5.1 1279.7 39.9 699.9 21.8 CD14 ++ LPS #X 21,4 40.8 1.9 338.2 15.8 518.0 24.2 CD14 +, 24 hours, inactive # A39 26.3 86.8 3.3 297.4 11.3 480.6 18.3 CD14 + 24 hour activated # A39 16.6 12.5 0.8 210.0 12.7 406.4 24.5 HL60, at rest 161.2 0.2 0,0 214.2 1.3 264.0 1,6 HL60 + PMA 23.6 2,8 0.1 372.5 15.8 397.5 16.8 U937, at rest 246.7 0,0 0,0 449.4 1.8 362.5 1,5 U937 + PMA 222.7 0,0 0,0 379.2 1.7 475.9 2.1 Jurkat, at rest 241.7 103.0 0.4 327.7 1.4 36.1 0.1 Jurkat activated 130.7 143.2 1,1 Colo205 88.8 43.5 0.5 HT-29 26.5 30.5 1,2

Table 19 HPRT
stand. reject IL-28RA
stand. reject Monocytes, 24 hours, inactive # A38 0.6 2,4 Monocytes, 24 hours, activated # A38 0.7 0.2 Monocytes, 24 hours, inactive # A112 2.0 0.7 Monocytes, 24 hours, activated # A112 0.3 0.1 Monocytes at rest #X 5.7 2.2 Monocytes + LPS #X 0.5 1,0 Monocytes, 24 hours, inactive # A39 0.7 0.8 Monocytes, 24 hours, activated # A39 0.1 0.7 HL60, at rest 19.7 0.1 HL60 + PMA 0.7 0.4 U937 at rest 7.4 0,0 U937 + PMA 7.1 0,0 Jurkat, at rest 3,7 1,1 Jurkat activated 2,4 1.8 Colo205 1.9 0.7 HT-29 2,3 1.7 Table 20 Avg
Hprt Avg
IFNAR2 Avg
IL-28RA Avg
CRF CD3 + / CD4 + 0 10.1 85.9 9.0 294.6 CD3 + / CD4 +, inactive, 18 hours 12.9 108.7 20.3 170.4 CD3 + / CD4 +, + Poly I / C, 18 hours 24.1 108,5 52.1 121.8 CD3 + / CD4 +, + PMA / Iono, 18 hours 47.8 83.7 16.5 40.8 CD3 neg., 0 15.4 111.7 24.8 706.1 CD3 neg., Inactive, 18 hours 15.7 206.6 37.5 263.0 CD3 neg., + Poly I / C, 18 hours 9.6 67.0 54.7 289.5 CD3 neg., + LPS, 18 hours 14.5 173.2 44.6 409.3 CD8 +, inactive, 18 hours 6.1 29.7 11.1 79.9 CD8 +, + PMA / Iono, 18 hours 78,4 47.6 26.1 85.5 12.8.1 - NHBE inactive 47.4 81.1 76.5 415.6 12.8.1 - NHBE + TNF-alpha 42.3 238.8 127.7 193.9 Saec 15.3 49.9 63.6 426.0

Table 21 IL-28RA
normal. CRF
normal. IFNAR2
normal. IL-28RA
stand. off CRF
stand. off IFNAR2
stand. off CD3 + / CD4 + 0 0.9 29.1 8.5 0.1 1,6 0.4 CD3 + / CD4 +, inactive, 18 hours 1,6 13,2 8.4 0.2 1,6 1.4 CD3 + / CD4 +, + Poly I / C, 18 hours 2.2 5.1 4,5 0.1 0.3 0.5 CD3 + / CD4 +, + PMA / Iono, 18 hours 0.3 0.9 1.8 0,0 0.1 0.3 CD3 neg., 0 1,6 46.0 7.3 0.2 4.7 1.3 CD3 neg., Inactive, 18 hours 2,4 16.8 13,2 0.4 2.7 2,3 CD3 neg., + Poly I / C, 18 hours 5.7 30,2 7.0 0.3 1.7 0.8 CD3 neg., + LPS, 18 hours 3,1 28,2 11.9 0.4 5,4 2.9 CD8 +, inactive, 18 hours 1.8 13.1 4.9 0.1 1,1 0.3 CD8 +, + PMA / Iono, 18 hours 0.3 1,1 0.6 0,0 0.1 0,0 12.8.1 - NHBE inactive 1,6 8.8 1.7 0.1 0.4 0.1 12.8.1 - NHBE + TNF-alpha 3.0 4.6 5.7 0.1 0.1 0.1 Saec 4.1 27.8 3.3 0.2 1,1 0.3 Table 22 Hprt
stand. off IFNAR2
stand. off IL-28RA
stand. off CRF
stand. off CD3 + / CD4 + 0 0.3 3,5 0.6 12.8 CD3 + / CD4 +, inactive 18 hours 1.4 13.7 1,1 8.5 CD3 + / CD4 + + Poly I / C, 18 hours 1.3 9.8 1,6 3.4 CD3 + / CD4 + + PMA / Iono, 18 hours 4.0 10.3 0.7 3,7 CD3 neg., 0 1.4 16.6 1,6 28.6 CD3 neg., Inactive 18 hours 2,4 16,2 2.7 12.6 CD3 neg., + Poly I / C, 18 hours 0.5 7.0 1,0 8.3 CD3 neg., + LPS 18 hours 1,0 39.8 5,6 73.6 CD8 +, inactive 18 hours 0.2 1,6 0.5 6.1 CD8 ++ PMA / Iono 18 hours 1.3 1.7 0.2 8.1 12.8.1 - NHBE inactive 2,4 5,6 2.7 2,8

12.8.1 - NHBE + TNF-alpha 0.5 3.4 3,5 3.4 Saec 0.5 4.8 1.8 9.9

Example 24

Murine IL-28 Does Not Affect Daudi Cell Proliferation

Human Daudi cells are suspended in RPMI + 10% FBS culture medium at 50,000 cells / ml and 5,000 cells per well are placed on a 96-well plate. IL-29-CEE (labeled glu conjugated IL-29) was added to each well, IFN-γ or IFN-α2a was added in 2-fold dilution series. IL-29-CEE is used in a concentration range from 1000 ng / ml to 0.5 ng / ml. IFN-γ is used in a concentration range from 125 ng / ml to 0.06 ng / ml. IFN-α2a is used in a concentration range from 62 ng / ml to 0.03 ng / ml. Cells are incubated for 72 hours at a temperature of 37 ° C. After 72 hours, Alamar Blue (Accumed, Chicago, Illinois) was added in an amount of 20 μl per well. The plates are further incubated for 24 hours at 37 ° C in an atmosphere of 5% CO. The tablets are read on an Fmax ™ plate reader (Molecular Devices, Sunnyvale, CA) using the SoftMax ™ Pro program at a wavelength of 544 (excitation) and 590 (radiation). Alamar Blue gives a fluorometric response in accordance with the metabolic activity of cells and, thus, allows a direct measurement of cell proliferation in comparison with the negative control. The results show that IL-29-CEE, unlike IFN-α2a, does not have a significant effect on the proliferation of Daudi cells.

Example 25

Murine IL-28 has no antiproliferative effect on murine B cells

Mouse B cells were isolated from the spleen of 2 Balb / C mice (7 months old) by elimination of CD43 + cells using MACS magnetic beads. Purified B cells are grown in vitro in the presence of LPS, monoclonal antibodies against IgM or against CD40. Murine IL-28 or murine IFNα was added to the cultures, after ³ hours ³ H-thymidine was added and the incorporation of ³ H-thymidine into the cells was measured after 72 hours.

IFNα at a concentration of 10 ng / ml inhibits the incorporation of ³ N-thymidine by mouse B cells activated either by LPS or by an anti-IgM antibody. However, murine IL-28 at any of the tested concentrations, up to 1000 ng / ml, does not inhibit the incorporation of ³ H-thymidine. In contrast, both mIFNα and murine IL-28 increase ³ H-thymidine incorporation by murine B cells activated by anti-CD40 monoclonal antibody.

The data obtained show that murine IL-28, unlike IFNα, does not exhibit antiproliferative activity even at high concentrations. In addition, zcyto24 enhances proliferation in the presence of anti-CD40 monoclonal antibodies. The results show that murine IL-28 differs from IFNα in that murine IL-28 does not exhibit antiproliferative activity against B cells even at high concentrations. In addition, murine IL-28 enhances proliferation in the presence of monoclonal antibodies against CD40.

