IL174797A

Homogeneous preparation of il-29 polypeptide and method for producing such

Abstract

This record has no abstract on file.

Term

No projected expiry on record.

24 claims: 8 independent, 16 dependent

1
5 ^CLAIMS:I 1. An isolated polypeptide consisting of an amino acid sequence having at least 98% sequence identity to amino acid residues 1-176 of SEQ ID NO: 159;wherein the polypeptide has anti-viral activity.
3
The polypeptide of either of claims 1 or 2 wherein the polypeptide has antihepatitis activity.
6
The polypeptide of any of claims 1-5 wherein the polypeptide further comprises a polyethylene glycol moiety covalently attached N־terminally to the polpeptide.
10
An isolated polynucleotide encoding the polypeptide of any of claims 1-3.
13
An expression vector comprising the following operably linked elements:י a transcription promoter;25 : a DNA segment encoding a polypeptide of any of claims 1-5;and a transcription terminator.
15
A method of producing a polypeptide comprising:30 culturing a cell into which has been introduced an expression vector of claim 13, wherein the cell expresses the polypeptide encoded by the DNA segment;and 01664457\47-01 recovering the expressed polypeptide.
16
A fusion protein comprising a polypeptide according to any of claims 1-5;and a polyalkyl oxide moiety.
22
An isolated polynucleotide encoding a fusion protein comprising a first portion and a second portion joined by a covalent bond, wherein the first portion comprises a polypeptide according to any of claims 1-5;and wherein the second portion comprises a polyethylene glycol.
23
A formulation comprising:an isolated polypeptide according to any of claims 15;and a pharmaceutically acceptable vehicle.

Independent claims9

4,322 paragraphs in 31 sections, as filed

BACKGROUND OF THE INVENTION

Cytokines play important roles in the regulation of hematopoiesis and immune responses, and can influence lymphocyte development. The human class II cytokine family includes interferon־a (IFN-a) subtypes, interferon-β (IFN- β), interferon-γ (IFN-γ), IL-10, IL-19 (U.S. Patent 5, 985,614), MDA-7 (Jiang et al, 10 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 transduce signals through either Class I or Class II cytokine receptors. Members of human class II cytokine receptor family include interferon-aRl (IFN-aRl), interferon-y-R2 (IFN-γ15 R2), interferon-γ R1 (IFN-γ Rl), interferon-yR2 (IFN-yR2), 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 (U.S. Patent 5,945,511) and CRF2-8 (Kotenko et al. Oncogene 19, 2557-2565, (2000)), IL 20Κβ (Blumberg et al, ibid, (2001)) (also known as DIRSI (PCT WO 99/46379)), IL 20 22RA1 (IL-22 receptor-al, submitted to HUGO for approval) (also known as IL-22R (Xie et al, J. Biol. Chem. 275, 31335-31339, (2000)), zcytorll (U.S. Patent 5,965,704) and CRF2-9 (Kotenko et al. Oncogene 19, 2557- 2565, (2000)), and tissue factor.

Class II cytokine receptors are typically heterodimers composed of two distinct receptor chains, the a and β receptor subunits (Stahl et al. Cell 74, 587-590, 25 (1993)). In general, the a subunits are the primary cytokine binding proteins, and the β subunits are required for formation of high affinity binding sites, as well as for signal transduction. An exception is the IL-20 receptor in which both subunits are required for IL-20 binding (Blumberg et al, ibid, (2001)).

The class II cytokine receptors are identified by a conserved cytokine30 binding domain of about 200 amino acids (D200) in the extracellular portion of the receptor. This cytokine-binding domain is comprised of two fibronectin type III (Fnlll) domains of approximately 100 amino acids each (Bazan J.F. Proc. Natl. Acad. Set USA 87,6934-6938, (1990); Thoreau et al., 282,16-31, (1991)). Each FnlH domain contains conserved Cys, Pro, and Trp residues that determine a characteristic folding pattern of seven β-strands similar to the constant domain of immunoglobulins (Uze et al., J. Interferon Cytokine Res. 15, 3-26, (1995)). The conserved structural elements of the class Π cytokine receptor family make it possible to identify new members of this family on the basis of primary amino acid sequence homology.

The interleukins are a family of cytokines that mediate immunological responses, including inflammation. Central to an immune response is the T cell, which 10 produce many cytokines and adaptive immunity to antigens. Cytokines produced by the T cell have been classified as type 1 and type 2 (Kelso, A. Tmmun. Cell B1.0L 76:300317, 1998). Type 1 cytokines include IL-2, interferon-gamma (IFN-γ), LT-a, and are involved in inflammatory responses, viral immunity, intracellular parasite immunity and allograft rejection. Type 2 cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13, and 15 are involved in humoral responses, helminth immunity and allergic response. Shared cytokines between Type 1 and 2 include IL-3, GM-CSF and TNF-a. There is- some evidence to suggest that Type 1 and Type 2 producing T cell populations preferentially migrate into different types of inflamed tissue.

Of particular interest, from a therapeutic standpoint, are the interferons 20 (reviews on interferons am provided by De Maeyer and De Maeyer-Guignard, “Interferons,” in The Cytokine Handbook, 3rd Edition, Thompson (ed.), pages 491-516 (Academic Press Ltd. 1998), and by Walsh, Biopharmaceuticale: Biochemistty and Biotechnology, pages 158-188 (John Wiley & Sons 1998». Interferons exhibit a variety of biological activities, and are useful for the treatment of certain autoimmune diseases, 25 particular cancers, and the enhancement of the immune response against infectious agents, including viruses, bacteria, fungi, and protozoa. To date, six forms of interferon have been identified, which have been classified into two major groups. The so-called “type Γ IFNs include IFN-a, ΚΝ-β. IPN־®, IFN-δ, and interferon-τ. Currently, IFN-γ and one subclass of IFN-α are the only type H IFNs.

30 Type i IFNs, which are thought to be derived from the same ancestral gene, have retained sufficient similar structure to act by the same cell surface receptor.

The a-chain of the human IFN-α/β receptor comprises an extracellular N-terminal domain, which has the characteristics of a class II cytokine receptor. IFN-γ does not share significant homology with the type IIFN or with the type II IFN-a subtype, but shares a number of biological activities with the type I IFN.

Clinicians are taking advantage of the multiple activities of interferons by using the proteins to treat a wide range of conditions. For example, one form of IFN-a has been approved for use in more than 50 countries for the treatment of medical conditions such as hairy cell leukemia, renal cell carcinoma, basal cell carcinoma, malignant melanoma, AIDS- related Kaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia, non-Hodgkin's lymphoma, laryngeal papillomatosis, mycosis fungoides, condyloma acuminate, chronic hepatitis B. hepatitis C, chronic hepatitis D, and chronic non-A, non-B/C hepatitis. The U.S. Food and Drug Administration has approved the use of IFN-β to treat multiple sclerosis, a chronic disease of the nervous system. IFN-γ is used to treat chronic granulomatous diseases, in which the interferon enhances the patient's immune response to destroy infectious bacterial, fungal, and protozoal pathogens. Clinical studies also indicate that IFN-γ may be useful in the treatment of AIDS, leishmaniasis, and lepromatous leprosy.

IL-28A, IL-28B, and IL-29 comprise a recently discovered new family of proteins that have sequence homology to type I interferons and genomic homology to IL-10. This new family is fully described in co־owned PCT application WO 02/086087 and Sheppard et al. Nature Immunol. 4:63-68, 2003; both incorporated by reference herein. Functionally, IL-28 and IL- 29 resemble type I INFs in their ability to induce an antiviral state in cells but, unlike type I IFNs, they do not display 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 present application teaches an IL-29 polypeptide having enhanced activity relative to the IL-29 polypeptide of WO 02/086087 and having amino acid differences relative to the IL-29 polypeptide sequence of WO 02/086087, i.e., these differences include an additional Methionine at the amino terminus, a deletion of amino acid residues 2-7, an Aspartic to Asparagine mutation at position 164 of SEQ ID NO: 159, and a Cysteine to Serine, Alanine, Threonine, Valine or Asparagine mutation at the fifth Cysteine from the N-terminus.

Expression of recombinant IL 28 and IL-29 can result in a heterogeneous mixture of proteins composed of intramolecular disulfide bonding in multiple conformations. The separation of these forms can be difficult and laborious. It is therefore desirable to provide IL-28 and IL-29 molecules having a single intramolecular disulfide bonding pattern upon expression and methods for refolding and purifying these preparations to maintain homogeneity. Thus, the present invention provides for compositions and methods to produce homogeneous preparations of IL-29.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

In the description that follows, a number of terms are used extensively. Hie following definitions are provided to facilitate understanding of the invention.

Unless otherwise specified, a, an, the, and at least one are used interchangeably and mean one or more than one.

The term affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly histidine tract, 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 (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 4, 1985), substance P. Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107, 1991. DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ).

The term allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or a 174797/1 may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.

The terms amino-terminal and carboxyl-terminal are used herein to denote positions within polypeptides. Where the context allows, these terms are used

PCT7US2004/025864 with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.

The term “complement/anti-complement pair” denotes non-identical moieties that form a non-covalentiy associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair. Other exemplary complement/anti-complement 10 pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the Eke. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of <109 M1־.

The term “degenerate nucleotide sequence” denotes a sequence of 15 nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term expression vector is used to denote a DNA molecule, Enear 20 or circular, that comprises a segment encoding a polypeptide of interest operably Enked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of repEcation, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may 25 contain elements of both.

The term “isolated, when appEed to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within geneticaUy engineered protein production systems. Such isolated molecules are those 30 that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3’ untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan. Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. 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 alternatively glycosylated or derivatized forms.

The term “level” when referring to immune cells, such as NK: cells, T cells, in particular cytotoxic T cells, B cells and the like, an increased level is either increased number of cells or enhanced activity of cell function.

The term “level” when referring to viral infections refers to a change m the level of viral infection and includes, but is not limited to, a change in the level of CTLs or NK cells (as described above), a decrease in viral load, an increase antiviral antibody titer, decrease in serological levels of alanine aminotransferase, or 20 improvement as determined by histological examination of a target tissue or organ.

Determination of whether these changes in level are significant differences or changes is well within the skill of one in the art.

The term “operably linked, when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended 25 purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from one species that is die functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.

PCT7US2004/025864 “Paralogs” are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example. » globin, β-globin, and myoglobin are paralogs of each other.

A polynucleotide is a single- or double-stranded polymer of 5 deoxyribonucleotide or ribonucleotide bases read from the 5 to the 3 end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated “bp׳.), nucleotides (“nt), or kilobases (“kb). Where the context allows, the latter two terms may describe 10 polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term “base pairs. It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all 15 nucleotides within a double-stranded polynucleotide molecule may not be paired.

A polypeptide is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as peptides.

The term promoter is used herein for its art-recognized meaning to 20 denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5’ non-coding regions of genes.

A protein is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate 25 groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their, amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

30 The term receptor denotes a cell-associated protein that binds to a bioactive molecule (i.e, a ligand) and mediates the effect of the ligand on the cell.

Membrane-bound receptors are characterized by a multi-peptide structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector 5 domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. In 10 general, receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).

The term secretory signal sequence denotes a DNA sequence that 15 encodes a polypeptide (a. secretory peptide) that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell m which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.

The , term “splice variant” is used herein to denote alternative forms of 20 RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a 25 protein encoded by a splice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as “about” X or “approximately” X, the stated value of X will be understood to be accurate to ±10%.

30 “zcyto20”, “zcyt021”, “zcyt022” are the previous designations for human IL-28A, human IL-29, and human IL-28B, respectively. The nucleotide and /2 amino acid sequence for IL-28A are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The nucleotide and amino acid sequences for IL-29 are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively. The nucleotide and amino acid sequence for IL28B are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively. These sequences are fully described in PCT application WO 02/086087 commonly assigned to ZymoGenetics, Inc., incorporated herein by reference.

zcyt024 and zcyt025 are the previous designations for mouse IL- 28, and are shown in SEQ ID NOs: 7, 8, 9, 10, respectively. The polynucleotide and polypeptides are fully described in PCT application WO 02/086087 commonly assigned 10 to ZymoGenetics, Inc., incorporated herein by reference.

zcytorl9 is the previous designation for IL-28 receptor a- subunit, and is shown in SEQ ID NO:11. The polynucleotides and polypeptides are described in PCT application WO 02/20569 on behalf of Schering, Inc., and WO 02/44209 assigned to ZymoGenetics, Inc and incorporated herein by reference. IL-28 receptor denotes the IL-28 a-subunit and CRF2-4 subunit forming a heterodimeric receptor.

The present invention provides polynucleotide molecules, including DNA and RNA molecules, that encode Cysteine mutants of IL-29 that result in expression of a recombinant IL-29 preparation that is a homogeneous preparation. For the purposes of this invention, a homogeneous preparation of IL-29 is a preparation in which comprises at least 98% of a single intramolecular disulfide bonding pattern in the purified polypeptide. In other embodiments, the single disulfide conformation in a preparation of purified polypeptide is at 99% homogeneous. In general, these Cysteine mutants will maintain some biological activity of the wildtype IL-29, as described herein. For example, the molecules of the present invention can bind to the IL-29 receptor with some specificity. Generally, a ligand binding to its cognate receptor is specific when the Kd falls within the range of 100 nM to 100 pM. Specific binding in the range of 100 mM to 10 nM Kd is low affinity: binding. Specific binding in the range of 2.5 pM to! 100 pM Kd is high affinity binding. In another example, biological activity of IL-29 Cysteine mutants is present when the molecules are capable of some level of antiviral activity associated with Wildtype IL-29. Determination of the level of antiviral activity is described in detail herein.

When referring to IL-28, the term shall mean both IL-28A and IL-28B. Previously IL-28A was designated zcyto20 (SEQ ID NOs: 1 and 2), IL-29 was designated zcyt021 (SEQID NOs:3 and 4), and IL-28B was designated zcyt022 (SEQ ID NOs:5 and 6). (See, PCT application WO 02/086087 and Sheppard et al., supra.) The mouse orthologs for IL-28 were previously designated as zcyt024 (SEQID NOs:7 and 8), zcyt025 (SEQID NOs:9 and 10).

Wildtype IL-28A gene encodes a polypeptide of 200 amino acids, as shown in SEQ ID- NO:2. The signal sequence for IL-28A can be predicted as comprising amino acid residue -25 (Met) through amino acid residue -1 (Ala) of SEQ ID NO:2. The mature peptide for IL-28A begins at amino acid residue 1 (Vai) of SEQ ID NO.2־. IL-28A helices are predicted as follow: helix A is defined by amino acid residues 31 (Ala) to 45 (Leu); helix B by amino acid residues 58 (Tier) to 65 (Gin); helix C by amino acid residues 69 (Arg) to 86 (Ala); helix D by amino acid residues 95 (Vai) to 114 (Ala); helix E by amino acid residues 126 (Tier) to 142 (Lys); and helix F by amino acid residues 148 (Cys) to 169 (Ala); as shown in SEQID NO: 2.

Wildtype IL-29 gene encodes a polypeptide of 200 amino acids, as shown in SEQ ID NO:4. The signal sequence for IL-29 can be predicted as comprising amino acid residue -19 (Met) through amino acid residue -1 (Ala) of SEQ ID NO:4, SEQ ID NO:119, or SEQID NO:121. The mature peptide for IL-29 begins at amino acid residue 1 (Gly) of SEQ ID NO:4. IL-29 has been described in PCT application WO 02/02627. IL-29 helices are predicted as follows: helix A is defined by amino acid residues 30 (Ser) to 44 (Leu); helix B by amino acid residues 57 (Asn) to 65 (Vai); helix C by amino acid residues 70 (Vai) to 85 (Ala); helix D by amino acid residues 92 (Glu) to 111 (Gin); helix E by amino acid residues 118 (Tier) to 139 (Lys); and helix F by amino acid residues 144 (Gly) to 170 (Leu); as shown in SEQID NO:4.