Example 26

Bone marrow propagation

Healthy bone marrow mononuclear cells (Poietic Technologies, Göttersberg, Maryland) are allowed to adhere to the polymer substrate in αMEM, 10% FBS, 50 mM β-mercaptoethanol, 2 ng / ml FLT3L at 37 ° C for 2 hours. Non-adherent cells are then transferred onto plates (96-well tissue culture plates) in an amount of 25,000 to 45,000 per well in αMEM, 10% FBS, 50 mM β-mercaptoethanol, 2 ng / ml FLT3L in the presence or absence of 1000 ng / ml IL-29-CEE, 100 ng / ml IL-29-CEE, 10 ng / ml IL-29-CEE, 100 ng / ml IFN-α2a, 10 ng / ml IFN-α2a or 1 ng / ml IFN -α2a. These cells are incubated with various cytokines in order to study the reproduction or differentiation of hematopoietic bone marrow cells (20 ng / ml IL-2, 2 ng / ml IL-3, 20 ng / ml IL-4, 20 ng / ml IL-5, 20 ng / ml IL-7, 20 ng / ml IL-10, 20 ng / ml IL-12, 20 ng / ml IL-15, 10 ng / ml IL-21 or without cytokine addition). After 8 to 12 days, Alamar Blue (Accumed, Chicago, Illinois) is added in an amount of 20 μl per well. The plates are further incubated for 24 hours at 37 ° C in an atmosphere of 5% CO. The tablets are read on an Fmax ™ plate reader (Molecular Devices, Sunnyvale, Calif.) Using the SoftMax ™ Pro program at a wavelength of 544 (excitation) and 590 (emission). Alamar Blue gives a fluorometric response in accordance with the metabolic activity of cells and, thus, allows a direct measurement of cell proliferation in comparison with the negative control.

IFN-α2a causes a significant inhibitory effect on bone marrow growth under all conditions studied. In contrast, IL-29 has no significant effect on bone marrow cell proliferation in the presence of IL-3, IL-4, IL-5, IL-7, IL-10, IL-12, IL-21 or without cytokine addition. A slight inhibitory effect on bone marrow growth is observed in the presence of IL-2 and IL-15.

Example 27

Inhibition of IL-28 or IL-29 signal transduction by soluble receptor (zcytoR19 / CFR2-4)

A. Reporter analysis of signal transduction

Reporter signal transduction assays can be used to detect the inhibitory properties of the zcytor19-Fc4 homodimeric and zcytor19-Fc / CRF2-4-Fc heterodimeric soluble receptors for zcyto20, zcyto21 and zcyto24 signaling. Human embryonic kidney cells (HEK), excessively producing the zcytor19 receptor, are transfected with a reporter plasmid containing an interferon-induced response element (ISRE), which causes transcription of the luciferase reporter gene. The luciferase activity after activation of cells transfected with cells under the influence of ligands (including zcyto20 (SEQ ID NO: 2), zcyto21 (SEQ ID NO: 4), zcyto24 (SEQ ID NO: 8)) reflects the interaction of the ligand with a soluble receptor.

B. Cell Transfection

HEK 293 cells excessively producing zcytor19 are transfected as follows: 700,000 293 cells per well (on a 6-well plate) are placed 18 hours before transfection on a plate with 2 ml DMEM + 10% fetal calf serum. For each well, 1 μg of pISRE luciferase DNA (Stratagene) and 1 μg of pIRES2-EGFP DNA (Clontech) were added to 6 μl of Fugene 6 reagent (Roche Biochemicals) in a total volume of 100 μl of DMEM. Said transfection mixture was added after 30 minutes to 293 cells previously plated on the plate. After 24 hours, the transfected cells were removed from the plate using a trypsin-EDTA mixture and reapplied in an amount of approximately 25,000 cells per well in 96 well microtiter plates. About 18 hours before ligand activation, the medium is changed to DMEM + 0.5% FBS.

C. Reporter signal transduction assays

Reporter signal transduction assays are performed as follows: after incubation for 18 hours at 37 ° C in DMEM + 0.5% FBS growth medium, transfected cells are activated with 10 ng / ml zcyto20, zcyto21 or zcyto24 and 10 μg / ml of the following soluble receptors : homodimeric human zcyto19-Fc, human heterodimeric zcyto19-Fc / human CRF2-4-Fc; homodimeric human CRF2-4-Fc; homodimeric murine zcyto19-Ig. After 4 hours of incubation at 37 ° C, the cells are lysed and relative light units (RLU) are measured using a luminometer after adding the luciferase substrate. The results are presented as percent inhibition of ligand-activated signal transmission in the presence of a soluble receptor, compared with signal transmission in the presence of only PBS. Table 23 shows that the heterodimeric soluble human zcyto19-Fc receptor / human CRF2-4 is capable of inhibiting, from 16% to 45%, the inducible zcyto20, zcyto21 and zcyto24 signal transduction, compared to the control. The homodimeric soluble human zcyto19-Fc receptor is also able to inhibit zcyto21-induced signal transmission by 45%. No noticeable effects were found for homodimeric soluble huCRF2-4-Fc or muzcytor19-Ig receptors.

Table 23
The percentage inhibition of ligand-activated signal transduction by an interferon-induced response element (ISRE) by soluble receptors Ligand Huzcyto19-Fc / huCRF2-4-Fc Huzcyto19-Fc HuCRF2-4-Fc Muzcytor19-Ig Zcyto20 16% 92% 80% 91% Zcyto21 16% 45% 79% 103% Zcyto24 47% 90% 82% 89%

Example 28

IL-28 and IL-29 inhibit HIV replication in healthy human PBMCs

Human Immunodeficiency Virus (HIV) is a pathogenic retrovirus that infects cells of the immune system. CD4 T cells and monocytes are the types of cells that are infected first. In order to investigate the ability of IL-28 and IL-29 to inhibit HIV replication in vitro, PBMCs taken from normal donors are infected with HIV in the presence of IL-28, IL-29 and MetIL-29C172S-PEG.

Healthy human peripheral blood mononuclear cells (PBMCs) are isolated from whole blood from selected donors having a seronegative response to HIV and HBV. The peripheral blood cells are precipitated 2-3 times by centrifugation at low speed, resuspended and washed in PBS, in order to remove platelets contaminating them. The washed blood cells are diluted 1: 1 with phosphate buffered saline Dulbecco's saline (D-PBS) and placed in 14 ml of lymphocyte separation medium ((LSM; Cellgro ™ from Mediatech, Inc., Herndon, Virginia); density 1,078 + / - 0.002 g / ml) in a 50 ml centrifuge tube and centrifuged for 30 min with an acceleration of 600 × g. Bound PBMCs are carefully aspirated from the interface and then washed twice with PBS and low speed centrifugation. After the final wash, the number of cells was counted out using trypan blue dye and resuspended in an amount of 1 × 10 ⁷ cells / ml in RPMI 1640 growth medium supplemented with 15% fetal calf serum (FBS), 2 mM L-glutamine, 4 μg / ml PHA -P. Cells are incubated for 48-72 hours at a temperature of 37 ° C. After incubation, PBMCs are centrifuged and resuspended in RPMI 1640 medium with 15% FBS, 2 mM L-glutamine, 100 units / ml penicillin, 100 μg / ml streptomycin, 10 μg / ml gentamicin and 20 units / ml recombinant human IL-2 . The concentration of PBMCs is maintained in the culture medium at a level of 1-2 × 10 ⁶ cells / ml and the medium is replaced twice a week until the cells are used in accordance with the analysis procedure. Monocytes are purified from the culture by adhesion to the walls of the flask with tissue culture.

To conduct a standard PBMC analysis, cells obtained from at least two donors and activated using PHA-P are combined, diluted with fresh medium to a final concentration of 1 × 10 ⁶ cells / ml and applied to the inner walls of a 96-well microplate with round-bottom wells in an amount of 50 μl per well (5 × 10 ⁴ cells per well). Test concentrations are obtained by 2X dilution in microtiter tubes and 100 μl of a mixture of each concentration is placed in the appropriate wells in a standard format. IL-28, IL-29 and MetIL-29C172S-PEG are added at concentrations in the range of 0-10 μg / ml, usually in the form of semi-log dilutions. 50 μl of a predetermined dilution of the mother liquor of the virus is placed in each test well (final MOI is 0.1). Wells, to which only cells and the virus are added, are used for viral control. Separately, using the MTS analytical system, virus-free tablets are prepared to study the cytotoxicity of the drug. The PBMC cultures were incubated for seven days, after which samples of cell-free supernatant fluids were collected and analyzed for reverse transcriptase activity and p24 antigen levels.

The decrease in reverse transcriptase activity and p24 antigen levels by IL-28, IL-29, and MetIL-29C172S-PEG is an indicator of antiviral activity. The results show that IL-28 and IL-29 may have therapeutic value in the treatment of HIV and AIDS.

Example 29

IL-28 and IL-29 inhibit GBV-B replication in monkey liver cells

HCV is a member of the Flaviviridae family of RNA viruses. HCV is difficult to replicate in both ex-vivo and in vitro cultures, and therefore there are no satisfactory systems for testing the anti-HCV activity of molecules in vitro. GB virus B (GBV-B) is an attractive surrogate model for use in the development of anti-HCV antiviral agents because its sequences have a relatively high degree of identity with the HCV sequence and it is a hepatotrophic virus. Currently, the virus can only be grown in the primary hepatocytes of some non-human primates. Cultivation is carried out either by isolating hepatocytes in vitro and infecting them with GBV-B, or by isolating hepatocytes from GBV-B-infected monkeys and using them directly with antiviral compounds.

The effects of IL-28, IL-29, and MetIL-29C172S-PEG were analyzed by extracellular RNA production in GBV-B using TaqMan RT-PCR and by cytotoxicity using CellTiter96 ^® reagent (Promega, Madison, Wisconsin) in three series of six semilogarithmic dilutions polypeptides IL-28, IL-29 and MetIL-29C172S-PEG. Untreated cultures serve as cell and viral controls. Both RIBAVIRIN® (with a maximum test concentration of 200 μg / ml) and IFN-α (with a maximum test concentration of 5000 international units / ml) are included as positive control compounds. Primary hepatocyte cultures are isolated and placed on collagen-coated plates. The next day, cultures are treated with test compounds (IL-28, IL-29, MetIL-29C172S-PEG, IFN-α, or RIBAVIRIN®) 24 hours before exposure to GBV-B virions or directly treated with test samples when infected hepatocytes are used in vivo . Test samples and media are added the next day and replaced after three days. After another three or four days (on day 6-7, the test sample addition field), the supernatant was collected and the number of cells was counted using CellTiter96® reagent. Viral RNA is extracted from the supernatant and quantified in three series by quantification using TaqMan RT-PCR using an in vitro transcribed RNA containing the RT-PCR target as a standard. The average value for a series of samples is calculated. Inhibition of virus production is analyzed graphically by plotting the average of the RNA amount and the number of cells obtained from three series of samples in comparison with untreated viral and cell controls. The inhibitory concentration of the drug, leading to a 50% suppression of GBV-B RNA production (IC50), and the toxic concentration, leading to the destruction of 50% of the number of cells, relative to the control values (TC50), are calculated by interpolation from the graphs constructed on the basis of the obtained data.

Inhibition of GBV-B RNA production by IL-28 and IL-29 is an indicator of the antiviral properties of IL-28 and IL-29 against this virus, which resembles hepatitis C virus, in hepatocytes, the main target of hepatitis C infection, and positive results indicate that IL-28 or IL-29 can be used in the treatment of HCV infection in humans.

Example 30

IL-28, IL-29 and MetIL-29C172S-PEG inhibit HBV replication in WT10 cells

Chronic hepatitis B (HBV) is one of the most common and severe viral infections in humans and belongs to the Hepadnaviridae virus family. In order to study the antiviral activity of IL-28 and IL-29 against HBV, IL-28, IL-29 and MetIL-29C172S-PEG, they are tested against HBV in the in vitro infection system using various human HepG2 cell lines. IL-28, IL-29 and MetIL-29C172S-PEG inhibit the replication of the virus in this system, and this indicates their therapeutic value in the treatment of HBV in humans.

WT10 cells are derived from the human HepG2 2.2.15 cell line. WT10 cells are stably transfected with the HBV genome, which ensures stable expression of HBV transcripts in the indicated cell line (Fu and Cheng, Antimicrobial Agents Chemother. 44 (12): 3402-3407, 2000). In a WT10 cell assay, the drug in question and 3TC used as control were tested in five concentrations each, obtained by a series of semi-log dilutions. Ending points are TaqMan PCR results for HBV extracellular DNA (IC50) and cell counts determined using CellTiter96 reagent (TC50). The analysis procedure is similar to that given by Korba et al., Antiviral Res. 15 (3): 217-228, 1991 and Korba et al., Antiviral Res. 19 (1): 55-70, 1992. Briefly, WT10 cells are placed on 96-well microtiter plates. After 16-24 hours, the confluent monolayer of HepG2-2.2.15 cells is washed, the medium is replaced with a complete nutrient medium containing various concentrations of test samples, and three series of experiments are performed. 3TC is used as a positive control, while the nutrient medium itself is added to the cells as a negative control (viral control, VC). Three days later, the culture medium is replaced with fresh culture medium containing appropriately diluted test samples. Six days after the initial addition of the test compounds, the culture supernatant was collected, treated with pronase and DNase and used for real-time quantitative TaqMan PCR analysis. PCR-amplified HBV DNA is detected in real time by monitoring amplification of fluorescence signals resulting from exonucleolytic degradation of the captured fluorescent probe molecule, which hybridizes with the amplified HBV DNA. For each PCR amplification, a standard curve is constructed simultaneously using dilutions of purified HBV DNA. Antiviral activity is calculated by decreasing HBV DNA levels (IC ₅₀ ). After that, a dye capture method is used to assess cell viability, which is used to calculate cytotoxicity (TC ₅₀ ). The therapeutic index (TI) is calculated as the ratio of TC ₅₀ / IC ₅₀ .

IL-28, IL-29, and MetIL-29C172S-PEG inhibit HepB virus replication in WT10 cells with an IC50 value of <0.032 μg / ml. This proves the antiviral activity of IL-28 and IL-29 against HBV grown in the liver cell line, and indicates the therapeutic value of IL-28 and IL-29 in the treatment of HBV infection in humans.

Example 31

IL-28, IL-29, and MetIL-29C172S-PEG inhibit BVDV replication in bovine kidney cells

HCV is a member of the Flaviviridae family of RNA viruses. Other viruses belonging to this family are bovine viral diarrhea virus (BVDV) and yellow fever virus (YFV). HCV is difficult to replicate in both ex vivo and in vitro cultures, so there is no system for studying in vitro activity against HCV. BVDV and YFV are used instead of HCV as surrogate viruses to study antiviral activity against the Flaviviridae virus family.

The antiviral effects of IL-28, IL-29, and MetIL-29C172S-PEG are studied by inhibition when tested for cytotoxic effect (CPE). In this assay, the death of cells in a Madina-Darby bovine kidney cell line (MDBK) after infection with the BVDV cytopathic virus and inhibition of cell death upon administration of IL-28, IL-29 and MetIL-29C172S-PEG are determined. MDBK cells are propagated in Dulbecco's modified basic medium (DMEM) containing phenol red together with 10% horse serum, 1% glutamine and 1% penicillin-streptomycin. CPE inhibition assays are performed in DMEM without phenol red in the presence of 2% FBS, 1% glutamine and 1% penicillin-streptomycin. The day before the analysis, the cells are trypsinized (1% trypsin-EDTA), washed, counted and placed at 10 ⁴ cells per well in 96-well BioCoat ^® plates (Fisher Scientific, Pittsburgh, PA) with a flat bottom in a volume of 100 μl per hole. The next day, the medium is removed and pre-titrated aliquots of the virus are added to the cells. The amount of virus corresponds to the maximum dilution, which leads to complete cell death (> 80%) by the time the CPE is fully developed (7th day for BVDV). Cell viability is determined using CellTiter96 ^® reagent (Promega), in accordance with the manufacturer's recommendations, and a Vmax plate reader (Molecular Devices, Sunnyvale, California). Each test sample was tested in six concentrations obtained by hemogarithmic dilutions using an assay medium. As positive controls, IFNα and RIBAVIRIN® are used. Test samples are administered with a viral infection. The average background and the data corrected for staining the sample to determine the percentage reduction in CPE and the percentage of cell viability for each concentration are determined in comparison with the controls and the IC ₅₀ value relative to TC _{50 is} calculated.

IL-28, IL-29, and MetIL-29C172S-PEG inhibit cell death induced by BVDV in bovine kidney MDBK cells. IL-28 inhibits cell death with an IC ₅₀ value of 0.02 μg / ml, IL-29 inhibits cell death with an IC ₅₀ value of 0.19 μg / ml, and MetIL-29C172S-PEG inhibits cell death with an IC value ₅₀ , equal to 0.45 μg / ml. The result proves that IL-28 and IL-29 have antiviral activity against the Flaviviridae virus family.

Example 32

Induction of interferon-activated genes by IL-28 and IL-29

A. Mononuclear cells of human peripheral blood

Freshly isolated human peripheral blood mononuclear cells are grown in the presence of IL-29 (20 ng / ml), IFN? 2a (2 ng / ml) (PBL Biomedical Labs, Piscataway, New Jersey), or just nutrient medium. Cells are grown for 6, 24, 48 or 72 hours, and then the complete RNA is isolated and treated with DNase that does not contain RNase. 100 ng of total RNA is used as a template for the one-step semi-quantitative RT-PCR method together with Taqman One-Step RT-PCR Master Mix® reagent and gene-specific primers, in accordance with the manufacturer's recommendations (Applied Biosystems, Branchburg, NJ). The results obtained are normalized with respect to HPRT and presented for each time point in the form of the multiplicity of induction in comparison with the nutrient medium alone, which is used as a control. Table 24 shows that IL-29 in all studied time intervals induces interferon-activated gene expression in mononuclear cells of human peripheral blood.