Wildtype IL-28B gene encodes a polypeptide of 200 amino acids, as shown in SEQ ID NO:6. The signal sequence for IL-28B can be predicted as comprising amino acid residue -21 (Met) through amino acid residue -1 (Ala) of SEQ ID NO:6. The mature peptide for IL-28B begins at amino acid residue 1 (Vai) of SEQ ID NO:6. IL-28B helices are predicted as follow: helix A is defined by amino acid residues 31 (Ala) to 45 (Leu); helix B by amino acid residues 58 (Tier) to 65 (Gin); helix C by amino acid residues 69 (Arg) to 86 (Ala); helix D by amino acid residues 95

174797/2 (Gly) to 114 (Ala); helix E by amino acid residues 126 (Tier) to 142 (Lys); and helix F is by amino acid residues 148 (Cys) to 169 (Ala); as shown in SEQ D) NO:6.

The present invention provides mutations in the IL-29 wildtype sequences that result in expression of single forms of the L-29 molecule. Because the 5 heterogeneity of forms is believed to be a result of multiple intramolecular disulfide bonding patterns, 10 specific embodiments of the present invention includes mutations to the cysteine residues within the wildtype IL-29 sequences. When IL-28 and IL- 29 are expressed in E. coli, an N-terminal or amino-terminal Methionine is present. SEQ ID NOs: 12-17, for example, show the nucleotide and amino acid residue numbering for 10 IL 28A, IL-29 and IL-28B when the N-terminal Met is present. Table 1 shows the possible combinations of intramolecular disulfide bonded cysteine pairs for wildtype IL-28A, IL-; 128B, and IL- 29.

Table

The polynucleotide and polypeptide molecules of the present invention have a mutation at one or more of the Cysteines present in the wildtype IL-29 molecule, yet retain some biological activity as described herein.

All the members of the family have been shown to bind to the same class II cytokine receptor, IL-28R. IL-28 a-subunit was previously designated zcytorl9 receptor. While not wanting to be bound by theory, these molecules appear to all signal through IL-28R receptor via the same pathway. IL-28 receptor is described in a commonly assigned PCT patent application WO 02/44209, incorporated by reference herein; Sheppard et al., supra; Kotenko et al., Nature Immunol. 4:69-77, 2003; and PCT WO/03/040345. IL-28R is a member of the Class II cytokine receptors which is characterized by the presence of one or more cytokine receptor modules (CRM) in their extracellular domains. Other class It cytokine receptors include zcytorll (commonly owned US Patent No. 5,965, 704), CRF2-4 (Genbank Accession No. Z17227), IL-10R (Genbank Accession No.s U00672 and NM_001558), DIRSI, zcytor7 (commonly owned US Patent No. 5, 945,511), and tissue factor. IL-28 receptor, like all known class II receptors except interferon-alpha/beta receptor alpha chain, has only a single class II CRM in its extracellular domain.

Four-helical bundle cytokines are also grouped by the length of their component helices. Long-helix form cytokines generally consist of between 24-30 residue helices, and include IL-6, ciliary neutrotrophic factor (CNTF), leukemia inhibitory factor (LIF) and human growth hormone (hGH). Short-helix form cytokines generally consist of between 1821־ residue helices and include IL-2, IL-4 and GM-CSF. Studies using CNTF and IL-6 demonstrated that a CNTF helix can be exchanged for the equivalent helix in IL-6, conferring CTNF-binding properties to the chimera. Thus, it appears that functional domains of four-helical cytokines are determined on the basis of structural homology, irrespective of sequence identity, and can maintain functional integrity in a chimera (Kallen et al., J. Biol. Chem. 274:11859 11867, 1999). Therefore, Cysteine mutants IL-28 and IL-29 polypeptides will be useful for preparing chimeric fusion molecules, particularly with other interferons to determine and modulate receptor binding specificity. Of particular interest are fusion proteins that combine helical and loop domains from interferons and cytokines such as INF-a, IL-10, human growth hormone.

The present invention provides polynucleotide molecules, including DNA and RNA molecules, that encode, Cysteine mutant IL-29 polypeptides. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic /2 code, considerable sequence variation is possible among these polynucleotide molecules. SEQ ID NOs:30, 31, 32, 33, 34, and 35 are a degenerate DNA sequences that encompasses all DNAs that encode IL-28A C48S, Met IL-28A C49S, IL-28A C50S, Met IL-28 A C51S, IL-29 Cl 71S and Met IL-29 C172S, respectively. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NOs: 30, 31,

32, 33, 34, and 35 also provides all RNA sequences encoding SEQ ID NOs: 30, 31, 32,

33, 34, and by substituting U for T.

IL-28A polypeptides also include a mutation at the second cysteine, C2, of the mature polypeptide. For example, C2 from the N-terminus or amino-terminus of the polypeptide of SEQ ID NO:2 is the cysteine at amino acid position 48, or position 49 (additional N-terminal Met) if expressed in E coli (see, for example, SEQ ID NO: 13). This second cysteine (of which there are seven, like IL-28B) or C2 of IL-28A can be mutated, for example, to a serine, alanine, threonine, valine, or asparagine. IL28A C2 mutant molecules of the present invention include, for example, polynucleotide molecules as shown in SEQ ID NOs:20 and 22, including DNA and RNA molecules, that encode IL-28A C2 mutant polypeptides as shown in SEQID NOs:21 and 23, respectively. SEQID NOs:36 and 37 are additional IL-28A C2 polypeptides of the present invention.

In addition to the IL-28A C2 mutants, also described are IL-28A polypeptides comprising a mutation at the third cysteine position, C3, of the mature polypeptide. For !example, C3 from the N-terminus or amino-terminus of the polypeptide of SEQ ID NO:2, is the cysteine at position 50, or position 51 (additional i'

N-terminal Met) if expressed in E. coli (see, for example, SEQ ID NO: 13). IL-28A C3 mutant molecules include, for example, polynucleotide molecules as shown in SEQ ID NOs:24 and 26, including DNA and RNA molecules, that encode IL-28A C3 mutant polypeptides as shown in SEQ ID NOs:25 and 27, respectively. SEQ ID NOs:38 and 39 are additional IL-28A C3 polypeptides.

The L-28A polypeptides include, for example, SEQ ID N0s:2, 13, 19, 21, 23, and 25, which are encoded by IL- 28A polynucleotide molecules as shown in SEQ ID NOs:l, 12, 18, 20, 22, and 24, respectively. Further L 28A polypeptides include, for example, SEQID NOs:36, 37, 38,and39.

IL-28B polypeptides also include a mutation at the second cysteine, C2, of the mature polypeptide. For example, C2 from the N-terminus or amino-terminus of the polypeptide of SEQID NO:6 is the cysteine at amino acid position 48, or position 49 (additional N־terminal Met) if expressed in E coli (see, for example, SEQID NO: 17). This second cysteine (of which there are seven, like IL 28A) or C2 of IL-28B can be mutated, for example, to a serine, alanine, threonine, valine, or asparagine. IL-28B C2 mutant molecules include, for example, polynucleotide molecules as shown in SEQ ID NOs:122 and 124, including DNA and RNA molecules, that encode IL-28B C2 mutant polypeptides as shown in SEQID NOs:123 and 125, respectively. Additional IL-28B C2 mutant molecules include polynucleotide molecules as shown in SEQ ID NOs:130 and

132 including DNA and RNA molecules, that encode IL-28B C2 mutant polypeptides as shown in SEQID NOs:131 and 133, respectively (PCT publication WO 03/066002 (Kotenko et al.)).

In addition to the IL-28B C2 mutants, also described are IL-28B polypeptides comprising a mutation at the third cysteine position, C3, of the s mature polypeptide. For example, C3 from the N-terminus or amino-terminus of the polypeptide of SEQ ID NO:6, is the cysteine at position 50, or position 51 (additional 5 N-terminal Met) if expressed in E. coli (see, for example, SEQID NO: 17). IL-28B C3 mutant molecules include, for example, polynucleotide molecules as shown in SEQ ID NOs:126 and 128, including DNA and RNA molecules, that encode IL-28B C3 mutant polypeptides as shown in SEQ ID NOs:127 and 129, respectively. Additional IL-28B C3 mutant molecules include polynucleotide molecules as shown in SEQ ID NOs:134 and 136 including DNA and RNA molecules, that encode IL-28B C3 mutant polypeptides as shown in SEQ ID NOs: 135 and 137, respectively (PCT publication WO 03/066002 (Kotenko et al.)).

The IL-28B polypeptides include, for example, SEQ ID NOs:6, 17, 123, 125, 127, 129, 131, 133, 135, and 137, which are encoded by IL-28B polynucleotide 15 molecules as shown in SEQ ID NOs:5, 16, 122, 124, 126, 128, 130, 132, 134, and 136, respectively.

IL-29 polypeptides of the present invention also include, for example, a mutation at the fifth cysteine, C5, of the mature polypeptide. For example, C5 from the N-terminus of the polypeptide of SEQ ID NO:4, is the cysteine at position 171, or position 172 (additional N-terminal Met) if expressed in E. coli. (see, for example, SEQ 5 ID NO: 15). This fifth cysteine or C5 of IL-29 can be mutated, for example, to a serine, alanine, threonine, valine, or asparagine. These IL-29 C5 mutant polypeptides have a disulfide bond pattern of Cl(CyslS of SEQ ID NO:4)/C3(Cysl 12 of SEQ1D NO:4) and C2(Cys49 of SEQ ID NO:4)/C4(Cysl45 of SEQ ID NO:4). IL-29 C5 mutant molecules of the present invention include polynucleotide molecules as shown in SEQ ID NO: 158, 10 including DNA and RNA molecules, that encode IL-29 C5 mutant polypeptides as shown in SEQ ID NO: 159, respectively.

Thus, the present invention provides an isolated polypeptide consisting of an amino acid sequence having at least 98% sequence identity to amino acid residues 1-176 of SEQ ID NO:159; wherein the polypeptide has anti-viral activity. The invention also provides an isolated polynucleotide encoding the polypeptide of the invention.

The IL-29 polypeptides of the present invention, may further include a signal sequence as shown in SEQ ID NO:119 or a signal sequence as shown in SEQ ID NO:121.

Within another aspect the present invention provides a fusion protein comprising a polypeptide according to the invention and a polyalkyl oxide moiety. The 10 polyalkyl oxcide moiety may optionally be polyethylene glycol, such as a 20kD: mPEG propionaldehyde or a 30kD mPEG propionaldehyde. The polyethylene glycol may be linear or branched. The polyethylene glycol may be covalently attached N terminally or C-terminally to the polypeptide.

- 174797/2

Table 3 sets forth the one-letter codes used within SEQ ID NOs:30, 31, 32, 33, 34, and 35 to denote degenerate nucleotide positions. Resolutions are the nucleotides denoted by a code letter. Complement indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G. with A being complementary to T. and G being complementary to C.

PCI7US2004/025864

Table 3

<td> Nucleoti de</td><td> Resolutio n</td><td> Compleme nt</td><td> Resolutio n</td>

5 The degenerate codons used in SEQ ID NOs:30,31,32,33, 34, and 35, encompassing all possible codons for a given amino acid, are set forth in Table 4.

<td></td><td> Table 4</td><td></td>

<td> One Amino Letter Acid Code</td><td> Codons</td><td> Degenerate Codon</td>

<td> Any</td><td colspan="2"> z. One of ordinary skill in the art</td><td> will appreciate that</td><td> some ambiguity is</td>

introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY). A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to /2 the amino acid sequence, for example, of SEQ ID NOs: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 can be readily tested for functionality as described herein.

One of ordinary skill in the art will also appreciate that different species can exhibit preferential codon usage. In general, 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 preferential codon usage or preferential codons is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 4). For example, the amino acid Threonine (Tier) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential. Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NOs:30, 31, 32, 33, 34, and 35 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.

As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of Cysteine mutant IL-28 or IL- 29 RNA. Such tissues and cells are identified by Northern blottilig (Thomas, Proc. Natl. Acad. Sei. USA 77:5201, 1980), or by screening conditioned medium from various cell types for activity on target cells or tissue. Once the activity or RNA producing cell or tissue is identified, total RNA can be prepared using guanidinium isothiocyanate extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18:52 94, 1979). Poly (A)+ RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sei. USA 69:1408-12, 1972). Complementary DNA (cDNA) is prepared from poly(A)+ RNA using known methods. In the alternative, genomic DNA can be isolated. Polynucleotides encoding Cysteine mutant IL-28 or IL-29 polypeptides are then identified and isolated by, for example, hybridization or PCR.

A full-length clones encoding Cysteine mutant IL-28 or IL-29 can be obtained by conventional cloning procedures. Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron. Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library. Expression libraries can be probed with antibodies to IL-28 receptor fragments, or other specific binding partners.

Those skilled in the art will recognize that the sequence disclosed in, for example, SEQ ID NOs:l, 3, and 5, respectively, represent mutations of single alleles of human IL-28 and IL-29 bands, and that allelic variation and alternative splicing are expected to occur. For example, an IL-29 variant has been identified where amino acid 5 residue 169 (Asn) as shown in SEQ ID NO:4 is an Arg residue, as described in WO 02/086087. Such allelic variants are included in the present invention. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO:1, 3 and 5, including those containing silent mutations and those 10 in which mutations result in amino acid sequence changes, in addition to the cysteine mutations, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NOs:2, 4, and 6. cDNAs generated from alternatively spliced mRNAs, which retain the properties of Cysteine mutant IL-28 or IL-29 polypeptides, are included within the scope of the present invention, as are polypeptides 15 encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art, and mutations to the polynucleotides encoding cysteines or cysteine residues can be introduced as described herein.

Isolated variant or Cysteine mutant IL-28- and IL-29-encoding nucleic acid molecules can! hybridize under stringent conditions to nucleic acid molecules having the nucleotide sequence of SEQ ID NOs: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, 5 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, and 160 or to nucleic acid molecules having a nucleotide sequence complementary to SEQ ID NOs: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 general, stringent 10 conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and 15 DNA-RNA, can hybridize if the nucleotide sequences have some degree of complementarity. Hybrids can tolerate mismatched base pairs in the double helix, but the stability of the hybrid is influenced by the degree of mismatch. The Tm of the mismatched hybrid decreases by 1°C for every 1-1.5% base pair mismatch. Varying the stringency of the hybridization conditions allows control over the degree of mismatch 5 that wiU be present in the hybrid. The degree of stringency increases as the hybridization temperature increases and the ionic strength of the hybridization buffer decreases. !

It is well within the abilities of one skilled in the art to adapt these conditions for use with a particular polynucleotide hybrid. The Tm for a specific target 10 sequence is the temperature (under defined conditions) at which 50% of the target sequence wffl hybridize to a perfectly matched probe sequence. Those conditions which influence the Tm include, the size and base pair content of the polynucleotide probe, the ionic strength of the hybridization solution, and the presence of destabilizing agents in the hybridization solution. Numerous equations for calculating Tm are known 15 in the art, and are specific for DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences of varying length (see, for example, Sambrook et al., Molecular cinning: 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, 20 (Academic Press, hie. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake, MN) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto, CA), as well as sites on the Internet, are available tools for analyzing a given sequence and calculating Tmbased on user defined criteria. Such programs can also analyze a given sequence under 25 defined conditions arid identify suitable probe sequences. Typically, hybridization of longer polynucleotide sequences, >50 base pairs, is performed at temperatures of about 20-25°C below the calculated Tm. For smaller probes, <50 base pairs, hybridization is typically earned out at the Tm or 5-10°C below the calculated Tm. This allows for the maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids.