Table 24 MxA Induction multiplicity Pkr Multiplicity of induction OAS Multiplicity of induction 6 hours, IL29 3,1 2.1 2,5 6 hours, IFNα2a 17,2 9.6 16,2 24 hours, IL29 19,2 5,0 8.8 24 hours, IFNα2a 57.2 9,4 22.3 48 hours, IL29 7.9 3,5 3.3

48 hours, IFNα2a 18.1 5,0 17.3 72 hours, IL29 9,4 3,7 9.6 72 hours, IFNα2a 29.9 6.4 47.3

B. Activated Human T Cells

Human T cells are isolated by negative selection from freshly grown peripheral blood mononuclear cells using a Pan T-cell Isolation® reagent kit, in accordance with the manufacturer's recommendations (Miltenyi, Auburn, California). T cells are then activated and propagated for 5 days along with anti-CD3, soluble anti-CD28 (0.5 μg / ml) immobilized on a plate (Pharmingen, San Diego, California) and interleukin 2 (IL-2; 100 units / ml) (R&D Systems, Minneapolis, Minnesota), washed and propagated for another 5 days along with IL-2. After activation and propagation, cells are stimulated by the action of IL-28A (20 ng / ml), IL-29 (20 ng / ml) or medium alone for 3, 6 or 18 hours. All RNA is isolated and treated with a RNase-free DNase. A one-step semi-quantitative RT-PCR® method is carried out as described in the previous example. The results obtained are normalized with respect to HPRT and presented for each time point in the form of the multiplicity of induction in comparison with the nutrient medium alone, which is used as a control. Table 25 shows that IL-28 and IL-29 in all studied time intervals induces interferon-activated gene expression in activated human T cells.

Table 25 MxA Induction multiplicity Pkr Multiplicity of induction OAS Multiplicity of induction Donor # 1, 3 hours, IL28 5.2 2,8 4.8 Donor # 1, 3 hours, IL29 5,0 3,5 6.0

Donor # 1, 6 hours, IL28 5.5 2.2 3.0 Donor # 1, 6 hours, IL29 6.4 2.2 3,7 Donor # 1, 18 hours, IL28 4.6 4.8 4.0 Donor # 1, 18 hours, IL29 5,0 3.8 4.1 Donor # 2, 3 hours, IL28 5.7 2.2 3,5 Donor # 2, 3 hours, IL29 6.2 2,8 4.7 Donor # 2, 6 hours, IL28 7.3 1.9 4.4 Donor # 2, 6 hours, IL29 8.7 2.6 4.9 Donor # 2, 18 hours, IL28 4.7 2,3 3.6 Donor # 2, 18 hours, IL29 4.9 2.1 3.8

C. Primary human hepatocytes

Freshly isolated human hepatocytes from two different donors (Cambreax, Baltimore, Maryland and CellzDirect, Tusson, Arizona) are activated for 24 hours using IL-28A (50 ng / ml), IL-29 (50 ng / ml), IFNα2a (50 ng / ml) or just a nutrient medium. After activation, complete RNA is isolated and treated with a RNAse-free DNase. The one-stage semi-quantitative RT-PCR method is carried out as described in the previous example. The results are normalized relative to HPRT and presented for each time point in the form of the multiplicity of induction compared with only the nutrient medium, which is used as a control. Table 26 shows that after 24-hour activation, IL-28 and IL-29 induce interferon-activated gene expression in human hepatocytes.

Table 26 MxA Induction multiplicity Pkr Multiplicity of induction OAS Multiplicity of induction Donor # 1, IL28 31,4 6.4 30,4 Donor # 1, IL29 31.8 5.2 27.8 Donor # 1, IFN-α2a 63,4 8.2 66.7 Donor # 2, IL28 41.7 4.2 24.3 Donor # 2, IL29 44.8 5.2 25,2 Donor # 2, IFN-α2a 53,2 4.8 38.3

D. HepG2 and HuH7: Human Liver Hepatoma Cell Lines

HepG2 and HuH7 cells (ATCC No. 8065, Manassas, Virginia) are activated by IL-28A (10 ng / ml), IL-29 (10 ng / ml), IFN? 2a (10 ng / ml) IFNB (1 ng / ml) (PBL Biomedical, Labs, Piscataway, New Jersey) or only a culture medium for 24 or 48 hours. In a separate culture, HepG2 cells are activated, as indicated above, by the action of 20 ng / ml MetIL-29C172S-PEG or MetIL-29-PEG. All RNA is isolated and treated with a RNase-free DNase. 100 ng of total RNA is used as a template for the implementation of the one-step semi-quantitative RT-PCR method, as indicated previously. The results are normalized relative to HPRT and presented for each time point in the form of the multiplicity of induction compared with only the nutrient medium, which is used as a control. Table 27 shows that IL-28 and IL-29 induce ISG expression in HepG2 and HuH7 cells of liver hepatoma cell lines after 24 and 48 hours.

Table 27 MxA Induction multiplicity Pkr Multiplicity of induction OAS Multiplicity of induction HepG2, 24 hours, IL28 12,4 0.7 3.3 HepG2, 24 hours, IL29 36.6 2.2 6.4 HepG2, 24 hours, IFNα2a 12,2 1.9 3.2 HepG2, 24 hours, IFNβ 93.6 3.9 19.0 HepG2, 48 hours, IL28 2.7 0.9 1,1 HepG2, 48 hours, IL29 27,2 2.1 5.3 HepG2, 48 hours, IFNα2a 2,5 0.9 1,2 HepG2, 48 hours, IFNβ 15.9 1.8 3.3 HuH7, 24 hours, IL28 132.5 5,4 52.6 HuH7, 24 hours, IL29 220,2 7.0 116.6 HuH7, 24 hours, IFNα2a 157.0 5.7 67.0 HuH7, 24 hours, IFNβ 279.8 5,6 151.8 HuH7, 48 hours, IL28 25.6 3.4 10.3 HuH7, 48 hours, IL29 143.5 7.4 60.3 HuH7, 48 hours, IFNα2a 91.3 5.8 32,3 HuH7, 48 hours, IFNβ 65.0 4.2 35.7

Table 28 MxA Induction multiplicity OAS Multiplicity of induction Pkr Multiplicity of induction MetIL-29-PEG 36.7 6.9 2.2 MetIL-29C172S-PEG 46.1 8.9 2,8

Data are presented for 20 ng / ml of MetIL-29-PEG and MetIL-29C172S-PEG versions of IL-29 after growing for 24 hours.

The data presented are normalized with respect to HPRT and are presented as the multiplicity of induction compared with non-activated cells.

Example 33

Antiviral activity of IL-28 and IL-29 in the HCV replicon system

The ability of antiviral drugs to inhibit HCV replication can be tested in vitro in the HCV replicon system. The replicon system consists of Huh7 cells of the human hepatoma cell line transfected with subgenomic RNA replicons that direct constitutive replication of HCV genomic RNA molecules (Blight, K.J. et al., Science 290: 1972-1974, 2000). Processing replicon clones using TNFα in the amount of 10 int. units / ml reduces the amount of HCV RNA by 85%, compared with untreated control cells. The ability of IL-28A and IL-29 to reduce the amount of HCV RNA produced after 72 hours by replicon clones indicates that the antiviral status conferred on Huh7 cells by treatment with IL-28A / IL-29 effectively inhibits the replication of HCV replicon and, thus, it is very likely effective in inhibiting HCV replication.

The ability of IL-28A and IL-29 to inhibit HCV replication, which is determined using the Bayer Branched chain DNA reagent kit, can be tested under the following conditions:

1. IL28 individually with increasing concentrations (6) * up to 1.0 μg / ml

2. IL29 individually with increasing concentrations (6) * up to 1.0 μg / ml

3. PEGIL29 individually with increasing concentrations (6) * up to 1.0 μg / ml

4. IFNα2A individually with concentrations of 0.3, 1.0 and 3.0 int. units / ml

5. Ribavirin individually

The positive control is IFNα, and the negative control is ribavirin.

To determine their viability, cells are stained with Alomar Blue after 72 hours.

* Concentrations for conditions 1-3 are as follows:

1.0 μg / ml, 0.32 μg / ml, 0.10 μg / ml, 0.032 μg / ml, 0.010 μg / ml, 0.0032 μg / ml.

The replicon clone (BB7) is treated 1X per day for three consecutive days with the above dose. Complete HCV RNA was determined after 72 hours.

Example 34

IL-28 and IL-29 have antiviral activity against pathogenic viruses

Two methods are used for in vitro analysis of the antiviral activity of IL-28 and IL-29 against a panel of pathogenic viruses, including, but not limited to, adenovirus, parainfluenza virus, respiratory syncytial virus, rhinovirus, Coxsackie virus, influenza virus, vaccinia virus, West Nile virus, dengue virus, equine encephalomyelitis virus, Pichinde virus and poliomyelitis virus. These two methods are suppression of the virus-induced cytopathic effect (CPE), which is determined by visual (under a microscope) observation of the cells, and increased cell uptake of the neutral red (NR) dye. In the CPE inhibition method, the effect of seven concentrations of the test drug is evaluated (using logarithmic dilutions such as 1000, 100, 10, 1, 0.1, 0.01, 0.001 ng / ml) against each virus in a 96-well flat plate format bottom containing host cells. The compounds are added 24 hours before the introduction of the virus, which is used, depending on the virus, with a concentration of from about 5 to 100 doses infecting the cell culture per well, which corresponds to a multiplicity of infection (MOI) in the range from 0.01 to 0.0001 infectious particles per cell. Tests are read after incubation at 37 ° C for a given time, sufficient to allow the development of an adequate cytopathic effect of the virus. In a capture assay, NR dye (at a concentration of 0.34% in culture medium) was added to the same set of plates to conduct a visual assessment. After 2 hours, the intensity of the dye absorbed by the cells and then eluted from the cells is determined using a microplate reader. Antiviral activity is expressed as a 50% effective (virus inhibitory) concentration (EC50), which is determined graphically by plotting data on semilogarithmic paper according to the concentration versus percent inhibition. In some cases, for the purpose of conducting a regression analysis, EC50 / IC50 values may be determined. In general, the EC50 values determined by the NR analysis method are two times higher than the EC50 values obtained by the CPE method.

Table 29
Visual analysis Virus Cell line Medicine EC50 visual IC50 visual SI (IC50 / EC50 visual) Adenovirus A549 IL-28A > 10 mcg / ml > 10 mcg / ml 0 Adenovirus A549 IL-29 > 10 mcg / ml > 10 mcg / ml 0 Adenovirus A549 MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 Parainfluenza virus MA-104 IL-28A > 10 mcg / ml > 10 mcg / ml 0 Parainfluenza virus MA-104 IL-29 > 10 mcg / ml > 10 mcg / ml 0 Parainfluenza virus MA-104 MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 Respiratory syncytial virus MA-104 IL-28A > 10 mcg / ml > 10 mcg / ml 0 Respiratory syncytial virus MA-104 IL-29 > 10 mcg / ml > 10 mcg / ml 0 Respiratory syncytial virus MA-104 MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 Rhinovirus 2 HF IL-28A > 10 mcg / ml > 10 mcg / ml 0 Rhinovirus 2 HF IL-29 > 10 mcg / ml > 10 mcg / ml 0 Rhinovirus 2 HF MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 Rhinovirus 9 Hela IL-28A > 10 mcg / ml > 10 mcg / ml 0 Rhinovirus 9 Hela IL-29 > 10 mcg / ml > 10 mcg / ml 0 Rhinovirus 9 Hela MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 Coxsackie V4 virus HF IL-28A > 10 mcg / ml > 10 mcg / ml 0 Coxsackie V4 virus HF IL-29 > 10 mcg / ml > 10 mcg / ml 0 Coxsackie V4 virus HF MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 Influenza (Type A [H3N2]) Maden-Darby Horse Kidney Cells IL-28A > 10 mcg / ml > 10 mcg / ml 0 Influenza (Type A [H3N2]) Maden-Darby Horse Kidney Cells IL-29 > 10 mcg / ml > 10 mcg / ml 0 Influenza (Type A [H3N2]) Maden-Darby Horse Kidney Cells MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 Influenza (Type A [H3N2]) Vero IL-28A 0.1 μg / ml > 10 mcg / ml > 100 Influenza (Type A [H3N2]) Vero IL-29 > 10 mcg / ml > 10 mcg / ml 0 Influenza (Type A [H3N2]) Vero MetIL-29 C172S-PEG 0.045 mcg / ml > 10 mcg / ml > 222 Vaccinia virus Vero IL-28A > 10 mcg / ml > 10 mcg / ml 0 Vaccinia virus Vero IL-29 > 10 mcg / ml > 10 mcg / ml 0 Vaccinia virus Vero MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 West Nile Virus Vero IL-28A 0.00001 mcg / ml > 10 mcg / ml > 1,000,000 West Nile Virus Vero IL-29 0.000032 mcg / ml > 10 mcg / ml > 300,000 West Nile Virus Vero MetIL-29 C172S-PEG 0.001 mcg / ml > 10 mcg / ml > 10,000 Dengue virus Vero IL-28A 0.01 mcg / ml > 10 mcg / ml > 1000 Dengue virus Vero IL-29 0.032 mcg / ml > 10 mcg / ml > 312 Dengue virus Vero MetIL-29 C172S-PEG 0.0075 mcg / ml > 10 mcg / ml > 1330 Horse Encephalomyelitis Virus Vero IL-28A 0.01 mcg / ml > 10 mcg / ml > 1000 Horse Encephalomyelitis Virus Vero IL-29 0.012 mcg / ml > 10 mcg / ml > 833

Horse Encephalomyelitis Virus Vero MetIL-29 C172S-PEG 0.0065 mcg / ml > 10 mcg / ml > 1538 Pichinde virus Bsc-1 IL-28A > 10 mcg / ml > 10 mcg / ml 0 Pichinde virus Bsc-1 IL-29 > 10 mcg / ml > 10 mcg / ml 0 Pichinde virus Bsc-1 MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 Polio virus Vero IL-28A > 10 mcg / ml > 10 mcg / ml 0 Polio virus Vero IL-29 > 10 mcg / ml > 10 mcg / ml 0 Polio virus Vero MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 Table 30
Neutral Red Analysis Virus Cell line Medicine EC50 NR IC50 NR SI NR (IC50 / EC50) Adenovirus A549 IL-28A > 10 mcg / ml > 10 mcg / ml 0 Adenovirus A549 IL-29 > 10 mcg / ml > 10 mcg / ml 0 Adenovirus A549 MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 Parainfluenza virus MA-104 IL-28A > 10 mcg / ml > 10 mcg / ml 0 Parainfluenza virus MA-104 IL-29 > 10 mcg / ml > 10 mcg / ml 0 Parainfluenza virus MA-104 MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 Respiratory syncytial virus MA-104 IL-28A > 10 mcg / ml > 10 mcg / ml 0 Respiratory syncytial virus MA-104 IL-29 > 10 mcg / ml > 10 mcg / ml 0 Respiratory syncytial virus MA-104 MetIL-29 C172S-PEG 5.47 mcg / ml > 10 mcg / ml > 2 Rhinovirus 2 HF IL-28A > 10 mcg / ml > 10 mcg / ml 0 Rhinovirus 2 HF IL-29 > 10 mcg / ml > 10 mcg / ml 0 Rhinovirus 2 HF MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 Rhinovirus 9 Hela IL-28A 1.726 mcg / ml > 10 mcg / ml > 6 Rhinovirus 9 Hela IL-29 0.982 mcg / ml > 10 mcg / ml > 10 Rhinovirus 9 Hela MetIL-29 C172S-PEG 2.051 μg / ml > 10 mcg / ml > 5 Coxsackie V4 virus HF IL-28A > 10 mcg / ml > 10 mcg / ml 0 Coxsackie V4 virus HF IL-29 > 10 mcg / ml > 10 mcg / ml 0 Coxsackie V4 virus HF MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 Influenza (Type A [H3N2]) Maden-Darby Horse Kidney Cells IL-28A > 10 mcg / ml > 10 mcg / ml 0 Influenza (Type A [H3N2]) Maden-Darby Horse Kidney Cells IL-29 > 10 mcg / ml > 10 mcg / ml 0 Influenza (Type A [H3N2]) Maden-Darby Horse Kidney Cells MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 Influenza (Type A [H3N2]) Vero IL-28A 0.25 mcg / ml > 10 mcg / ml > 40 Influenza (Type A [H3N2]) Vero IL-29 2 mcg / ml > 10 mcg / ml > 5 Influenza (Type A [H3N2]) Vero MetIL-29 C172S-PEG 1.4 mcg / ml > 10 mcg / ml > 7 Vaccinia virus Vero IL-28A > 10 mcg / ml > 10 mcg / ml 0 Vaccinia virus Vero IL-29 > 10 mcg / ml > 10 mcg / ml 0 Vaccinia virus Vero MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 West Nile Virus Vero IL-28A 0.0001 mcg / ml > 10 mcg / ml > 100,000 West Nile Virus Vero IL-29 0,00025 mcg / ml > 10 mcg / ml > 40,000 West Nile Virus Vero MetIL-29 C172S-PEG 0,00037 mcg / ml > 10 mcg / ml > 27000 Dengue virus Vero IL-28A 0.1 μg / ml > 10 mcg / ml > 100 Dengue virus Vero IL-29 0.05 mcg / ml > 10 mcg / ml > 200 Dengue virus Vero MetIL-29 C172S-PEG 0.006 mcg / ml > 10 mcg / ml > 166

Horse Encephalomyelitis Virus Vero IL-28A 0.035 mcg / ml > 10 mcg / ml > 286 Horse Encephalomyelitis Virus Vero IL-29 0.05 mcg / ml > 10 mcg / ml > 200 Horse Encephalomyelitis Virus Vero MetIL-29 C172S-PEG 0.02 mcg / ml > 10 mcg / ml > 500 Pichinde virus Bsc-1 IL-28A > 10 mcg / ml > 10 mcg / ml 0 Pichinde virus Bsc-1 IL-29 > 10 mcg / ml > 10 mcg / ml 0 Pichinde virus Bsc-1 MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0 Polio virus Vero IL-28A > 1,672 mcg / ml > 10 mcg / ml > 6 Polio virus Vero IL-29 > 10 mcg / ml > 10 mcg / ml 0 Polio virus Vero MetIL-29 C172S-PEG > 10 mcg / ml > 10 mcg / ml 0

Example 35

IL-28, IL-29, metIL-29-PEG and metIL-29C172S-PEG stimulate ISG induction in the mouse liver AML-12 cell line

Interferon-activated genes (ISGs) are genes that are induced by type I interferons (IFNs), as well as molecules of the IL-28 and IL-29 family, indicating that IFN and IL-28 and IL-29 induce the same pathways leading to antiviral activity. Human type I interferons (IFNα1-4 and IFNβ) show little or no activity against murine cells, which is believed to be due to the lack of cross-species reactivity. To investigate whether IL-28 and IL-29 have an effect on mouse cells, ISG induction by IL-28 and IL-29 is evaluated by real-time PCR on mouse cells obtained from the AML-12 cell line.