30 Following hybridization, the nucleic acid molecules can be washed to remove non-hybridized nucleic acid molecules under stringent conditions, or under /2 highly stringent conditions. Typical stringent washing conditions include washing in a solution of 0.5x - 2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 55 - 65°C. That is, nucleic acid molecules encoding a variant or Cysteine mutant IL-28 or IL-29 polypeptides hybridize with a nucleic acid molecule having the nucleotide sequence of

SEQ ID NOs: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) under stringent washing conditions, in which the wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, including 0.5x SSC with

0.1%. 1% SDS at 55°C, or 2x SSC with 0.1% SDS at 65°C. One of skill in the art can readily devise equivalent conditions, for example, by substituting SSPE for SSC in the wash solution.

Typical highly stringent washing conditions include washing in a solution of O.lx - 0.2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 50 - 65°C. In other words, nucleic acid molecules encoding a variant of a Cysteine mutant IL-28 or IL-29 polypeptide hybridize with a nucleic acid molecule having the nucleotide 5 sequence of SEQ ID NOs: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 highly stringent washing conditions, in which the wash stringency is equivalent to O.lx - 0.2x SSC with 0.1% SDS at 50 - 65°C, including O.lx SSC with 10 0.1% SDS at 50°C, or 0.2x SSC with 0.1% SDS at 65°C.

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, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of

10, a gap extension penalty of 1, and the BLOSUM62 scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 4 (amino acids are indicated by the standard one letter codes).

Total number of identical matches t x 100

[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences] tn

Table

<td> |_1 ^1CMCOrHOCOCMv—1 Illi</td><td> CO H CO 1 1</td>

<td> i-h OOCOCOr-ICMr-ICMr־ICM h1 1 1 1 1 1 1 1 1־</td><td> CM CM CO 1 1</td>

<td> rh IDCM^I’I’CMCOCOCMOCM 1 . 1 1 ן I 1 I 1</td><td> CM co co ' 1 1</td>

<td> M inCMOCOCOrHCMCOvHOH 1 1 I 1 1 1 1</td><td> CO CM CM 1 1 1</td>

<td> m incMCMomcMHoroT-ioH ־י* III III</td><td> CM H CM 1 1 1</td>

<td> Γ) CTirO^rOCOv-lr-ICOr-1 CM CO Η H M 1 1 I 1 1 1 1 1 1 1 1 1</td><td> CM CM H 1 1 1</td>

<td> Q ID COOCMHHcO^HcOCOHOt-l I 1 1 1 1 1 1 1 1 1</td><td> <1 CO CO 1 1 1</td>

<td> 5־ lOH COOOOHCOCOOCMCOCMHO M I II 1 . 1 1</td><td> ςμ cm co 1 1 1</td>

<td> icC^tlHCMCMOHHOCMHrHvHHCMHHO 111 II 1 1 1 1 1 1 1</td><td> CO CM o 1 1</td>

<td> CrtSQUOlMOKHJWSfePHWFi</td><td> >H ></td>

o cm

Those skilled in the art appreciate that there are many established algorithms available ;to align two amino acid sequences. The “FASTA” similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and 5 the amino acid sequence of a putative variant IL-28 or IL-29. The FASTA algorithm is described by Pearson and Lipman, Proc. Nat’l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO:2) and a test sequence that have 10 either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity 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 15 contribute to the highest score. If there are several regions with scores greater than the “cutoff’ value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether'the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned 20 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 amino acid insertions and deletions. Preferred parameters for FASTA analysis are: ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA 25 program by modifying the scoring matrix file (“SMATRIX”), as explained in Appendix 2 of Pearson, Meth, Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from three to six, most 30 preferably three, with other parameters set as default.

Variant IL-28 or IL-29 Cysteine mutant polypeptides or polypeptides with substantially similar sequence identity are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 6) and other 5 substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and amino- or carboxyl terminal extensions, such as an amino- terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. The present invention thus includes polypeptides wherein the polypeptide has at least 99% sequence identity to 10 amino acid residues 1-176 of SEQ ID NO: 159. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the IL-28 and IL 29 polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.

Table

Conservative amino acid substitutions

<td> 5 Basic:</td><td> arginine lysine histidine</td>

<td> Acidic:</td><td> glutamic acid aspartic acid</td>

<td> 10 Polar:</td><td> glutamine asparagine</td>

<td> Hydrophobic:</td><td> leucine isoleucine valine</td>

<td> 15 Aromatic:</td><td> phenylalanine tryptophan tyrosine</td>

<td> Small: .</td><td> glycine alanine</td>

<td> 20</td><td> serine threonine methionine</td>

Determination of amino acid residues that comprise regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can determine specific residues that will be more or less tolerant of change and maintain the overall, tertiary structure of the molecule. Methods for analyzing sequence structure include, but are not limited to alignment of multiple sequences with high amino acid or nucleotide identity, secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al., Current Opin. Struct. BioL 6:3-10, 1996). In general, when designing modifications to molecules or identifying specific fragments determination of structure will be accompanied by evaluating activity of modified molecules.

Amino acid sequence changes are made in Cysteine mutant IL-28 or IL29 polypeptides so as to minimize disruption of higher order structure essential to biological activity. For example, where the Cysteine mutant IL-28 or IL-29 polypeptide comprises one or more helices, changes in amino acid residues will be made so as not 5 to disrupt the helix geometry and other components of the molecule where changes in conformation abate some critical function, for example, binding of the molecule to its binding partners. The effects of amino acid sequence changes can be predicted by, for example, computer modeling as disclosed above or determined by analysis of crystal structure (see, e.g., Lapthom et al., Nat. Struct. BipL. 2:266-268, 1995). Other 10 techniques , that are well known in the art compare folding of a variant protein to a standard molecule (e.g., the native protein). For example, comparison of the cysteine pattern in a variant and standard molecules can be made. Mass spectrometry and chemical modification using reduction and alkylation provide methods for determining cysteine residues which are associated with disulfide bonds or are free of such 15 associations (Bean et al., Anal, Biochem, 201:216-226, 1992; Gray, Protein S.cL 2:1732-1748, 1993; and Patterson et al., Anal. Chem, 66:3727-3732, 1994). It is generally believed that if a modified molecule does not have the same cysteine pattern as the standard molecule folding would be affected. Another well known and accepted method for measuring folding is circular dichrosism (CD). Measuring and comparing 20 the CD spectra generated by a modified molecule and standard molecule is routine (Johnson, Proteins 7:205-214, 1990). Crystallography is another well known method for analyzing folding and structure. Nuclear magnetic resonance (NMR), digestive peptide mapping !and epitope mapping are also known methods for analyzing folding and structurally similarities between proteins and polypeptides (Schaanan et al., Science 25 257:961-964,1992).

A Hopp/Woods hydrophilicity profile of the Cysteine mutant IL-28 or IL-29 protein sequence as shown in SEQ ID NOs: 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, 30 151, 153, 155159 ־157 ־ or 161 can be generated (Hopp et al., Proc. Natl. Acad.

Sci.78:3824-3828, 1981; Hopp, J. Immun, Meth. 88:1-18, 1986 and Triquier et al., /2

Protein Engineering 14:153 1697 1998). The profile is based on a sliding six-residue window. Buried G. S. and T residues and exposed Η. Y. and W residues were ignored. Those skilled in the art will recognize that hydrophilicity or hydrophobicity will be taken into account when designing modifications in the amino acid sequence of a Cysteine mutant IL-28 or IL-29 polypeptide, so as not to disrupt the overall structural and biological profile. Of particular interest for replacement are hydrophobic residues selected from the group consisting of Vai, Lou and He or the group consisting of Met, Gly, Ser. Ala, Tyr and Trp.

The identities of essential amino acids can also be inferred from analysis of sequence similarity between IFN־a and members of the family of IL-28A, IL-28B, and IL-29 (as shown in Tables 1 and 2). Using methods such as FASTA analysis described previously, regions of high similarity are identified within a family of proteins and used to analyze amino acid sequence for conserved regions. An alternative approach to identifying a variant polynucleotide on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant IL-28 or IL-29 gene can hybridize to a nucleic acid molecule as discussed above.

Other methods of identifying essential amino acids in the polypeptides of the present invention are procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et al., Proc. Natl Acad. Sei. 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 technique, single alanine mutations are introduced at every residue in the molecule, and the resultant Cysteine mutant molecules are tested for biological or biochemical activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699 (1996).

A functional Cysteine mutant IL-28 or IL-29 or fragment thereof as defined herein is characterized by its proliferative or differentiating activity, by its ability to induce or inhibit specialized cell functions, or by its ability to bind specifically to an anti-IL-28 or IL-29 antibody or IL-28 receptor (either soluble or immobilized). The specialized activities of Cysteine mutant IL-28 or IL-29 polypeptides /2 and how to test for them are disclosed herein. As previously described herein, IL-28 and IL-29 polypeptides are characterized by a six-helical-bundle.

The Cysteine mutant IL-29 polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides can be produced according to conventional techniques using cells into which have been introduced an expression vector encoding the polypeptide. As used herein, cells into which have been introduced an expression vector include both cells that have been directly manipulated by the introduction of exogenous DNA molecules and progeny thereof that contain the introduced DNA. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed 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.

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

Within another aspect the present invention provides a cultured cell comprising an expression vector as disclosed above.

Within another aspect the present invention provides a method of producing a protein comprising: culturing a cell as disclosed above under conditions wherein the DNA segment is expressed; and recovering the protein encoded by the DNA segment.

In general, a DNA sequence encoding a Cysteine mutant IL-29 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.

A wide variety of suitable recombinant host cells includes, but is not limited to, gram- negative prokaryotic host organisms. Suitable strains of E. coli include W3110, K12-derived strains MM294, TO-1, JM-107, BL21, and UT5600. Other suitable strains include: BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF', DH5IMCR, DH1OB, 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 Rk־M!c-, E. coli K12 RR1 (see, for example, Brown (ed.), Molecular Biology Labfax (Academic Press 1991)). Other gram-negative prokaryotic hosts can include Serratia, Pseudomonas, Caulobacter. Prokaryotic hosts can include gram-positive organisms such as Bacillus, for example, B. subtilis and B. thuringienesis, and B. thuringienesis var. israelensis, as well as Streptomyces, for example, S. lividans, S. ambofaciens, S. fradiae, and 5. griseofuscus. Suitable strains of Bacillus subtilus include BRI 51, YB886, Mil 19, Ml 120, and B170 (see, for example, Hardy, Bacillus Cloning Methods, in DNA Cloning: A Practical Approach, Glover (ed.) (LRL Press 1985)).

Standard techniques for propagating vectors in prokaryotic hosts are well-known to those of skill in the art (see, for example, Ausubel et al. (eds.), Short Protocols in Molecular Biology 3rd Edition (John Wiley & Sons 1995); Wu et al., Methods in Gene Biotechnology (CRC Press, Inc. 1997)). In one embodiment, the methods of the present invention use Cysteine mutant IL- 28 or IL-29 expressed in the W3110 strain, which has been deposited at the American Type Culture Collection (ATCC) as ATCC # 27325.

When large scale production of Cysteine mutant IL-29 using the expression system of the present invention is required, batch fermentation can be used. Generally, batch fermentation comprises that a first stage seed flask is prepared by growing E. coli strains expressing Cysteine mutant IL-29 in a suitable medium in shake flask culture to allow for growth to an optical density (OD) of between 5 and at 600 nm. A suitable medium would contain nitrogen from a source(s) such as ammonium sulfate, ammonium phosphate, ammonium chloride, yeast extract, hydrolyzed animal proteins, hydrolyzed plant proteins or hydrolyzed caseins. Phosphate will be supplied from potassium phosphate, ammonium phosphate, phosphoric acid or sodium phosphate. Other components would be magnesium chloride or magnesium sulfate, ferrous sulfate or ferrous chloride, and other trace elements. Growth medium can be supplemented with carbohydrates, such as fructose, glucose, galactose, lactose, and glycerol, to improve growth. Alternatively, a fed batch culture is used to generate a high yield of Cysteine mutant IL-29 protein. The Cysteine mutant IL-29 producing E. coli strains are grown under conditions similar to those described for the first stage vessel used to inoculate a batch fermentation.

Following fermentation the cells are harvested by centrifugation, resuspended in homogenization buffer and homogenized, for example, in an APV-Gaulin homogenizer (Invensys APV, Tonawanda, New York) or other type of cell disruption equipment, such as bead mills or sonicators. Alternatively, the cells are taken directly from the fermenter and homogenized in an APV-Gaulin homogenizer. The washed inclusion body prep can be solubilized using 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). The solutions can be prepared in Tris, phopshate, HEPES or other appropriate buffers. Inclusion bodies can also be solubilized with urea (2-4 M) /2 containing sodium lauryl sulfate (0.1- 2%). In the process for recovering purified Cysteine mutant IL-28 or L- 29 from transformed E. coli host strains in which the Cysteine mutant IL- 28 or IL-29 is accumulates as retractile inclusion bodies, the cells are disrupted and the inclusion bodies are recovered by centrifugation. The inclusion 5 bodies are then solubilized and denatured in 6 M guanidine hydrochloride containing a reducing agent. The reduced Cysteine mutant IL-29 is then oxidized in a controlled renaturation step. Refolded Cysteine mutant IL-29 can be passed through a filter for clarification and removal of insoluble protein. The solution is then passed through a filter for clarification and removal of insoluble protein. After the Cysteine mutant IL-29 10 protein is refolded and concentrated, the refolded Cysteine mutant IL-29 protein is captured in dilute buffer on a cation exchange column and purified using hydrophobic interaction chromatography.

Cultured mammalian cells are suitable hosts within the present invention. 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), DEAE-dextran mediated transfection (Ausubel et al., ibid. ), and 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, by Levinson et al., U.S. Patent No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. Suitable cultured mammalian cells include the 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 Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, VA. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No.

4,956,288. Other suitable promoters include those from metallothionein genes (U.S.

Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as transfectants. Cells that have been cultured in the presence of the selective agent and are able to pass the' gene of interest to their progeny are referred to as stable transfectants. A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as amplification. Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. : Other drug tp.r1 stance genes (e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as: CD4, CD8, Class IMHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed-by Guarino et al., U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa califomica 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, l aboratory Manual. New York, Oxford University Press., 1994; and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, /2

Humana Press, 1995. The second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, V.A, et al., J Virol 67:4566 79, 1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBacl™ (Life Technologies) containing a Tn7 transposon to move the DNA encoding the Cysteine mutant IL-29 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a bacmid. The pFastBacl™ transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case Cysteine mutant IL-29. However, pFastBacl™ can be modified to a considerable degree. The polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins, M.S. and Possee, 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 transfer vector constructs, a short or long version of the basic protein promoter can be used. Moreover, transfer vectors can be constructed which replace the native IL-28 or IL-29 secretory signal sequences with secretory signal sequences derived from insect proteins. For example, a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (vitrogen, Carlsbad, CA), or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native IL-29 secretory signal sequence. In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed Cysteine mutant IL-29 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4. 1985). Using techniques known in the art, a transfer vector containing Cysteine mutant IL-29 is transformed into E. coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacnid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus that expresses Cysteine mutant IL-29 is subsequently produced. Recombinant viral stocks are made by methods commonly used the art.

The recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994. Another suitable cell line is the High FiveO™ cell line (Ihvitrogen) derived from Trichoplusia ni (U.S. Patent No. 5,300,435).

Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming 5. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311, Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POTI vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-65, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533. The use of Pichia methanolica as host for the /2 production of recombinant proteins is disclosed in U.S. Patent Nos. 5,955,349,

5,888,768 and 6,001,597, U.S. Patent No. 5,965,389, U.S. Patent No. 5,736,383, and

U.S. Patent No. 5,854,039.

It is preferred to purify the polypeptides and proteins of the present invention to 280% purity, more preferably to >90% purity, even more preferably >95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified polypeptide or protein is substantially free of other polypeptides or proteins, particularly those of animal origin.