AML-12 cells were plated on 6-well plates in complete DMEM at a concentration of 2 × 10 ⁶ cells / well. 24 hours after the application of the cells, human IL-28 and IL-29 at a concentration of 20 ng / ml were added to the cultures. As a control, cells either activate murine IFNα (positive control) or do not activate (negative control). Cells after adding obtained from CHO IL-28A (SEQ ID NO: 2) or IL-29 (SEQ ID NO: 4) are grown for 8, 24, 48 and 72 hours. RNA was isolated from the cell mass using the RNAEasy-kit ^® reagent kit (Qiagen, Valencia, California). RNA is treated with DNase (Millipore, Billerika, Mass.) To purify any trace of DNA from RNA. cDNA generated using a mixture of Perkin-Elmer RT. The induction of ISG genes is evaluated by real-time PCR using primers and probes specific for murine OAS, Pkr, and Mx1. To obtain quantitative data, real-time PCR HPRT is reduplicated with ISG PCR. A standard curve is obtained using known amounts of RNA from IFN-stimulated murine PBLs. All data are shown as expression relative to internal expression of HPRT.

Human IL-28A and IL-29 activate ISG induction in the hepatocytes of AML-12 cell line mice, and the results show that, unlike type I interferons, the IL-28/29 family of proteins exhibit cross-species reactivity.

Table 31 Activation Oas Pkr Mx1 Absent 0.001 0.001 0.001 Human IL-28 0.04 0.02 0.06 Human IL-29 0.04 0.02 0,07 Mouse IL-28 0.04 0.02 0.08 IFNα mouse 0.02 0.02 0.01

All data are presented as the multiplicity of increase in induction with respect to HPRT gene expression:

As an example, the data for the time point 48 hours.

Table 32
AML12′s MxA Induction multiplicity OAS Multiplicity of induction Pkr Multiplicity of induction MetIL-29-PEG 728 614 8 MetIL-29C172S-PEG 761 657 8

Cells are activated by 20 ng / ml MetIL-29-PEG or MetIL-29C172S-PEG for 24 hours.

The data presented are normalized with respect to HPRT and are presented as the multiplicity of induction with respect to inactive cells.

Example 36

ISGs are effectively induced in the spleen of transgenic mice expressing human IL-29

Prepare transgenic (Tg) mice expressing human IL-29 under the control of the Eu-lck promoter. To study whether IL-29 has in vivo activity in mice, ISG expression is analyzed by real-time PCR using the spleens of transgenic IL-29 Eu-lck mice.

Transgenic mice (C3H / C57BL / 6) are prepared using a construct that expresses the human IL-29 gene under the control of the Eu-lck promoter. The specified promoter is active in T cells and B cells. Transgenic mice and their non-transgenic littermates (n = 2 / group) were killed at approximately 10 weeks of age. The spleens of mice are recovered. RNA was isolated from the cell mass using the RNAEasy-kit ^® reagent kit (Qiagen). RNA is treated with DNase to purify any trace of DNA from RNA. cDNA is generated using a mixture of Perkin-Elmer RT®. ISG gene induction is evaluated by real-time PCR using primers and probes (5 ′ FAM, 3 ′ NFQ) specific for mouse OAS, Pkr, and Mx1. In order to obtain quantitative HPRT data by real-time PCR, ISGs are reduced with PCR. In addition, a standard curve is obtained using known amounts of IFN-stimulated murine PBLs. All data are shown as expression relative to internal expression of HPRT.

Spleen isolated from IL-29 Tg mice showed a high induction of ISGs in OAS, Pkr, and Mx1 compared to nontransgenic odnokomnyh controls, indicating that human IL-29 in vivo is biologically active in mice.

Table 33 Mice Oas Pkr Mx1 Non transgenic 4,5 4,5 3,5 IL-29 Tg 12 8 21

All data are presented as the ratio of the increase in expression relative to the expression of the HPRT gene. Mean expression values were obtained from two mice.

Example 37

Human IL-28 and IL-29 proteins induce gene expression of ISG in the liver, spleen, and blood of mice

In order to determine whether human IL-28 and IL-29 can induce interferon-activated genes in vivo, CHO-derived human IL-28A and IL-29 proteins are injected into mice. In addition, in vivo assays also test for IL-29 derived from E. coli, as described above, using MetIL-29C172S-PEG and MetIL-29-PEG. At different time periods and with different doses, the gene induction of ISG in the blood, spleen and liver of mice is determined.

C57BL / 6 mice are injected intraperitoneally or intravenously with human CHO-derived IL-28A and IL-29 or MetIL-29C172S-PEG and MetIL-29-PEG in a dose range (10 μg - 250 μg). Mice are killed at different time periods (1 hour - 48 hours). The spleen and liver are extracted from mice and RNA is isolated. RNA is also isolated from blood cells. Tablets are prepared from cells and RNA is isolated from them using the RNAEasy-kit ^® reagent kit (Qiagen). RNA is treated with DNase (Amicon) to purify RNA of any DNA residues. cDNA was generated using a mixture of Perkin-Elmer RT (Perkin-Elmer). Gene induction of ISG is evaluated by real-time PCR using primers and probes specific for murine OAS, Pkr, and Mx1. In order to obtain quantitative HPRT data by real-time PCR, ISGs are reduced with PCR. A standard curve is calculated using known amounts of IFN-stimulated murine PBLs. All data are shown as expression relative to internal expression of HPRT.

Depending on the dose, human IL-29 induces gene expression of ISG (OAS, Pkr and Mx1) in the liver, spleen and blood of mice. The expression of ISG reaches a maximum value in the range of 1-6 hours after injection and shows prolonged, compared with control mice, expression up to 48 hours. In this experiment, human IL-28A does not induce gene expression of ISG.

Table 34 Injection OAS-1 hour OAS-6 hour OAS-24 hour OAS-48 hour Missing - Liver 1,6 1,6 1,6 1,6 IL-29, Liver 2,5 four 2,5 2,8 Missing - Spleen 1.8 1.8 1.8 1.8 IL-29, Spleen four 6 3.2 3.2 Missing - Blood 5 5 5 5 IL-29, Blood 12 eighteen eleven 10

The results are presented as the ratio of the increase in expression relative to the expression of the HPRT gene. As a sample, an example is given for induction in OAS by IL-29 as a single intraperitoneal injection of 250 μg. The data shown are mean expression values for 5 different animals / groups.

Table 35 Injection OAS (24 hours) Absent 1.8 IL-29 10 mcg 3,7 IL-29 50 mcg 4.2 IL-29 250 mcg 6 Table 36 MetIL-29-PEG MetIL-29C172S-PEG No medicine 3 hour 6 hour 12 hour 24 hour 3 hour 6 hour 12 hour 24 hour 24 hour PKR 18.24 13.93 4.99 3.77 5.29 5.65 3.79 3,55 3.70 Oas 91.29 65.93 54.04 20.81 13,42 13.02 10.54 8.72 6.60 Mx1 537.51 124,99 33.58 35.82 27.89 29.34 16.61 0.00 10.98

Mice are injected intravenously with 100 μg of protein. Data are presented as a multiplicity of increase in expression compared with the expression of HPRT in the liver of mice. Similar data were obtained for the spleen and blood of mice.

Example 38

IL-28 and IL-29 induce ISG protein in mice

In order to study the effect of human IL-28 and IL-29 on the induction of ISG protein (OAS), the serum and plasma of mice treated with IL-28 and IL-29 proteins are tested for OAS activity.

C57BL / 6 mice were given intravenous injections of human IL-28A and IL-29 as solutions in PBS in a concentration range (10 μg - 250 μg). Serum and plasma were taken from mice at different time periods and the OAS activity was determined using the OAS Radimimmunoassay (RIA) kit from Eiken Chemicals (Tokyo, Japan).

IL-28 and IL-29 induce OAS activity in the serum and plasma of mice, indicating that these proteins are biologically active in vivo.