Expressed recombinant Cysteine mutant IL-29 proteins (including chimeric polypeptides and multimeric proteins) are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See, in general, Affinity !Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice, Springer- Verlag, New York, 1994. Proteins comprising a polyhistidine affinity tag (typically about 6 histidine residues) are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bio/Technol. 6: 1321-1325, 1988. Proteins comprising a glu-glu tag can be purified by immunoaffinity chromatography according to conventional procedures. See, for example, Grussenmeyer et al., supra. Maltose binding protein fusions are purified on an amylose column according to methods known in the art.

Cysteine mutant IL-29 polypeptides can also be prepared through chemical synthesis according to methods known in the art, including exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd 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, 1RL Press, Oxford, 1989. In vitro synthesis is particularly advantageous for the preparation of smaller polypeptides.

Using methods known in the art, Cysteine mutant IL-29 proteins can be prepared as monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; fusion proteins; and may or may not include an initial methionine amino acid residue. Cysteine mutant IL-29 conjugates used for therapy may comprise pharmaceutically acceptable water-soluble polymer moieties. Conjugation of interferons with water-soluble polymers has been shown to enhance the circulating half-life of the interferon, and to 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-(Cl-C10)alkoxy-PEG, aryloxy-PEG, poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG, monomethoxy-PEG propionaldehyde, PEG propionaldehyde, bis-succinimidyl carbonate PEG, propylene; glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol), monomethoxy-PEG butyraldehyde, PEG butyraldehyde, monomethoxy-PEG acetaldehyde, PEG acetaldehyde, methoxyl PEG-succinimidyl propionate, methoxyl PEG-succinimidyl butanoate, polyvinyl alcohol, dextran, cellulose, or other carbohydrate-based polymers. Suitable PEG may have a molecular weight 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, which can be linear or branched. A Cysteine mutant IL-29 conjugate can also comprise a mixture of such water-soluble polymers.

One example of a Cysteine mutant IL-29 conjugate comprises a Cysteine mutant IL-29 moiety and a polyalkyl oxide moiety attached to the N 2s terminus of the Cysteine mutant IL-29 moiety. PEG is one suitable polyalkyl oxide. As an illustration, Cysteine mutant IL-29 can be modified with PEG, a process known as PEGylation. PEGylation of Cysteine mutant IL-29 can be carried out by any of the PEGylation reactions known in the art (see, for example, EP 0 154 316, 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)). Eor example, PEGylation can be performed by an acylation reaction or by an alkylation reaction with a reactive polyethylene glycol molecule. In an alternative approach, /2

Cysteine mutant IL-29 conjugates are formed by condensing activated PEG, in which a terminal hydroxy or amino group of PEG has been replaced by an activated linker (see, for example, Karasiewicz et al, U.S. Patent No. 5,382,657).

PEGylation by acylation typically requires reacting an active ester derivative of PEG with a Cysteine mutant IL-29 polypeptide. An example of an activated PEG ester is PEG esterified to N-hydroxysuccinimide. As used herein, the term acylation includes the following types of linkages between Cysteine mutant IL29 and a water-soluble polymer: amide, carbamate, urethane, and the like. Methods for preparing PEGylated Cysteine mutant IL-29 by acylation will typically comprise the steps of (a) reacting an Cysteine mutant IL-29 polypeptide with PEG (such as a reactive ester of an aldehyde derivative of PEG) under conditions whereby one or more PEG groups attach to Cysteine mutant IL-29, 3 IS and (b) obtaining the reaction product(s). Generally, the optimal reaction conditions for 3 acylation reactions will be determined based upon known parameters and desired results. For example, the larger the ratio of PEG: Cysteine mutant IL-29, the; greater the percentage of polyPEGylated Cysteine mutant IL-29 product.

PEGylation by alkylation generally involves reacting a terminal aldehyde, e.g, propionaldehyde, butyraldehyde, acetaldehyde, and the like, derivative of PEG with Cysteine mutant IL-29 in the presence of a reducing agent. PEG groups are preferably attached to the polypeptide via a -CH2-NH2 group.

Derivatization via reductive alkylation to produce a monoPEGylated product takes advantage of the differential reactivity of different types of primary amino; groups available for derivatization. Typically, the reaction is performed at a pH that allows one to take advantage of the pKa differences between the E-amino groups of the lysine residues and the of-amino group of the N-terminal residue of the protein. By such selective derivatization, attachment of a water-soluble polymer that contains a reactive group such as an aldehyde, to a protein is controlled. The conjugation with the polymer occurs predominantly at the JV-terminus of the protein without significant modification of other reactive groups such as the lysine side chain amino groups.

Reductive alkylation to produce a substantially homogenous population of monopolymer Cysteine mutant IL-29 conjugate molecule can comprise the steps of:

(a) reacting a Cysteine mutant IL-29 polypeptide with a reactive PEG under reductive alkylation conditions at a pH suitable to permit selective modification of the of-amino group at the amino terminus of the Cysteine mutant IL-29, and (b) obtaining the reaction product(s). The reducing agent used for reductive alkylation should be stable in aqueous solution and preferably be able to reduce only the Schiff base formed in the initial process of reductive alkylation. Preferred reducing agents include sodium borobydride, sodium cyanoborohydride, dimethylamine borane, 10 trimethylamine borane, and pyridine borane.

For a substantially homogenous population of monopolymer Cysteine mutant IL-29 conjugates, the reductive alkylation reaction conditions are those that permit the selective attachment of the water-soluble polymer moiety to; the N-terminus of Cysteine mutant IL-29. Such reaction conditions generally provide for pKa differences between the lysine amino groups and the oc-amino group at the N terminus. The pH also affects the ratio of polymer to protein to be used. In general, if the pH is lower, a larger excess of polymer to protein will be desired because the less reactive the N-terminal of-group, the more polymer is needed to achieve optimal conditions. If the pH is higher, the polymer: Cysteine mutant IL-29 need not be as large because more reactive groups are available. Typically, the pH will fall within the range of 3 - 9, or 3 -

6. Another factor to consider is the molecular weight of the water-soluble polymer. Generally, the higher the molecular weight of the polymer, the fewer number of polymer molecules which may be attached to the protein. For PEGylation reactions, the typical molecular weight is about 2 kDa to about 100 kDa, about 5 kDa to about 50 kDa, about 12 kDa to about 40 kDa, or about 20kDa to about kDa. The molar ratio of water-soluble polymer to Cysteine mutant IL-28 or IL-29 will generally be in the range of 1:1 to 100:1. Typically, the molar ratio of water soluble polymer to Cysteine mutant IL-29 will be 1:1 to 20:1 for polyPEGylation, and 1:1 to 5:1 for monoPEGylation.

General methods for producing conjugates comprising interferon and water-soluble polymer moieties are known in the art. See, for example, Karasiewicz et /2 al., U.S. Patent No. 5,382,657, Greenwald et al., U.S. Patent No. 5,738, 846, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996), Monkarsh et al., Anal. Biochem. 247:434 (1997). PEGylated species can be separated from unconjugated Cysteine mutant IL-29 polypeptides using standard purification methods, such as dialysis, ultrafiltration, ion exchange chromatography, affinity chromatography, size exclusion chromatography, and the like.

The Cysteine mutant IL-29 molecules of the present invention are capable of specifically binding the IL-28 receptor and/or acting as an antiviral agent. The binding of Cysteine mutant IL-29 polypeptides to the IL-28 receptor can be assayed using established approaches. Cysteine mutant IL-29 can be iodinated using an iodobead (Pierce, Rockford, IL) according to manufacturer's directions, and the 125I-IL-29 can then be used as described below.

In a first approach fifty nanograms of 125I-IL-29 can be combined with lOOOng of IL-28 receptor human IgG fusion protein, in the presence or absence of possible binding competitors including unlabeled Cysteine mutant IL-28, Cysteine mutant IL-29, IL-28, or IL-29. The same binding reactions would also be performed substituting other cytokine receptor human IgG fusions as controlsfor specificity. Following incubation at 4°C, protein-G (Zymed, SanFransisco, CA) is added to the reaction, to capture the receptor-IgG fusions and any proteins bound to them, and the reactions are incubated another hour at 4°C. The protein-G sepharose is then collected, washed three times with PBS and 125I-IL-29 bound is measure by gamma counter (Packard Instruments, Downers Grove, IL).

In a second approach, the ability of molecules to inhibit the binding of 125I-IL-29 to plate bound receptors can be assayed. A fragment of the IL 28 receptor, representing the extracellular, ligand binding domain, can be adsorbed to the wells of a 96 well plate by incubating 100 μΐ of 1 g/mL solution of receptor in the plate overnight. In a second form, a receptor-human IgG fusion can be bound to the wells of a 96 well plate that has been coated with an antibody directed against the human IgG portion of the fusion protein. Following coating of the plate with receptor the plate is washed, blocked with SUPERBLOCK (Pierce, Rockford, IL) and washed again. Solutions containing a fixed concentration of 125I-IL-29 with or without increasing concentrations of potential binding competitors including, Cystein mutant IL-28, cysteine mutant IL-49174797/2

29, IL-28 and IL-29, and 100 pi of the solution added to appropriate wells of the plate.

> ל׳ 1

Following a one hour incubation at 4°C the plate is washed and the amount I-IL- 29 bound determined by counting (Topcount, Packard Instruments, Downers grove, IL). The specificity of binding of 125I-IL-29 can be defined by receptor molecules used in 5 these binding assays as well as by the molecules used as inhibitors.

Methods for detection and diagnosis of viral infections are well known to those skilled in the art. The exact method used for measuring a reduction in virus in response to administration of molecules of the present invention will be dependent upon the species of virus and whether the infection is in vitro or in viva. If the infection is in 10 viva, measurement and detection of infection and changes in the levels of infection, can vary depending on subject infected, type of viral infection, and the like. 3 For example, methods include, but are not limited to, measuring changes in CD4 cell counts, serologic tests, measuring the DNA of the virus and RNA of the virus by conventional and real-time quantitative polymerase chain reaction assays, viral induced antibody 15 levels, immunofluorescence and enzyme-linked immunosorbant assays, s cytopathic effects, and histology.

Antiviral effects may be direct or indirect. An example of a direct antiviral effect is, for example, where Cysteine mutant IL-28 or IL-29 polypeptide competes for a viral receptor or co-receptor to block viral infection. Cysteine mutant IL-28 or ILr29 may be given parentally to prevent viral infection or to reduce ongoing viral replication and re- infection (Gayowski, T. et al., Transplantation 64:422-426, 1997). An example of an indirect antiviral effect is, for example, where a Cysteine mutant IL-28 or IL-29 may bind CD4 or another leukocyte receptor and exhibit antiviral effects by modulating the effects of the immune response.

Additional types of viral infections for Cysteine mutant IL-28 or IL-29 use include, but are not limited to: infections caused by DNA Viruses (e.g., Herpes Viruses such as Herpes Simplex viruses, Epstein-Barr virus, Cytomegalovirus; Pox viruses such as Variola (small pox) virus; Hepadnaviruses (e.g. Hepatitis B virus); Papilloma viruses; Adenoviruses); RNA Viruses (e.g., HIV I, II; HTLV I, II; Poliovirus; Hepatitis A; coronoviruses, such as sudden acute respiratory syndrome (SARS); Orthomyxoviruses (e.g., Influenza viruses); Paramyxoviruses (e.g., Measles virus); Rabies virus; Hepatitis C virus), Flaviviruses, Influenza viruses; caliciviruses; rabies viruses, rinderpest viruses, Arena virus, and the like. Moreover, examples of the types of virus-related diseases for which Cysteine mutant IL-28 or IL-29 could be used include, but are not limited to: Acquired immunodeficiency; Severe Acute Respiratory Syndrome (SARS); Hepatitis; Gastroenteritis; Hemorrhagic diseases; Enteritis; Carditis; Encephalitis; Paralysis; Brochiolitis; Upper and lower respiratory disease; Respiratory Papillomatosis; Arthritis; Disseminated disease, Meningitis,

Mononucleosis. In addition, Cysteine mutant IL-28 or IL-29 can be used in various applications for antiviral immunotherapy, and in conjunction with other cytokines, other protein or small molecule antivirals, and the like.

Clinically, diagnostic tests for HCV include serologic assays for antibodies and molecular tests for viral particles. Enzyme immunoassays are available (Vrielink et al., Transfusion 37:845-849, 1997), but may require confirmation using additional tests such as an immunoblot assay (Pawlotsky et al., Hepatology 27:17001702, 1998). Qualitative and quantitative assays generally use polymerase chain reaction techniques, and are preferred for assessing 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, quantitative RT-PCR (Amplicor HCV Monitor™, Roche Molecular Systems, Branchburg, NJ) and a branched DNA (deoxyribonucleic acid) signal amplification assay (Quantiplex™ HCV RNA Assay [bDNA], Chiron Corp., Emeryville, CA). A non-specific laboratory test for HCV infection measures alanine aminotransferase level (ALT) and is inexpensive and readily available (National Institutes of Health Consensus Development Conference Panel, Hepatology 26 (Suppl, !):2S-10S, 1997). Histologic evaluation of liver biopsy is generally considered the most accurate means for determining HCV progression (Yano et al., Hepatology 23:1334-1340, 1996.) For a review of clinical tests for HCV, see, Lauer et al. N, Engl. J. Med. 345:41-52,2001.

There are several in vivo models for testing HBV and HCV that are known to those skilled in art. With respect to HCV, for example, the HCV Replicon model is a cell-based system to study the effectiveness of a drug to inhibit HCV replication (Blight et al., Science, 290(54981:1972-1974 (Dec. 8, 2000); and Lohmann et al., Science. 285(54241:110-113 (July 2,1999)). A well-known and accepted in vitro HBV model to one of skill in the art can be used to determine the anti-HBV activity of a test molecule is disclosed in Korba et al., Antiviral Res., 19(1):55-70 (1992) and Korba et al., Antiviral Res., 15(3):217-228 (1991).

For example, the effects of Cysteine mutant IL-28 or IL-29 on mammals infected with HBV can accessed using a woodchuck model. Briefly, woodchucks chronically infected with woodchuck hepatitis virus (WHV) develop hepatitis and /2 hepatocellular Carcinoma that is similar to disease in humans chronically infected with HBV. The model has been used for the preclinical assessment of antiviral activity. A chronically infected WHV strain has been established and neonates are inoculated with serum to provide 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). Chimpanzees may also be used to evaluate the effect of Cysteine mutant IL-28 or IL-29 on HBV infected mammals. Using chimpanzees, characterization of HBV was made and these studies demonstrated that the chimpanzee disease was remarkably similar to the disease: in humans (Barker et al., J. Infect. Dis. 132:451 458, 1975 and Tabor et al., J. Infect. Dots. 147:531 534, 1983.) The chimpanzee model has been used in evaluating vaccines i (Prince et al., In: Vaccines 97, Cold Spring Harbor Laboratory Press, 1997.) Therapies 3 for HIV are routinely tested using non-human primates infected with simian immunodeficiency viruses (for a review, see, Hirsch et al., Adv. Pharmcol. 49:437-477. 2000 and Nathanson et al., AIDS 13 (suppl. A):S 113-S120. 1999.) For a review of use of non- human primates in HIV, hepatitis, malaria, respiratory syncytial virus, and other diseases, see, Sibal et al., ILAR J. 42 (2):74-84. 2001. A recently developed transgenic mouse model (Guidotti et al., Journal of Virology 69:6158-6169, 1995) supports the replication of high levels of infectious HBV and has been used as a chemotherapeutic model for HBV infection. Transgenic mice are treated with antiviral drugs and the levels of HBV DNA and RNA are measured in the transgenic mouse liver and serum following treatment. HBV protein levels can also be measured in the transgenic mouse serum following treatment. This model has been used to evaluate the effectiveness of lamivudine and IFN-alpha in reducing HBV viral titers (Morrey et al., Antiviral Therapy 3:59-68,1998).