Table 37 Injection OAS-1 hour OAS-6 hour OAS-24 hour OAS-48 hour Absent 80 80 80 80 IL-29 80 80 180 200

OAS activity is given in pM / dl in plasma for a single concentration (250 μg) of human IL-29.

Example 39

IL-28 and IL-29 inhibit adenoviral pathology in mice

In order to study the antiviral activity of IL-28 and IL-29 against viruses that infect the liver, test samples were tested in mice against infectious adenoviral vectors expressing the internal gene for green fluorescent protein (GFP). When given intravenously, these viruses for gene expression primarily target the liver. Adenoviruses are not capable of replication, but lead to liver damage caused by inflammatory cell infiltration, which can be monitored by measuring serum levels of liver enzymes such as AST and ALT, or by direct observation of liver pathology.

C57BL / 6 mice were intraperitoneally injected once daily with 50 μg of murine IL-28 (zcyto24 as indicated in SEQ ID NO: 8) or MetIL-29C172S-PEG for 3 days. Control animals were injected with PBS. One hour after the 3rd dose, mice were injected with a single bolus injection of the adenoviral vector, AdGFP (1 × 10 ⁹ plaque forming units (bout)) in the tail vein. After that, an additional 4 doses of PBS, mouse IL-28 or MetIL-29C172S-PEG (a total of 7 doses) are administered daily to mice. One hour after the last dose of PBS, mouse IL-28, or MetIL-29C172S-PEG was injected from mice, blood was last taken for analysis, after which the animals were sacrificed. Serum and liver tissue are analyzed. Serum is analyzed for liver enzymes AST and ALT. The liver is removed and analysis of GFP expression and histology are performed. For histology, liver samples are fixed in formalin and then waxed followed by H&E staining. Slices of the liver that darken during processing are examined under an optical microscope. Changes are noted and scored on a scale designed to evaluate liver pathology and inflammation.

Murine IL-28 and IL-29 inhibit adenovirus infection and gene expression, as measured by liver fluorescence. In PBS-treated mice (n = 8), the average relative liver fluorescence is 52.4 (in relative units). In contrast, IL-28-treated mice (n = 8) had an average relative liver fluorescence of 34.5, and IL-29-treated mice (n = 8) had an average relative liver fluorescence of 38.9. A decrease in adenovirus infection and gene expression leads to a decrease in liver pathology, which is determined by serum AST and ALT levels and histology. In PBS-treated mice (n = 8), the mean serum AST was 234 u / L and the serum ALT was 250 u / L. In contrast, in IL-28 treated mice (n = 8), the mean serum AST was 193 units / L and the serum ALT was 216 units / L; in IL-29-treated mice (n = 8), the average value for serum AST was 162 units / L, and the value for serum ALT was 184 units / L. In addition, histology of the liver shows that mice injected with murine IL-28 or IL-29 have lower rates of liver pathology and inflammation than the PBS-treated group. In liver samples from the IL-29 group, there is also less proliferation of sinusoidal cells, fewer mitosis figures and less changes in hepatocytes (in particular, vacuolization, the presence of several nuclei, an increase in the size of hepatocytes) than in the PBS-treated group. These data indicate that murine IL-28 or IL-29 possess antiviral properties against trophic liver viruses.

Example 40

LCMV Models

Infections of the lymphocytic choriomeningitis virus virus (LCMV) in mice are an excellent model of acute and chronic infection. These models are used to assess the effect of cytokines on the antiviral immune response and the effect that IL-28 or IL-29 have on the viral load and antiviral immune response. The two models used are: LCMV Armstrong infection (acute) and clone 13 infection LCMV. (See, in particular, Wherry et al., J. Virol. 77: 4911-4927, 2003; Blattman et al., Nature Med. 9 (5): 540-547, 2003; Hoffman et al., J. Immunol 170: 1339-1353, 2003.) Three stages of development of CD8 T cells in response to the virus are observed: 1) reproduction, 2) contraction, and 3) memory (acute model). Injections of IL-28 or IL-29 are administered at each stage of both the acute and chronic models. In a chronic infection model, IL-28 or IL-29 is administered 60 days after infection to analyze the effect of IL-28 or IL-29 on persistent viral load. IL-28 or IL-29 is administered in both acute and chronic models and the viral load in the blood, spleen and liver is examined. Other parameters that can be studied include: staining the tetramer in a stream to calculate the number of LCMV-specific CD8 + T cells; the ability of tetramer + cells to produce cytokines when activated by their related LCMV antigen; and the ability of LCMV-specific CD8 + T cells to proliferate in response to their sister LCMV antigen. The phenotype of LCMV-specific T cells is determined by flow cytometry in order to analyze cell activation and differentiation status. In addition, the ability of LCMV-specific CTLs to lyse target cells carrying their related LCMV antigen is studied. Also analyze the number and function of LCMV-specific CD4 + T cells.

Determine the reduction in viral load after treatment with IL-28 or IL-29. Significant is a 50% reduction in viral load in any organ, in particular, the liver. For mice treated with IL-28 or IL-29, a 20% increase in the percentage of tetrameric positive T cells that proliferate, give cytokines or exhibit a mature phenotype is also considered significant, compared with untreated mice.

Injections of IL-28 or IL-29, which lead to a decrease in viral load, are the result of more effective control of viral infection, especially in the chronic model, where, in the absence of drug treatment, viral titers remain elevated for a long period of time. Significant is a two-fold reduction in viral titer compared to untreated mice.

Example 41

Influenza model for acute viral infection

A. Preliminary experiments to study antiviral activity

In order to determine the antiviral activity of IL-28 or IL-29 against acute influenza virus infection, an in vivo study of influenza-infected c57B1 / 6 mice is carried out according to the following protocol:

Animals: 148 female BALB / c mice (Charles River) at 6 weeks of age, 30 per group.

Groups:

(1) Absolute control group (non-infected animals) for parallel study of antibody titer and histopathology (2 animals in the group)

(2) Carrier (intraperitoneal) physiological control

(3) Amantadine (positive control) 10 mg / day for 5 days (by mouth), administered 2 hours before infection

(4) Animals treated with IL-28 or IL-29 (5 μg, intraperitoneally, administered 2 hours after infection)

(5) Animals treated with IL-28 or IL-29 (25 μg, intraperitoneally, administered 2 hours after infection)

(6) Animals treated with IL-28 or IL-29 (125 μg, intraperitoneally, administered 2 hours after infection)

Day 0 - Except for absolute controls, all animals are infected with the influenza virus

For viral load (10 for LD50)

For immunological training (LD30)

Day 0-9 - Daily Injection of IL-28 or IL-29 (Intraperitoneally)

Body weight and general condition are recorded (3 times a week)

Day 3 - Kill 8 animals in each group.

Viral load in the right lung (TCID50)

Histopathology of the left lung

Blood Samples for Antibody Titration

Day 10 - All surviving animals are killed, blood samples are taken for antibody titration, isolation of pulmonary lymphocytes (4 pools out of 3) for direct CTL analysis (in all 5 groups) and quantification of the immune phenotype for the following markers: CD3 / CD4, CD3 / CD8, CD3 / CD8 / CD11b, CD8 / CD44 / CD62L, CD3 / DX5, GR-1 / F480 and CD19.

Study No. 2

A study of the efficacy of IL-28 or IL-29 in C57B1 / 6 mice infected with a mouse-adapted virus is carried out on 8 week old female C57B1 / 6 (Charles River) mice.

Group 1: Carrier (intraperitoneal)

Group 2: Positive control: neutralizing anti-flu antibody (A / USSR (H1N1) goat anti-flu) (Chemicon International, Temecula, California); 40 μg / mouse 2 and 4 hours after infection (10 μl intranasally)

Group 3: IL-28 or IL-29 (5 mcg, ip)

Group 4: IL-28 or IL-29 (25 mcg, ip)

Group 5: IL-28 or IL-29 (125 mcg, ip)

After observations during life and immunological training receive:

Day 0 - All animals are infected with influenza virus (dose determined in experiment 2)

Day 0-9 - Daily Injection of IL-28 or IL-29 (Intraperitoneally)

Every two days, body weight and general condition are recorded.

Day 10 - Kill the surviving animals and conduct a virus analysis to determine the viral load in the lung.

Isolation of pulmonary lymphocytes (for direct CTL analysis in the lungs using EL-4 as targets at different E: T ratios (based on the best results from experiments 1 and 2).

Tetramer staining: When using MHC class I complexes with viral peptides: FLU-NP _366-374 / D ^b (ASNENMETM), (LMCV peptide / D ^b ), the number of CD8 + T cells that bind MHC class I tetramers that contain a nucleoprotein epitope is examined (NP) influenza A.

Quantification of the immunophenotype for: CD8, tetramer, intracellular IFNγ, NK1.1, CD8, tetramer, CD62L, CD44, CD3 (+ or -), NK1.1 (+), intracellular IFNγ, CD4, CD8, NK1.1, DX5 , CD3 (+ or -), NK1.1, DX5, tetramer, single color samples for cytometer calibration.