Moreover, Cysteine mutant IL-29 polyeptides and proteins of the present invention can be characterized by their activity, that is, modulation of the proliferation, differentiation, migration, adhesion, gene expression or metabolism of responsive cell types. Biological activity of Cysteine mutant IL-29 polypeptides and proteins is assayed using in vitro or in viva assays designed to detect i cell proliferation, differentiation, migration or adhesion; or changes in gene expression or cellular metabolism (e.g., production of other growth factors or other macromolecules). Many suitable assays are known in the art, and representative assays are disclosed herein. Assays using cultured /2 cells are most convenient for screening, such as for determining the effects of amino acid substitutions, deletions, or insertions.

Activity of Cysteine mutant IL-29 proteins can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to an appropriate animal model. Assays measuring cell proliferation or differentiation are well known in the art. For example, assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990), incorporation of radiolabelled nucleotides (as disclosed by, 10 e. g., Raines and Ross, Methods Enzymol. 109:7491985 ,773־; Wahl et al., Mol. Cell Biol. 8:5016-5025, 1988; and Cook et al., Analytical Biochem. 179:1-7. 1989), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J. Immunol. Methods 82:169-179, 1985), and use of tetrazolium salts (Mosmann, J. Immunol. 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. 48:48274833, 1988). Differentiation can be assayed using suitable precursor cells that can be induced to differentiate into a more mature phenotype. Assays measuring differentiation include, for example, measuring cell-surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 52:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all I incorporated herein by reference).

Cysteine mutant IL-29 polypeptide activity may also be detected using assays designed to measure IL-29-induced production of one or more additional growth factors or other macromolecules. Certain members of the protein family comprising IL29 have been shown to increase circulating monocyte numbers in vivo. Monocyte activation is important in both innate and adaptive immunity. For example, activation of monocytes has been shown to stimulate antigen presentation by several mechanisms. Antigen presentation promotes activation i and proliferation of T-cells, both cytotoxic and helper T cells. The maturation and activation of dendritic cells also promotes activation of T-cells and both innate and adaptive immunity. Increases in activated monocytes and macrophages have also been shown to increase cytolytic activity. Therefore, Cysteine mutant IL-29 will be useful as an anti- infectious agent, enhancing /2 innate cell- mediated and humoral immune responses. Increases in ICAM staining in CD 14+ monocytes was seen suggesting that IL-28 and 1 L-29 play a role in monocyte activation. While data show that family members promote an anti- viral response to virus, bacteria and parasites may also be affected.

Monocyte activation assays are carried out (1) to look for the ability of: Cysteine mutant IL-29 proteins to further stimulate monocyte activation, and (2) to examine the ability of Cysteine mutant IL-29 proteins to modulate i attachment-induced or endotoxin- induced monocyte activation (Fuhlbrigge et al., J. Immunol. 138: 37993802,1987). IL-la and TNFa levels produced in response to activation are measured by ELISA (Biosource, Inc. Camarillo, CA). Monocyte/macrophage cells, by virtue of CD 14 (LPS receptor), are exquisitely sensitive to endotoxin, and proteins with moderate levels of endotoxin-like activity will activate these cells.

Increased levels of monocytes suggest that Cysteine mutant IL-28 or IL 29 may have a direct effect on myeloid progenitor cells in the bone marrow. Increasing differentiation of myeloid progenitor cells to monocytes is essential in restoring immnunocompetency, for example, after chemotherapy. Thus, administration of Cysteine mutant IL-28 or IL-29 to patients receiving chemotherapy could promote their I recovery and ability to resist infection commonly associated with chemotherapy regimens. Thus, methods for expanding the numbers of monocytes or monocyte progenitor cells by either culturing bone marrow or peripheral blood cells with the molecules of the present invention such that there is an increase in the monocyte or monocyte progenitor cells for achieving this effect in vitro or ex viva. The present invention also provides for the in vivo administration of the molecules of the present invention to a mammal needing increased monocyte or monocyte progenitor cells. Increased monocyte and monocyte progenitor cells can be measured using methods well known to clinicians, physicians, and other persons skilled the art. Monocyte cells are included in the myeloid lineage of hematopoietic cells, so affects on other cells in that

PCT7US2004/025864 lineage would not be unusual. For example, when a factor facilitates the differentiation or proliferation of one type of cell in the myeloid or lymphoid lineage, this can affect production of other cells with a common progenitor or stem cell.

Hematopoietic activity of Cysteine mutant IL-28 or IL-29 proteins can be assayed on various hematopoietic cells in culture. Preferred assays include primary bone marrow colony assays and later stage lineage-restricted colony assays, which are known in the art (e.g., Holly et al., WIPO Publication WO 95/21920). Marrow cells plated on a suitable semi-solid medium (e.g., 50% methylcellulose containing 15% fetal bovine serum, 10% bovine serum albumin, and 0.6% PSN antibiotic mix) are incubated in the presence of test polypeptide, then examined microscopically for colony formation. Known hematopoietic factors are used as controls. Mitogenic activity of Cysteine mutant IL-28 or IL-29 polypeptides on hematopoietic cell lines can be measured as disclosed above.

Cell migration is assayed essentially as disclosed by Kahler et al. (Arteriosclerosis, Thrombosis, and Vascular Biology 17:932-939, 1997). A protein is considered to be chemotactic if it induces migration of cells from an area of low protein concentration to an area of high protein concentration. A typical assay is performed using modified Boyden chambers with a polystryrene membrane separating the two chambers (Transwell; Coming Costar Corp.). The test sample, diluted in medium containing 1% BSA, is added to the lower chamber of a 24-well plate containing Transwells. Cells are then placed on the Transwell insert that has been pretreated with 0.2% gelatin. Cell migration is measured after 4 hours of incubation at 37°C. Nonmigrating cells are wiped off the top of the Transwell membrane, and cells attached to the lower face of the membrane are fixed and stained with 0.1% crystal violet. Stained cells are then extracted with 10% acetic acid and absorbance is measured at 600 nm. Migration is then calculated from a standard calibration curve. Cell migration can also be measured using the matrigel method of Grant et al. ( Angiogenesis as a component of epithelial-mesenchymal interactions” in Goldberg and Rosen, Epithelial. Mesenchymal Interaction in Cancer, Birkhauser Verlag, 1995, 235-248, Baatout, Anticancer Research 17:451-456,1997).

Cell adhesion activity is assayed essentially as disclosed by LaFleur et al. (J. Biol. Chem. 272:32798-32803, 1997). Briefly, microtiter plates are coated with the test protein, non-specific sites are blocked with BS A, and cells (such as smooth muscle cells, leukocytes^ or endothelial cells) are plated at a density of approximately 1045 cells/well. The wells are incubated at 37°C (typically for about 60 minutes), then nonadherent cells are removed by gentle washing. Adhered cells are quantitated by conventional methods (e.g., by staining with crystal violet, lysing the cells, and determining the optical density of the lysate). Control wells are coated with a known adhesive protein, such as fibronectin or vitronectin.

Expression of Cysteine mutant IL-28 or IL-29 polynucleotides in animals provides models for further study of the biological effects of overproduction or inhibition of protein activity in vivo. IL-28- or IL-29-encoding polynucleotides and antisense polynucleotides can be introduced into test animals, such as mice, using viral vectors or naked DNA, or transgenic animals can be produced.

One in vivo approach for assaying proteins of the present invention utilizes viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acids. For review, see Becker et al., MetL Cell Biol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine 4:44-53,

1997. The adenovirus system offers several advantages. Adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. Also see, Wu et al., J. Biol, Chem. 263:14621-14624, 1988; Wu et al., J. Biol. Chem. 267:963-967,1992; and Johnston and Tang, Meth, Cell Biol. 43:353-365,1994.

Transgenic mice, engineered to express a Cysteine mutant IL-28 or IL29 gene, and mice that exhibit a complete absence of Cysteine mutant IL-28 or IL-29 gene function, referred to as knockout mice (Snouwaert et al., Science 257:1083, 1992), can also be generated (Lowell et al., Nature 366:740-742, 1993). These mice can be employed to study the Cysteine mutant IL-28 or IL29־ gene and the protein encoded thereby in an in vivo system. Preferred promoters for transgenic expression include promoters from 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. Cysteine mutant IL-28 or IL29 and inhibitors of their activity are expected to have a variety of therapeutic applications. These therapeutic applications include treatment of diseases which require immune regulation, including autoimmune diseases such as rheumatoid arthritis, multiple 1sclerosis, myasthenia gravis, systemic lupus erythematosis, and diabetes. IL-28 or IL-29 may be important in the regulation of inflammation, and therefore would be useful in treating rheumatoid arthritis, asthma and sepsis. There may be a role of IL-28 or IL-29 in mediating tumorgenesis, whereby a Cysteine mutant IL-28 or IL-29 antagonist would -be useful in the treatment of cancer. IL-28 or IL-29 may be useful in modulating the immune system, whereby Cysteine mutant IL-28 or IL29 antagonists may be used for reducing graft rejection, preventing graft-vs-host disease, boosting immunity to infectious diseases, treating immunocompromised patients (e.g, EHV+ patients), or in improving vaccines.

Members of the protein family of the present invention have been shown to have an antiviral effect that is similar to interferon-a. Interferon has been approved in the United States for treatment of autoimmune diseases, condyloma acuminatum, chrome hepatitis C, bladder carcinoma, cervical carcinoma, laryngeal papillomatosis, fungoides mycosis, chronic hepatitis B, Kaposi’s sarcoma in patients infected with human immunodeficiency virus, malignant melanoma, hairy cell leukemia, and multiple sclerosis. In addition, Cysteine mutant IL-28 or IL-29 may be used to treat forms of arteriosclerosis, such as atherosclerosis, by inhibiting cell proliferation. Accordingly, the present invention contemplates the use of Cysteine mutant IL-28 or IL-29 proteins, polypeptides, and peptides having IL-28 and IL-29 activity to treat such conditions, as well !as to treat retinopathy. The present invention also contemplates the use of Cysteine mutant IL-28 or IL-29 proteins, polypeptides, and peptides having IL-28 and IL-29 activity to treat lymphoproliferative disorders, including B-cell lymphomas,

PCI7US2004/025864 chronic lymphocytic leukemia, acute lymphocytic leukemia, Non-Hodkin s lymphomas, multiple myeloma, acute myelocytic leukemia, chronic myelocytic leukemia.

Interferons have also been shown to 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 augment the level of expression of human tumor antigens indicates that interferons can be useful in an adjuvant setting for immunotherapy or enhance immunoscintigraphy using anti-tumor antigen antibodies (Guadagni et al., Cancer Immunol. Immunother. 26:222 (1988); Gnadagni et al., Int. J. Biol. Markers 9:53 (1994)). Thus, the present invention includes the use of Cysteine mutant IL-28 or IL-29 proteins, polypeptides and peptides having IL-28 and IL-29 activity as an adjuvant for immunotherapy or to improve immunoscintigraphy using anti-tumor antigen antibodies.

The activity and effect of Cysteine mutant IL-28 or IL-29 on tumor progression and metastasis can be measured in vivo. Several syngeneic mouse models have been developed to study the influence of polypeptides, compounds or other treatments on tumor progression. In these models, tumor cells passaged in culture are implanted into mice of the same strain as the tumor donor. The cells will develop into tumors having similar characteristics in the recipient mice, and metastasis will also occur in some of the models. Appropriate tumor models for our studies include the Lewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No. CRL6323), amongst others. These are both commonly used tumor lines, syngeneic to the C57BL6 mouse, that are readily cultured and manipulated in vitro. Tumors resulting from implantation of either of these cell lines are capable of metastasis to the lung in C57BL6 mice. The Lewis lung carcinoma model has recently been used in mice to identify an inhibitor of angiogenesis (O'Reilly MS, et al. Cell 79: 315-328,1994). C57BL6/J mice are treated with an experimental agent either through daily injection of recombinant protein, agonist or antagonist or a one-time injection of recombinant adenovirus. Three days following this treatment, 105 to 106 cells are implanted under the dorsal skin. Alternatively, the cells themselves may be infected with recombinant

PCT7US2004/025864 adenovirus, such as one expressing Cysteine mutant IL-28 and IL-29, before implantation so that the protein is synthesized at the tumor site or intracellularly, rather than systemically The mice normally develop visible tumors within 5 days. The tumors are allowed to grow for a period of up to 3 weeks, during which time they may reach a size of 1500 - 1800 mm3 in the control treated group. Tumor size and body weight are carefully monitored throughout the experiment. At the time of sacrifice, the tumor is removed and weighed along with the lungs and the liver. The lung weight has been shown ,to correlate well with metastatic tumor burden. As an additional measure, lung surface metastases are counted. The resected tumor, lungs and liver are prepared for histopathological examination, immunohistochemistry, and in situ hybridization, using methods known in the art and described herein. The influence of the expressed polypeptide in question, e.g., Cysteine mutant IL-28 and IL-29, on the ability of the tumor to recruit vasculature and undergo metastasis can thus be assessed. In addition, aside from using adenovirus, the implanted cells can be transiently transfected with Cysteine mutant IL-28 and IL-29. Use of stable Cysteine mutant IL-28 or IL-29 transfectants as well as use of induceable promoters to activate Cysteine mutant IL-28 or IL-29 expression in vivo are known in the art and can be used in this system to assess induction of metastasis. Moreover, purified Cysteine mutant IL-28 or IL-29 conditioned media can be directly injected in to this mouse model, and hence be used in this system. For general reference see, O'Reilly MS, et al. Cell 79:315-328, 1994, and Rusciano D, et al. Murine Models of Liver Metastasis. Invasion Metastasis 14:349361,1995.

Cysteine mutant IL-28 or IL-29 can also be used to treat myocarditis, a disorder that arises when the heart is involved in an inflammatory process. The infiltration of lymphocytes and myocytolysis is thought to result after infection by virus, bacteria, fungi or parasites (see, for example, Brodison et al., J, Infection 37:99 (1998)). Cysteine mutant IL-28 or IL-29 can be injected intravenously or subcutaneously to treat infections associated with myocarditis. Cysteine mutant IL-28 or IL-29 can also be administered intravenously as an immunoregulatory cytokine in the treatment of autoimmune myocarditis. Interferon dosages can be extrapolated using a autoimmune /2 model of myocarditis in the A/J mouse (Donermeyer, et al., J. Exp. Med. 182:1291 (1995)).

Recent reports have highlighted the role of type I interferons in the prevention of viral-induced diabetes by inducing a strong antiviral state in pancreatic beta cells early during viral infection (Flodstroem et al., Nature Immunology 3, 373 382 (2002)). This prevents the loss of beta cells due to viral-induced cell death and autoimmunity that accompanies it. Cysteine mutant IL-28 or IL-29 also induce an antiviral state in cells that express the IL-28 receptor. IL-28 receptor is highly expressed in pancreatic tissue and therefore IL-28 and IL-29 may play a role in prevention of viral-induced diabetes due to beta cell death. In addition, the role of type I interferons in prevention of viral- induced diabetes may be extended to other viralinduced autoimmune diseases and therefore, IL-28 and IL-29 may also play a role in prevention of other diseases such as muscular sclerosis, lupus, and viral-induced autoimmune diseases in tissues that express the IL-28 receptor.

Cysteine mutant L-28 or IL-29 polypeptides can be administered alone or in combination with other vasculogenic or angiogenic agents, including VEGF. When using Cysteine mutant IL-28 or IL-29 in combination with an additional agent, the two compounds can be administered simultaneously or sequentially as appropriate for the specific condition being treated.

Cysteine mutant IL-28 or IL-29 will be useful in treating tumorgenesis, and therefore would be useful in the treatment of cancer. An IL-28 may inhibit B-cell, tumor lines suggesting that there may be therapeutic benefit in treating patients with Cysteine mutant IL-28 or IL- 29 in order to induce the B cell tumor cells into a less proliferative state. The ligand could be administered in combination with other agents already in use including both conventional chemotherapeutic agents as well as immune modulators such as interferon alpha. Alpha/beta interferons have been shown to be effective in treating some leukemias and animal disease models, and the growth inhibitory effects of interferon-alpha and Cysteine mutant IL-28 or IL-29 may be additive for B- cell tumor-derived cell lines.

Within another aspect, the present invention provides a pharmaceutical formulation comprising an isolated polypeptide according to the invention and a pharmaceutically acceptable vehicle.

174797/2

For pharmaceutical use, Cysteine mutant IL-29 proteins are formulated for topical or parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. In general, pharmaceutical formulations will include Cysteine mutant IL-29 polypeptide in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in 10 water, or the like. Formulations may further include one or more excipients, : preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial: surfaces, etc. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995. Cysteine mutant IL-29 will preferably be used in a concentration of about 10 to 100 pg/ml of total volume, although concentrations in the range of 1 ng/ml to 1000 pg/ml may be used. For topical application, such as for the promotion of wound healing, the protein will be applied in the range of 0.1-10 pg/cm2 of wound area, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art. Dosing is daily or intermittently over the period of treatment. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. Sustained release formulations can also be employed. In general, a therapeutically effective amount of IL29 Cysteine i mutant is an amount sufficient to produce a clinically significant change in the treated L condition, such as a clinically significant change in viral load or immune function, a significant reduction in morbidity, or a significantly increased histological score.

As an illustration, pharmaceutical formulations may be supplied as a kit comprising a container that comprises a IL29 polypeptide of the present invention. Therapeutic polypeptides can be provided in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection. Alternatively, such a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a therapeutic polypeptide. Such a kit may further comprise written information on indications and usage of the pharmaceutical composition. Moreover, such information may include a statement that the IL29

- 174797/2 polypeptide formulation is contraindicated in patients with known hypersensitivity to IL-28 or IL29 polypeptide.

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 may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally cloaking them with a human-like surface by replacement of exposed residues, wherein the result is a veneered antibody). In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. One skilled in the art can generate humanized antibodies with specific and different constant domains (i.e., different Ig subclasses) to facilitate or inhibit various immune functions associated with particular antibody constant domains. Antibodies are defined to be specifically binding if they bind to Cysteine mutant IL-28 or IL-29 polypeptide or protein with an affinity at least 10-fold greater than the binding affinity to control (non-Cysteine mutant IL-28 and IL-29) polypeptide or protein. The affinity of a monoclonal antibody can be readily determined by one of ordinary skill in the art (see, for example, Scatchard, Ann. NY Acad. Sei. 51: 660-672, 1949).

Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see for example, Burrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982, which is incorporated herein by reference). The polypeptide immunogen may be a full length molecule or a portion thereof. If the polypeptide portion is hapten-like, such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

A variety of assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to Cysteine mutant IL-28 or IL- 29 polypeptides. Exemplary assays are described in detail in Using Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press,

1999. Representative examples of such assays include: concurrent /2 immunoelectrophoresis, radio-immunoassays, radio- immunoprecipitations, enzymelinked immunosorbent assays (ELISA), dot blot assays, Western blot assays, inhibition or competition assays, and sandwich assays.

For certain applications, including in vitro and in vivo diagnostic uses, it is advantageous to employ labeled antibodies. Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti-complement pairs as intermediates. Antibodies of the present invention may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications^.g., inhibition of cell proliferation). See, in general, Ramakrishnan et al. Cancer Res. 56: 1324-1330,1996.

Passages of the description which are outside the scope of the claims do not constitute part of the claimed invention.

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

EXAMPLES

Example

Mammalian Expression plasmids

An expression plasmid containing zcyto20 and zcyt021 was constructed via homologous recombination. Fragments of zcyto20 and zcyt021 cDNA were generated using PCR amplification. The primers for PCR were as follows:

zcyto20/pZMP21: zc40923, and zc43152 SEQ ID NOs:42 and 43, respectively; and zcyt021/pZMP21: zc40922, and zc43153 SEQ ID NOs:2 and 73, respectively.

The PCR reaction mixture was run on a 1% agarose gel and a band corresponding to the size of the insert was gel-extracted using a QIAquick™ Gel

Extraction Kit (Qiagen, Valencia, CA).

The plasmid pZMP21, which was cut with BglU, was used for recombination with the PCR insert fragment. Plasmid pZMP21 is a mammalian expression vector containing an expression cassette having the MPSV promoter, and multiple restriction sites for insertion of coding sequences; an E. coli origin of replication; a mammalian selectable marker expression unit comprising an SV40 promoter, enhancer and origin of replication, a DHFR gene, and the SV40 terminator; and URA3 and CEN-ARS sequences required for selection and replication in S. cerevisiae. It was constructed from pZP9 (deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, under Accession No. 98668) with the yeast genetic elements taken from pRS316 (deposited.at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 201102209, under Accession No. 77145), an internal ribosome entry site (IRES) element from poliovirus, and the extracellular domain of CD8 truncated at the C-tenninal end of the transmembrane domain.

One hundred microliters of competent yeast (5. cerevisiae) cells were independently combined with 10 pl of the insert DNA and lOOng of the cut pZMP21 vector above, and the mix was transferred to a 0.2-cm electroporation cuvette. The yeast/DNA mixture was electropulsed using power supply (BioRad Laboratories, Hercules, CA) settings of 0.75 kV (5 kV/cm), °־> ohms, and 25 pF. Six hundred pl of 1.2 M sorbitol was added to the cuvette, and the yeast was plated in a 100-pl and 300pl aliquot onto two tJRA-D plates arid incubated at 30°C. After about 72 hours, the Ura+ yeast transformants from a single plate were resuspended in 1 ml ¾0 and spun briefly to pellet the yeast cells. The cell pellet was resuspended in 0.5 ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). The five hundred microliters of the lysis mixture was added to an Eppendorf tube containing 250 pl acid-washed glass beads and 300 pl phenol-chlorofonn, was vortexed for 3 minutes, and spun for 5 minutes in an Eppendorf centrifuge at maximum speed. Three hundred microliters of the aqueous phase was transferred to a fresh tube, and the DNA was precipitated with 600 pl ethanol (EtOH) and 30pl 3M sodium acetate, followed by centrifugation for 30 minutes at maximum speed. The DNA pellet was resuspended in 30 pl TE.

Transformation of electrocompetent E. coli host cells (MC1061) was done using 5 pl of the yeast DNA prep and 50 μΐ of cells. The cells were electropulsed at 2.0 kV, 25 pF, and 400 ohms. Following electroporation, 1 ml SOC (2% Bacto™ Tryptone (Difcb, Detroit, MI), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KC1, 10 mM MgC12, 10 mM MgSO4, 20 mM glucose) was added and then the cells were plated in a 50 pl and 200 pl aliquot on two LB AMP plates (LB broth (Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/L Ampicillin).

The inserts of three clones for each construct were subjected to sequence analysis and one clone for each construct, containing the correct sequence, was selected. Larger scale plasmid DNA was isolated using a commercially available kit (QIAGEN Plasmid Mega Kit, Qiagen, Valencia, CA) according to manufacturer’s instructions. The correct constructs were designated zcyto20/pZMP21 and zcyt021/pZMP21.

Example

Expression of Mammalian Constructs in CHO cells

200pg of a zcyto20/pZMP21 and zcyt021/pZMP21 construct were digested with 200 units of Pvu I at 37°C for three hours and then were precipitated with IPA and spun down in a 1.5 mL microfuge tube. The supernatant was decanted off the pellet, and the pellet was washed with 1 mL of 70% ethanol and allowed to incubate for 5 minutes at room temperature. The tube was spun in a microfuge for 10 minutes at 14,000 RPM and the supernatant was aspirated off the pellet. The pellet was then resuspended in 750 pl of PF-CHO media in a sterile environment, and allowed to incubate at 60°C for 30 minutes. CHO cells were spun down and resuspended using the DNA-media solution. The DNA/cell mixture was placed in a 0.4 cm gap cuvette and electroporated using the following parameters: 950 pF, high capacitance, and 300 V. The contents of the cuvette were then removed and diluted to 25 mLs with PF-CHO media and placed in a 125 mL shake flask. The flask was placed in an incubator on a shaker at 37°C, 6% CO2, and shaking at 120 RPM.

Example

Purification and Analysis of zcyto20-CHQ Protein

A. Purification ofZcyto20-CHO Protein

Recombinant zcyto20 (IL-28A) protein was produced from a pool of DXB11-CHO cell, lines. Cultures were harvested, and the media were sterile filtered using a 0.2 pm filter.

The purification of zcyto20-CHO protein was achieved by the sequential use of a Poros HS 50 column (Applied Biosystems, Framingham, MA), a Monolithic WCX column (Isco, Inc., Lincoln, NE), a ToyoPearl Butyl 650S column,.־ (TosoH, Montgomeryville, PA), and a Superdex 75 column (Amersham Biosciences, Piscataway, NJ). Culture media from DXB111-CHO were adjusted to pH 6.0 before loading onto a Poros 50 HS column. The column was washed with 50 mM MES (2Morpholinoethanesulfonic acid), 100 mM NaCl, pH 6 and the bound protein was eluted with a 10 column volumes (CV) linear gradient to 60% of 50 mM MES, 2 M NaCl, pH

6. The eluting fractions were collected and the presence of zcyto20 protein was confirmed by SDS-PAGE with a Coomassie staining. This fractions containing zcyto20 protein were pooled, diluted with double distilled water to a conductivity of about 20 mS, and loaded onto a Monolithic WCX column. The column was washed with 93% of 50 mM MES, 100 mM NaCl, pH 6, and 7% of 50 mM MES, 2 M NaCl, pH 6. The bound protein was eluted with a 25-CV linear gradient from 7% to 50% of 50 mM MES, 2 M NaCl, pH 6. The eluting fractions were collected and the presence of zcyto20 protein was confirmed by SDS-PAGE with a Coomassie staining. The fractions containing zcyto20 protein were pooled, adjusted to 1 M ammonium sulfate and loaded onto a ToyoPearl Butyl 650S column. Zcyto20 was eluted with a decreasing ammonium sulfate gradient and the fractions containing the pure zcyto20 were pooled and concentrated for injection into a Superdex 75 column. Fractions containing zcyto20 protein from the gel filtration column was pooled, concentrated, filtered

PCT7US2004/025864 through a 0.2 pm filter and frozen at -80°C. The concentration of the final purified protein was determined by a BCA assay (Pierce Chemical Co., Rockford, IL) and

HPLC-amino acid analysis.

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

Recombinant zcyto20 protein was analyzed by SDS-PAGE (Nupage 412% Bis-Tris, Invitrogen, Carlsbad, CA) and Western blot using rabbit anti-zcyt021CEE-BV IgG as the primary antibody that cross-reacts to zcyto20-CHO protein. The gel was electrophoresed using Invitrogen’s Xcell H mini-cell (Carlsbad, CA) and transferred to a 0.2 pm nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA) using Invitrogen’s Xcell Π blot module according to directions provided in the instrument manual. The transfer was run at 500 mA for 50 minutes in a buffer containing 25 mM Tris base, 200 mM glycine, and 20% methanol. The membrane was blocked with 10% non-fat dry milk in lx PBS for 10 minutes then probed with the primary antibody in lx PBS containing 2.5% non-fat dry milk. The blot was labeled for one hour at room temperature while shaking. For the secondary antibody labeling, blot was washed three times for 10 minutes each with PBS and then probed with goat antirabbit IgG-HRP (Pierce Chemical Co., Rockford, IL) for one hour. The blot was washed three times with lx PBS for 10 minutes each and developed using a 1:1 mixture of SuperSignal® ULTRA reagents (Pierce Chemical Co., Rockford, IL) and the signal was captured using a Lumi-hnager (Boehringer Mannheim GmbH, Germany).

C. Summary of protein purification and analysis

The purified zcyto20 protein from the CHO media migrated predominantly as a doublet at approximately 20 kDa and a minor triplet dimer at about 38 kDa on a 4-12% Bis-Tris gel under non-reducing conditions. They all collapsed into a single 20 kDa band under reducing conditions. MS peptide mapping indicated a mixture of two isomers with respect to disulfide linkage and the presence of O-linked glycosylation site.

Example

Purification and Analysis of zcvto21-CHO Protein

A. Purification ofZcyto21-CHO Protein

Recombinant zcyt021 was produced from stable DXB11-CHO cell lines. Cultures were harvested, and the media were sterile filtered using a 0.2 μπι filter. Proteins were purified from the conditioned media by starting with a combination of cationic and anionic exchange chromatography followed by a hydrophobic interaction chromatography and a size exclusion chromatography. DXB111-CHO culture media were adjusted to pH 6.0 before loading onto a Poros 50 HS column (Applied Biosystems, Framingham, MA). The column was washed with lx PBS, pH 6 and the bound protein was eluted with 5x PBS, pH 8.4. The eluting fraction was collected and the presence of zcyt021 protein was confirmed by SDS-PAGE with a Coomassie stain. This fraction was then diluted to a conductivity of 13 mS and its pH adjusted to.8.4 and flowed through a Poros 50 HQ column (Applied Biosystems, Framingham, MA). The flow-through containing zcyt021 protein were then adjusted to about 127 mS with ammonium sulfate and loaded onto a Toyopearl Phenyl 650S column (TosoH, Montgomeryville, PA). Zcyt021 protein was eluted with a decreasing ammonium sulfate gradient and the fractions containing the pure zcyt021 were pooled and ׳ concentrated for injection into a Superdex 75 column (Amersham Biosciences, Piscataway, NJ). The concentration of the final purified protein was determined by a BCA assay (Pierce Chemical Co., Rockford, IL) and HPLC-amino acid analysis. i:

B. SDS-PAGE and Western blotting analysis of zcyto21-CHO protein

Recombinant zcyt021 protein was analyzed by SDS-PAGE (Nupage 412% Bis-Tris, Invitrogen, Carlsbad, CA) and Western blot using rabbit anti-zcyt021CEE-BV IgG as the primary antibody. The gel was electrophoresed using Invitrogen’s Xcell Π mini-cell (Carlsbad, CA) and transferred to a 0.2 μπι nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA) using Invitrogen’s Xcell Π blot module annording to directions provided in the instrument manual. The transfer was run at 500 mA for 50 minutes in a buffer containing 25 mM Tris base, 200 mM glycine, and 20% methanol. The transferred blot was blocked with 10% non-fat dry milk in lx PBS for minutes then probed with the primary antibody in lx PBS containing 2.5% non-fat dry milk. The blot was labeled for one hour at room temperature while shaking. For the secondary antibody labeling, blot was washed three times for 10 minutes each with PBS and then probed with goat anti-rabbit IgG-HRP (Pierce Chemical Co., Rockford, IL) for one hour. The blot was washed three times with lx PBS for 10 minutes each and developed using a 1:1 mixture of SuperSignal® ULTRA reagents (Pierce Chemical Co., Rockford, IL) and the signal was captured using a Lumi-Imager (Boehringer Mannheim GmbH, Germany).

C. Summary of protein purification and analysis

The purified zcyt021 protein from the CHO media migrated as two or more approximately 28 kDa bands on a 4-12% Bis-Tris gel under both reducing and non-reducing conditions. MS peptide mapping indicated a mixture of two isomers .with respect to disulfide linkage and the presence of one N-linked glycosylation and several O-linked glycosylation sites.