Survival / Investigation with the re-introduction of a provocative test

Day 30: Survival studies in mice were performed for 9 days with different doses of IL-28 or IL-29 or with a positive control of anti-flu antibody. Determines body weight and production of antibodies against influenza in individual serum samples (total, IgG1, IgG2a, IgG2b).

Repeated challenge test

Day 0: Both groups are infected with A / PR virus (1LD30)

Group 6 not treated

Group 7 is treated for 9 days with a dose of 125 μg of IL-28 or IL-29.

Day 30: Survival study.

Body weight and production are measured in individual serum samples (total, IgG1, IgG2a, IgG2b).

Day 60: Conduct a re-challenge study.

Surviving animals in each group are divided into 2 subgroups.

In group 6A and 7A, a repeat provocative assay of the A / PR virus (1 LD30) was administered.

In group 6B and 7B, a repeat provocative assay of the A / PR virus (1 LD30) is administered.

Observe both groups and determine the day when the animals are killed. Body weight is measured and antibody production is determined in individual serum samples (total, IgG1, IgG2a, IgG2b).

Example 42

IL-28 and IL-29 have in vivo antiviral activity against hepatitis B virus (HBV)

The transgenic mouse model (Guidotti et al., J. Virology 69: 6158-6169, 1995) supports the replication of high levels of HBV infectious and is used as a chemotherapeutic model for HBV infection. Transgenic mice are treated with antiviral drugs and after the experiment, the levels of DNA and HBV RNA in the liver and serum of transgenic mice are determined. After the experiment, HBV protein levels in transgenic mice can also be determined. This model was used to evaluate the efficacy of lamivudine and TNF-α for lowering HBV viral titers.

Male HBV TG mice are intraperitoneally injected daily for 14 days with 2.5, 25 or 250 μg IL-28 or IL-29 (a total of 8 doses). Mice were bled for analysis of serum on the day treatment is started (day 0) and on day 7. One hour after the last blood sample was taken for analysis of IL-29, the animals were euthanized. Serum and liver are analyzed for HBV DNA in the liver, HBV RNA in the liver, HBV DNA in serum, HBc in the liver, HBe in serum and HBs in serum.

The decrease in the amount of HBV DNA in the liver, HBV RNA in the liver, HBV DNA in serum, HBc in the liver, HBe in serum and HBs in serum in response to IL-28 or IL-29 reflects the antiviral activity of these compounds against HBV.

Example 43

IL-28 and IL-29 inhibit the replication of human herpes-8 virus (HHV-8) in BCBL-1 cells

The antiviral activity of IL-28 and IL-29 against HHV-8 was examined in an in vitro infection system using a B-lymphoid cell line, BCBL-1.

In the HHV-8 assay, the test compound and ganciclovir, which is used as a control, are each tested in five concentrations, obtained as a series of semilogarithmic dilutions. The points of closure are TaqMan PCR results for extracellular HHV-8 DNA (IC50) and cell numbers using a reagent CellTiter96 ^® (T50; Promega, Madison, WI). Briefly, BCBL-1 cells are plated on 96-well microtiter plates. After 16-24 hours, the cells are washed and the medium is replaced with a complete nutrient medium containing various concentrations of the test compounds; conduct three series of experiments. Ganciclovir is used as a positive control, and the culture medium itself serves as a negative control (viral control, VC). Three days later, the culture medium is replaced with fresh culture medium containing an appropriately diluted test compound. Six days after the initial administration of the test compound, the culture supernatant was collected, treated with pronase and DNase, and then used for real-time TaqMan PCR quantification. PCR-amplified HHV-8 DNA is detected in real time by monitoring amplification of fluorescence signals resulting from exonucleolytic degradation of the captured fluorescent probe molecule, which hybridizes with the amplified HHV-8 DNA. For each PCR amplification, a standard curve is constructed simultaneously using dilutions of purified HHV-8 DNA. Antiviral activity is calculated by decreasing HHV-8 DNA levels (IC ₅₀ ). After that, another dye capture method is used to assess cell viability, which is used to calculate cytotoxicity (TC ₅₀ ). The therapeutic indicator (TI) is calculated as the ratio of TC ₅₀ / IC ₅₀ .

IL-28 and IL-29 inhibit HHV-8 virus replication in BCBL-1 cells. The IC ₅₀ value for IL-28A is 1 μg / ml, and the TC ₅₀ value is> 10 μg / ml (TI> 10). The IC ₅₀ value for IL-29 is 6.5 μg / ml, and the TC ₅₀ value is> 10 μg / ml (TI> 1.85). The IC ₅₀ value for MetIL-29C172S-PEG is 0.14 μg / ml, and the TC ₅₀ value is> 10 μg / ml (TI> 100).

Example 44

IL-28 and IL-29 have antiviral activity against Epstein-Barr virus (EBV)

The antiviral activity of IL-28 or IL-29 against EBV was investigated in an in vitro infection system using a B-lymphoid cell line, P3HR-1. In an EBV assay, the test and control compounds are each examined in five concentrations, obtained as a series of semi-log dilutions. The points of closure are TaqMan PCR for extracellular DNA EBV (IC50) and cell numbers using a reagent CellTiter96 ^® (T50; Promega). Briefly, P3HR-1 cells are plated on 96-well microtiter plates. After 16-24 hours, the cells are washed and the medium is replaced with a complete nutrient medium containing various concentrations of the test compounds; conduct three series of experiments. In addition to the positive control, the nutrient medium itself is added to the cells as a negative control (viral control, VC). Three days later, the culture medium is replaced with fresh culture medium containing an appropriately diluted test compound. Six days after the initial administration of the test compound, the culture supernatant was collected, treated with pronase and DNase, and then used for real-time TaqMan PCR quantification. PCR-amplified EBV DNA is detected in real time by monitoring the amplification of fluorescence signals resulting from exonucleolytic degradation of the captured fluorescent probe molecule, which hybridizes with the amplified EBV DNA. For each PCR amplification, a standard curve is constructed simultaneously using dilutions of purified EBV DNA. Antiviral activity is calculated by decreasing EBV DNA levels (IC ₅₀ ). After that, another dye capture method is used to assess cell viability, which is used to calculate cytotoxicity (TC ₅₀ ). The therapeutic indicator (TI) is calculated as the ratio of TC ₅₀ / IC ₅₀ .

Example 45

Antiviral activity of IL-28 and IL-29 against herpes simplex virus-2 (HSV-2)

The antiviral activity of IL-28 and IL-29 against HSV-2 was examined in an in vitro infection system in Vero cells. The antiviral effects of IL-28 and IL-29 are investigated by cytotoxic effect inhibition assay (CPE). This analysis includes the death of Vero cells under the action of the cytopathic virus HSV-2 and inhibition of cell death under the action of IL-28 and IL-29. Vero cells are propagated in Dulbecco's modified basic medium (DMEM) containing phenol red together with 10% horse serum, 1% glutamine and 1% penicillin-streptomycin, and CPE inhibition assays are performed in DMEM without phenol red in the presence of 2% FBS. 1% glutamine and 1% penicillin-streptomycin. The day before the analysis, the cells are trypsinized (1% trypsin-EDTA), washed, counted and placed at 10 ⁴ cells per well in 96-well BioCoat ^® plates (Fisher Scientific, Pittsburgh, PA) with a flat bottom in a volume of 100 μl per hole. The next morning, the medium is removed and pre-titrated aliquots of the virus are added to the cells. The amount of virus used corresponds to the maximum dilution, which leads to complete cell death (> 80%) by the time of maximum development of CPE. Cell viability is determined using CellTiter96 ^® reagent (Promega), in accordance with the manufacturer's recommendations, and a Vmax plate reader (Molecular Devices, Sunnyvale, California). Each test sample was tested in six concentrations obtained by hemogarithmic dilutions using an assay medium. Acyclovir is used as a positive control. The compounds are administered together with a viral infection. The average background and the data corrected for staining the sample to determine the percentage reduction in CPE and the percentage of cell viability for each concentration are determined in comparison with the controls and the IC ₅₀ value relative to TC _{50 is} calculated.

In this assay, IL-28, IL-29, and MetIL-29C172S-PEG did not inhibit cell death (IC ₅₀ > 10 μg / ml). IFNα also does not show antiviral activity in this assay.

A full description of patents, patent applications and publications, as well as freely available electronic materials (for example, depositors of amino acid and nucleotide sequences in the GenBank database) provided herein are incorporated by reference. The above detailed description and examples are provided only to facilitate understanding of the invention. They do not limit the present invention in any way. The invention is not limited to the specific details given and described, since the variations obvious to those skilled in the art, which are defined in the claims below, should be included.

Claims (3)

1. The use of the polypeptide characterized by the amino acid sequence of SEQ ID NO: 159, with a polyethylene glycol group at the N-terminus, in the manufacture of a medicament for the treatment of hepatitis C. 2. The use according to claim 1, where the polyethylene glycol group is mPEG / propionic aldehyde. 3. The use according to claim 1, where the polyethylene glycol group has a molecular weight of approximately 20 kDa.

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