Example

Identification of ]L-29 Forms

Peak fractions from purified pools of IL-29 were digested overnight at 37°C with sequencing grade trypsin (Roche Applied Science, Indianapolis, IN) in phosphate buffer at approximately pH 6.3 to limit disulfide re-arrangement. Each digest was analyzed by reversed-phase HPLC (Agilent, Palo Alto, CA) connected inline to a quadrupole-time of flight hybrid mass spectrometer (Micromass, Milford MA). Spectra were collected, converted from mass to charge ratio to mass, and compared to all theoretical peptides and disulfide-linked peptide combinations resulting from trypsin digestion of IL-29. Disulfides were assigned by comparing spectra before and after reduction with assignment of appropriate masses to disulfide linked peptides in IL-29. The material from fraction #20 showed the disulfide pattern C15 — C112 and C49 — C145 with C171 observed as a S-glutathionyl cysteine (all referring to SEQ ID NO:4).

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The material from fraction #51 showed the disulfide pattern C49 — C145 and Cl 12 —

C171 with C15 observed as an S-glutathionyl cysteine (referring to SEQ ID NO:4).

Example

E, coli Expression Plasmids

Construction of expression vector, pTAP237

Plasmid pTAP237 was generated by inserting a PCR-generated linker into the Smal site of pTAP186 by homologous recombination. Plasmid pTAP186 was derived from the plasmids pRS316 (a Saccharomyces cerevisiae shuttle vector) and pMAL-c2, an E. coli expression plasmid derived from pKK223-3 and comprising the tac promoter and the rmB terminator. Plasmid pTAP186 contains a kanamycin resistance gene in which the Sma I site has been destroyed and has Nori and Sfil sites flanking the yeast ARS-CEN6 and URA3 sequences, facilitating their removal from the plasmid by digestion with Noll. The PCR-generated linker replaced the expression coupler sequence in pTAP186 with the synthetic RBS H sequence. It was prepared from 100 pmoles each of oligonucleotides zc29,740 and zc29,741, as shown in SEQ ID NOS: 44 and 45, respectively, and approximately 5 pmoles each of oligonucleotides zc29,736 and zc29,738, as shown in SEQ ID NOs:46 and 47, respectively. These oligonucleotides were combined by PCR for ten cycles of 94°C for 30 seconds, 50 C for 30 seconds, and 72°C for 30 seconds, followed by 4°C soak. The resulting PCR י products were concentrated by precipitation with two times the volume of 100% ethanol. Pellet was resuspended in 10 pL water to be used for recombining into the recipient vector pTAP186 digested with Smal to produce the construct containing the synthetic RBS Π sequence. Approximately 1 pg of the PCR-generated linker and 100 ng of pTAP186 digested with Smal were mixed together and transformed into competent yeast cells (S. cerevisiae). The yeast was then plated onto -URA D plates and left at room temperature for about 72 hours. Then the Ura+ transformants from a single plate were resuspended in 1 mL H2O and spun briefly to pellet the yeast cells. The cell pellet was resuspended in 0.5 mL of lysis buffer. DNA was recovered and transformed into E. coli MCI061. Clones were screened by colony PCR as disclosed above using pmoles each of oligonucleotides zc29,740 and zc29,741, as shown in SEQ ID NOS: 44 and 45, respectively. Clones displaying the correct size band on an agarose gel were subject to sequence analysis. The correct plasmid was designated pTAP237.

Example

Codon Optimization of IL-29 Cysteine mutant

A. Codon Optimization Generation of the IL-29 ־wildtype expression construct

Native human IL-29 gene sequence was not well expressed in E. coli strain W3110. Examination of the codons used in the IL-29 coding sequence indicated that it contained an excess of the least frequently used codons in E. coli with a CAI value equal to 0.206. The CAI is a statistical measure of synonymous codon bias and can be used to predict the level of protein production (Sharp et al., Nucleic Acids Res. 15(31:1281-95, 1987). Genes coding for highly expressed proteins tend to have high CAI values (> 0.6), while proteins encoded by genes with low CAI values (< 0.2) are generally inefficiently expressed. This suggested a reason for the poor production of IL-29 in E. coli. Additionally, the rare codons are clustered in the second half of the message leading to higher probability of translational stalling, premature termination of \ translation, and amino acid misincorporation (Kane JF. 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 dramatically improved when the level of certain rare tRNAs is increased within the host (Zdanovsky et al., Applied Enviromental Microb. 66:31663173, 2000; You et al,. Biotechniques 27:950-954, 1999). The pRARE plasmid carries genes encoding the tRNAs for several codons that are rarely used E. coli (argU, argW, leuW, proL, ileX and glyT). The genes are under the control of their native promoters (Novy, ibid.). Co-expression with pRARE enhanced IL-29 production in E. coli and yield approximately 200 mg/L. These data suggest that re-resynthesizing the gene coding for IL-29 with more appropriate codon usage provides an improved vector for expression of large amounts of IL-29.

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The codon optimized IL-29 coding sequence was constructed from sixteen overlapirig oligonucleotides: zc44,566 (SEQ ID NO:48), zc44,565 (SEQ ED NO:49), zc44,564 (SEQ ED NO:50), zc44,563 (SEQ ID NO:51), zc44,562 (SEQ ED NO:52), zc44,561 (SEQ ID NO:53), zc44,560 (SEQ ID NO:54), zc244,559 (SEQ ID NO:55), zc44,558 (SEQ ED NO:56), zc44,557 (SEQ ID NO:57). Primer extension of these overlapping oligonucleotides followed by PCR amplication produced a full length IL-29 gene with codons optimized for expression in E. col. The final PCR product was inserted into expression vector pTAP237 by yeast homologous recombination. The expression construct was extracted from yeast and transformed into competent E. coli MC1061. Clones resistance to kanamycin were identified by colony PCR. A positive clone was verified by sequencing and subsequently transformed into production host strain W3110. The expression vector with the optimized IL-29 sequence was named pSDH184. The resulting gene was expressed very well in E. coli. expression, levels with the new construct increased to around 250 mg/L.

B. Generation of the codon optimized zcyt021 C172S Cysteine mutant expression construct

The strategy used to generate the zcyt021 C172S Cysteine mutant is based on the QuikChange Site-Directed Mutagenesis :Kit (Stratagene). Primers were designed to introduce the C172S mutation based on manufacturer’s suggestions. These primers were designated ZG44,340 (SEQ ID NO:58) and ZG44.341 (SEQ ID NO:59). PCR was performed to generate the zcyt021 C172S Cysteine mutant according to QuikChange Mutagenesis instructions. Five identical 50 μΐ reactions were set-up. 2.5 μΐ pSDH175 (missing yeast vector backbone sequence) DNA was used as template per reaction. A PCR cocktail was made up using the following amounts of reagents: 30 μΐ lOx PCR buffer, 125 ng (27.42 μΐ) ZG44.340,125 ng (9.18 μΐ) ZG44,341, 6 μΐ dNTP, 6 μΐ Pfu Turbo polymerase (Stratagene, La Jolla, CA), and 206.4 μΐ water. 47.5 μΐ of the cocktail was aliqubtted into each reaction. The PCR conditions were as follows: 1 cycle of 95°C for 30 seconds followed by 16 cycles of 95°C for 30 seconds, 55°C for 1 minute, 68°C for 7 minutes, followed by 1 cycle at 68°C for 7 minutes, and ending with a 4°C hold. All five PCR reactions were consolidated into one tube. As per

PCI7US2004/025864 manufacturer’s instructions, 5 pl Dpnl restriction enzyme was added to the PCR reaction and incubated at 37°C for 2 hours. DNA was precipitated my adding 10% 3 Molar Sodium Acetate and two volumes of 100% ethanol. Precipitation was carriedout at -20°C for 20 minutes. DNA was spun at 14,000 rpm for 5 minutes and pellet was speed-vac dried. DNA pellet was resuspended in 20 μΐ water. DNA resulting from PCR was transformed into E.coli strain DH10B. 5μ1 DNA was mixed with 40 μΐ ElectroMAX DH10B cells (Invitrogen). Cells and DNA mixture were then electroporated in a 0.1cm cuvette (Bio-Rad) using a Bio-Rad Gene Pulser Π™ set to 1.75 kV, 100 Ω, and 25 pF. Electroporated cells were then outgrown at 37°C for 1 hour. Mixture was plated on an LB + 25 pg/ml kanamycin plate and incubated at 37°C overnight. Ten clones were screened for presence of zcyt021 C172S insert. DNA was isolated from all ten clones using the QIAprep™ Spin Miniprep Kit (Qiagen, Valencia, .CA) and analyzed for presence of insert by cutting with Xbal and PstI restriction enzymes. Nine clones contained insert and were sequenced to insure the zcyt021 C172S mutation had been introduced. A clone was sequence verified and was subsequently labeled pSDH188.

Example

E, coli IL-29 expression construct

A DNA fragment of IL-29 containing the wildtype sequence was isolated using PCR. Primers zc41,212 (SEQ ID NO: 60) containing 41 base pair (bp) of vector flanking sequence and 24 bp corresponding to the amino terminus of IL-29, and primer zc41,041 (SEQ ID NO:61) contained 38 bp corresponding to the 3’ end of the vector which contained the zcyt021 insert were used in the reaction. The PCR conditions were as follows: 25 cycles of 94°C for 30 seconds, 50 C for 30 seconds, and 72°C for 1 minute; followed by a 4°C soak. A' small sample (2-4 pL) of the PCR sample was run on a 1% agarose gel with IX TBE buffer for analysis, and the expected band of approximately 500 bp fragment was seen. The remaining volume of the 100 pL reaction was precipitated with 200 pL absolute ethanol. The pellet was resuspended in 10 pL water to be used for recombining into recipient vector pTAP238 cut with Smal to produce the construct encoding the zcyt021 as disclosed above. The clone with correct sequence was designated as pTAP377. Clone pTAP377 was digested with Notl/Ncol (10μ1 DNA, 5μ1 buffer 3 New England BioLabs, 2 μΕ Not 1, 2 μΕ Ncol, 31 pL water for 1 hour at 37°C) and religated with T4 DNA ligase buffer (7 μΕ of the previous digest, 2 pL of 5X buffer, 1 μΕ of T4 DNA ligase). This step removed the yeast sequence, CEN-ARS, to streamline the vector. The pTAP337 DNA was diagnostically digested with Pvu2 and Pstl to confirm the absence of the yeast sequence. P/taP377 DNA was transformed into E. coli strain W3U0/pRARE, host strain carrying extra copies of rare E. coli tRNA genes.

Example

E. coli IL-28 A expression construct

A DNA fragment containing the wildtype sequence of zcyto20 (as 15 shown in SEQ ID NO: 1) was isolated using PCR. Primers zc43,431 (SEQ ID NO:62) containing 41 bp of vector flanking sequence and 24 bp corresponding to the amino terminus of zcyto20, and primer zc43,437 (SEQ ID NO:63) contained 38 bp corresponding to the 3’ end of the vector which contained the zcyto20 insert. The PCR conditions were as follows: 25 cycles of 94°C for 30 seconds, 50°C for 30 seconds, and 20 72°C for 1 minute; followed by a 4°C soak. A small sample (2-4 μΕ) of the PCR sample was run on a 1% agarose gel with IX TBE buffer for analysis, and the expected band of approximately 500 bp fragment was seen. The remaining volume of the 100 μΕ reaction was precipitated with 200 pL absolute ethanol. The pellet was resuspended in 10 μΕ water to be used for recombining into recipient vector pTAP238 cut with Smal to produce the construct encoding the zcyto20 as disclosed above. The clone with correct sequence was designated as pYEL7. It was digested with Notl/Ncol (10μ1 DNA, 5μ1 buffer 3 New England BioLabs, 2 pL Notl, 2 pL Ncol, 31 pL water for 1 hour at 37°C) and religated with T4 DNA ligase buffer (7 pL of the previous digest, 2 pL of 5X buffer, 1 pL of T4 DNA ligase). This step removed the yeast sequence, CEN-ARS, to 30 streamline the vector. Hie relegated pYEL7 DNA was diagnostically digested with

Pvu2 and Pstl to confirm the absence of the yeast sequence. PYEL7 DNA was transformed, into E. coli strain W3110/pRARE.

Example zcvt021 C172S Cysteine mutant expression construct

The strategy used to generate the zcyt021 C172S Cysteine mutant (SEQ ID NO: 28) is based on the QuikChange® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Primers were designed to introduce the C172S mutation based on manufacturer’s suggestions. These primers were designated ZG44.327 and ZG44,328 (SEQ ID NOs:64 and 65, respectively). PCR was performed to generate the zcyt021 C172S Cysteine mutant according to QuikChange Mutagenesis instructions. Five identical 50 μΐ reactions were set-up. 2.5 μ1 pTAP377 (missing yeast vector backbone sequence) DNA was used as template per reaction. A PCR cocktail was made up using the following amounts of reagents: 30 μΐ lOx PCR buffer, 125 ng (27.42 μΐ) ZG44,327 (SEQ ID NO: 64), 125 ng (9.18 pl) ZG44,328 (SEQ ID NO: 65), 6 μΐ dNTP, 6 μΐ Pfu ־ Turbo polymerase (Strategene), and 206.4 μΐ water. 47.5 μ1 of the cocktail was aliquotted into each reaction. The PCR conditions were as follows: 1 cycle of 95°C for , 30 seconds followed by 16 cycles of 95°C for 30 seconds, 55°C for 1 minute, 68°C for Ί minutes, followed by 1 cycle at 68°C for ר minutes, and ending with a 4°C hold. All five PCR reactions were consolidated into one tube. As per manufacturer’s .

instructions, 5 μΐ Dpnl restriction enzyme was added to the PCR reaction and incubated at 37°C for 2 hours. DNA was precipitated my adding 10% 3 Molar Sodium Acetate and two volumes of 100% ethanol (Aaper Alcohol, Shelbyville, KY). Precipitation was carried-out at -20°C for 20 minutes. DNA was spun at 14,000 rpm for 5 minutes and pellet was speed-yac dried. DNA pellet was resuspended in 20 μΐ water. DNA resulting from PCR was transformed into E.coli strain DH10B. 5 μΐ DNA was mixed with 40 μΐ

ElectroMAX DH10B cells (Invitrogen, Carlsbad, CA). Cells and DNA mixture were then electroporated in a 0.1cm cuvette (Bio-Rad, Hercules, CA) using a Bio-Rad Gene

Pulser D™ set to 1.75kV, 100Ω, and 25 μΓ. Electroporated cells were then outgrown at

37°C for 1 hour.

Mixture was plated on an LB + 25 μg/ml kanamycin plate and incubated at 37 °C overnight. Ten clones were screened for presence of IL-29 insert. DNA was isolated from all ten clones using the QIAprep™ Spin Miniprep Kit (Qiagen) and analyzed for presence of insert by cutting with Xbal (Roche) and PstI (New England Biolabs) restriction enzymes. Nine clones contained insert and were sequenced to insure the zcyt021 C172S mutation had been introduced. A clone (isolet #6) was sequence verified and was subsequently labeled pSDH171. A similar strategy can be implemented to generate a zcyt021 C15S mutant.

Example zcyto20 C49S Cysteine mutant expression construct

The zcyto20 C49S Cysteine mutant coding sequence was generated by overlap PCR (SEQ ID NO:20). The first 187 bases of the wildtype IL-28A sequence (SEQ ID NO:1) was generated by PCR amplification using pYEL7 (SEQ ID NO:67) as template and oligonucleotide primers zc43,431 (SEQ ID NO: 62) and zc45,399' (SEQ ID NO:66). The second DNA fragment from base 105 to 531 was generated by PCR amplification using pYEL7 (SEQ ID NO:67) as template and oligonucleotide primers zc45,398 (SEQ ID NO: 68) and zc43,437 (SEQ ID NO:63). Primers zc45,399 (SEQ ID t NO:66) and zc45,398 (SEQ ID NO:68) contained the specific modified sequence which changed the cysteine 49 to a serine. These two PCR products were combined and PCR overlap amplified using oligonucleotide primers zc43,431 (SEQ ID NO:62) and zc43,437 (SEQ ID NO:63). The final PCR product was inserted into expression vector pTAP238 by yeast homologous recombination (Raymond et al. Biotechniques. Jam 26(1):134-8, 140-1, 1999). The expression construct was extracted from yeast and transformed into competent E. coli DH10B. Kanamycin resistant clones were screened by colony PCR. A positive clone was verified by sequencing and subsequently transformed into production host strain W3U0/pRARE. The expression construct with the zcyto20 C49S Cysteine mutant coding sequence was named pCHAN9.

Example 12 zcyto20 C51S Cysteine mutant expression construct

The zcyto20 C51S Cysteine mutant coding sequence was generated by overlap PCR (SEQ ID NO124). The first 193 bases of the wildtype IL-28A sequence was generated by PCR amplification using pYEL7 (SEQ ID NO:67) as template and oligonucleotide primers zc43,431 (SEQ ID NO:62) and zc45,397 (SEQ ID NO:63). The second DNA fragment from base 111 to 531 was generated by PCR amplification using pYEL7 (SEQ ID NO:67) as template and oligonucleotide primers zc45,396 (SEQ ID NO:70) and zc43,437 (SEQ ID NO:63). Primers zc45,397 (SEQ ID NO:69) and zc45,396 (SEQ ID NO:70) contained the specific modified sequence which changed the cysteine51 to a serine. These two PCR products were combined and PCR overlap amplified using oligonucleotide primers zc43,431 (SEQ ID NO:62) and zc43,437 (SEQ ID NO:63). The final PCR product was inserted into our in-house expression vector pTAP238 by yeast homologous recombination (Raymond et al. supra). The expression construct was extracted from yeast and transformed into competent E. coli DH10B. Kanamycin resistant clones were screened by colony PCR. A positive clone was verified by sequencing and subsequently transformed into production host strain W3110/pRARE The expression construct with the zcyto20 C50S Cysteine mutant coding sequence was named pCHANlO.

Example

Expression of Π-28Α, IL-29 and Cvs to Ser Cysteine mutants in E. coli

In separate experiments, E. coli transformed with each of the expression vectors described jn Examples 6-9 were inoculated into 100 mL Superbroth Π medium (Becton Dickinson, San Diego, CA) with 0.01% Antifoam 289 (Sigma Aldrich, St. Louis, MO), 30 pg/ml kanamycin , 35 μg/ml chloramphenicol and cultured overnight at 37°C. A 5 mL inoculum was added to 500 mL of same medium in a 2 L culture flask which was shaken at 250 rpm at 37°C until the culture attained an OD600 of 4. IPTG was then added to a final concentration of 1 mM and shaking was continued for another

2.5 hours. The cells were centrifuged at 4,000 x g for 10 min at 4 °C. The cell pellets were frozen at -80°C until use at a later time.

Example

Refolding and Purification of IL-28

A. Inclusion body preparation

Human wildtype IL-29 was expressed in E. coli strain W3110 as inclusion bodies as described above. A cell pellet from a fed-batch fermentation was resuspended in 50 mM Tris, pH 7.3. The suspension was passed through an APVGaulin homogenizer (Invensys APV, Tonawanda, New York) three times at 8000 psi. The insoluble material was recovered by centrifugation at 15,000 g for 30 minutes. The pellet was washed consecutively with 50 mM Tris, 1% (v/v) Triton X100, pH 7.3 and 4 M Urea. The inclusion body was then dispersed in 50 mM Tris, 6 M guanidine hydrochloride, 5 mM DTT at room temperature for 1 hour. The material , was then centrifuged at 15,000 g for 1 hour. The supernatant from this step contains reduced soluble IL-29.

B. Refolding

The solubilized EL-29 was diluted slowly into 50 mM Tris, pH 8,0.75 M , Arginine, 0.05% PEG3350, 2 mM MgC12, 2 mM CaCl2, 0.4 mM KC1,10 mM NaCl, 4 mM reduced Glutathione, 0.8 mM oxidized Glutathione at room temperature while stirring. The final concentration of IL-29 in the refolding buffer was 0.1 mg/ml. The refolding mixture was left at room temperature overnight. Concentrated acetic acid was then used to adjust the pH of the suspension to 5. The suspension was then filtered through a 0.2 pm filter. RP-HPLC analysis of the refolding mixture showed two prominent peaks.

C. Purification

The refolding mixture was in-line diluted (1:2) with 50 mM NaOAc at pH 5 and loaded onto a Pharmacia SP Sepharose Fast Flow cation exchange column (North Peapack, NJ). The column was washed with 3 column volumes of 50 mM NaOAc, 400 mM NaCl, pH 5. The bound IL-29 was eluted with 50 mM NaOAc, 1.4 M

NaCl, pH 5. Solid (NH4)2SO4 was added to the elute pool of the cation exchange step so that the final concentration of (NH4)2SO4 was 0.5 M. The material was then loaded onto a ToyoPearl Phenyl 650S HIC column (Tosoh Biosep, Montgomery, PA). The column was then washed with 3 column volumes of 50 mM NaOAc, 1 M (NH4)2SO4, pH 5. A linear gradient of 10 column volumes from 50 mM NaOAc, 1 M (NH4)2SO4, pH 5 to 50 mM NaOAc, pH 5 was used to elute the bound zcyt021. Fractions were collected of the elute. Two prominent peaks were observed in this step. RP-HPLC analysis of the elute fractions was performed. Two products corresponding to two disulfide bond isomers were produced after final buffer exchange into PBS, pH 7.3.

Example

Refolding and Purification of IL-29 Cysteine mutant

As described in Example 3, purification of EL-29 produced two disulfide bond isomers. A HIC FPLC step was employed to separate the two forms.,. The separation was not baseline resolved. Severe “Peak Shaving” had to be used to obtain substantially pure isomers (>95%). The yield for this step and by extension for the whole process suffered. The final yields were 8% and 9% for the C15-C112 form and C112-C171 form respectively. Wildtype IL-29 produced in CHO and baculovirus (BV) systems also showed similar phenomena. It was established that the C15-C112 form of the isomer is homologous in disulfide bond patterns to type I INF’s. The C15-C112 form also demonstrated 30-fold higher bioactivity than the C112-C171 form in an ISRE assay (see below).

Refolding and purification 0fzcyt021 Cysl72Ser mutein

The inclusion body preparation, refolding and purification of zcyto21 C172S polypeptide (SEQ ID NO:29) is essentially the same as those of IL-29 wild-type (SEQ ED NO :4). RP-HPLC analysis of the refolding mixture of the mutein showed only one prominent peak corresponding to the C15-C112 form of the wild-type IL-29. Subsequent HIC chromatography show only a single peak. It was therefore unnecessary to employ severe “peak shaving”. The final yield for the entire process is close to 50%.

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The zcyt021 Cysl72Ser polypeptide (SEQ ID NO:29) showed equivalent bioactivity to the C15-C112 form of wild-type IL-29 in ISRE assay shown in Example 16.

Example

Antiviral Activity: Cytopathic Effect in Hela and L929 cells

Initial functional assays for antiviral activity were conducted using conditioned media from transiently transfected human embryonal kidney (HEK) cells. Production of this conditioned medium is described as follows. A full-length cDNA for human or murine IL-28A, IL-28B, or JL-29 was cloned into the pzp7Z vector using standard procedures. The human or murine IL-28 A, IL-28B, or IL-29 constructs were transfected into 293 HEK cells. Briefly, for each construct 700,000 cells/well (6 well plates) were plated approximately 18h prior to transfection in 2 milliliters DMEM + 10% fetal bovine serum. Per well, 1.5 micrograms human or murine IL-28A, IL-28B, or IL-29 DNA and 0.5 micrograms pIRES2-EGFP DNA (Clontech) were added to 6 microliters Fugene 6 reagent (Roche Biochemicals) in a total of 100 microliters DMEM. Two micrograms pIRES2-EGFP DNA alone was used as a negative control. These transfection mixtures were added 30 minutes later to the pre-plated 293 cells. Twenty-four hours later the cell media were removed and DMEM + 0.1% bovine serum albumin was added. Conditioned media was collected after 48 hours, filtered through a 0.45 micron filter and used for antiviral and reporter assays.

Antiviral Assays were carried out using human cervical carcinoma cells (HeLa) and mouse fibroblast cells (L929). On the first day, conditioned medium containing human or murine IL-28A, IL-28B, or IL-29 was diluted and plated with 50,000 cells in a 96-well flat bottom microtiter plate. Following a 24-hour incubation at 37°C, the medium was removed and replaced with medium containing encephelomyocarditis virus at a multiplicity of infection of 0.1. The cells were again incubated for 24 hours at 37°C. Culture wells were then scored visually on a 4-point scale for the presence of cytopathic effect, which was then converted to %CPE as shown in Table 7. Conditioned medium from cells transfected with GFP alone and purified human interferon-a-2a or murine interferon-alpha were included as controls.

Table 7: Determination of Cytopathic Effect

<td> Designation</td><td> Observation of Cytopathic Effect (CPE)</td>

<td> +/-</td><td> Possible CPE (about 1% of monolayer surface)</td>

<td> +</td><td> CPE limited to one plaque (about 5% of the surface)</td>

<td> +1</td><td> CPE is; limited to three plaques, affecting less than 25% of the monolayer</td>

Table 8 shows that conditioned medium containing human or murine IL28A, 1L-28B, or IL-29 inhibited viral infection (%CPE) in HeLa cells in a dose5 dependent manner, while control GFP conditioned medium failed to significantly block ; the appearance of cytopathic effect. As shown in Table 9, conditioned medium containing human or murine IL-28 A, IL-28B, or IL-29 did not inhibit viral infection in L929 cells. In both experiments purified interferon showed positive antiviral activity.

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Table 8: Percentage Cytopathic Effect of human or murine IL-28 A, IL28B, or IL-29 in HeLa Cells using Conditioned Medium (CM)_____________________

Relative CM Control zcyto20 zcyt021 zcyt022 zcyt024 zcyt025 hffN- hIFN-a-2a

<td colspan="2" rowspan="2"> Concentration GFP</td><td colspan="2"> IL-28A IL-29</td><td rowspan="2"> IL-28B (CM)</td><td rowspan="2"> mouse IL-28 (CM)</td><td rowspan="2"> mouse IL-28 (CM)</td><td rowspan="2"> a-2a</td><td rowspan="2"> Concentration</td>

Table 9: Percentage Cytopathic Effect of human or murine JL-28A, IL5 28B, or IL-29 in L929 Cells using Conditioned Medium (CM)_______________________

<td rowspan="2"> Relative CM Cone.</td><td rowspan="2"> Control GFP</td><td rowspan="2"> zcyt02C (CM)</td><td rowspan="2"> l zcyt02 1 (CM)</td><td colspan="2"> zcyt02 zcyt024</td><td rowspan="2"> zcyt025 (CM)</td><td rowspan="2"> mlFNalpha</td><td rowspan="2"> mIFN-alpha Cone.</td>

Example

Signaling Via Interferon-Response Pathway

Interaction of type 1 interferons with their specific receptor leads to induction of a number of genes responsible for their antiviral/antiproliferative activity.

These include 2’-5’ oligoadenylate synthetase (2-5 OAS), double-stranded RNA dependent Pkr kinase (Pkr), phospholipid scramblase, and intercellular adhesion molecule-1 (ICAM-1). Induction of genes with as yet unknown function, such as a 56kDa interferon stimulated gene product (DSG-56k), also occurs. To determine if some or all of these genes are induced upon treatment of cells with IL-28A, human Daudi B lymphoid cells were treated for 72 hours with conditioned medium from Sf9 cells infected with baculovirus expressing IL-28A. Conditioned medium from Sf9 cells infected with wild-type baculovirus was used as a negative control. Following treatment cells were collected and lysed for isolation of total RNA. One microgram of total RNA was converted to cDNA using reverse transcriptase and used as a template for polymerase chain reaction using oligonucleotide primers specific for the human interferon-stimulated genes described above. Oligonucleotide primers for human glycerol-3-phosphate dehydrogenase (G3PDH) were used as a non-interferon stimulated gene control. The results show clear induction of ISG-56k, Pkr, 2-5 OAS and phospholipid scramblase following treatment of cells with 1L-28A. No induction was seen for ICAM-1 or the non-interferon stimulated gene control, G3PDH.

Example

Signal Transduction Reporter Assay

A signal transduction reporter assay can be used to determine the functional interaction of human and mouse IL-28 and IL-29 with the IL-28 receptor. Human embryonal kidney (HEK) cells are transfected with a reporter plasmid containing an interferon-stimulated response element (ISRE) driving transcription of a luciferase reporter gene' in the presence or absence of pZP7 expression vectors containing cDNAs. for class Π cytokine receptors (including human DIRSI, IFNaRl, IFNaR2 and IL-28 receptor). Luciferase activity following stimulation of transfected cells with class Π ligands (including 1L-28A (SEQ ID NO:2), IL-29 (SEQ ID NO:4), IL28B (SEQ ID NO:6), zcytolO, huILlO and huIFNa-2a) reflects the interaction of the ligand with transfected and native cytokine receptors on the cell surface. The results and methods are described below.

Cell Transfections

293 HEK cells were transfected as follows: 700,000 293 cells/well (6 well plates) were plated approximately 18h prior to transfection in 2 milliliters DMEM + 10% fetal bovine serum. Per well, 1 microgram pISRE-Luciferase DNA (Stratagene), 1 microgram cytokine receptor DNA and 1 microgram pIRES2-EGFP DNA (Clontech,) were added to 9 microEters Fugene 6 reagent (Roche Biochemicals) in a total of 100 microEters DMEM. Two micrograms pIRES2-EGFP DNA was used when cytokine receptor DNA was not included. This transfection mix was added 30 minutes later to the pre-plated 293 cells. Twenty-four hours later the transfected ceUs were removed from the plate using trypsin-EDTA and replated at approximately 25,000 ceUs/well in 96 well microtiter plates. Approximately 18 h prior to Egand stimulation, media was changed to DMEM + 0.5%FBS.

Signal Transduction Reporter Assays

The signal transduction reporter assays were done as foUows: Following an 18h incubation at 37°C in DMEM + 0.5%FBS, transfected cells were stimulated with dilutions (in DMEM + 0.5%FBS) of the foUowing class H Egands; IL-28A, IL-29, IL-28B, zcytolO, huILlO and huIFNa-2a. Following a 4-hour incubation at 37°C, the ceUs were lysed, and the relative Eght units (RLU) were measured on a luminometer after addition of a luciferase substrate. The results obtained are shown as the fold induction of the RLU of the experimental samples over the medium alone control (RLU of experimental samples/RLU of medium alone = fold induction). Table 10 shows that IL-28A, IL-29, and IL-28B induce ISRE signaEng in 293 cells transfected with ISREluciferase giving a 15 to 17-fold induction in luciferase activity over medium alone. ׳The addition of IL-28 receptor alpha subunit DNA (SEQ ID NO: 11), using the endogenous CRF2-4 (SEQ ID NO:71) to the transfection mix results in a 6 to 8-fold further induction in ISRE signaEng by IL-28A, IL-29, and IL-28B giving a 104 to 125fold total induction. None of the other transfected class Π cytokine receptor DNAs resulted in increased ISRE signaEng. These results indicate that IL-28A, IL-29, and IL28B functionally interact with the IL-28 cytokine receptor. Table 10 also shows that huIFNa-2a can induce ISRE signaling in ISRE-luciferase transfected 293 cells giving a 205-fold induction of luciferase activity compared to medium alone. However, the addition of IL-28 receptor DNA to the transfection leads to an 11-fold reduction in ISRE-signaling (compared to ISRE-luciferase DNA alone), suggesting that IL-28 receptor over-expression negatively effects interferon signaling, in contrast to the positive effects of IL-28 receptor over-expression on IL-28A, IL-29, and IL-28B signaling.

Table 10

Interferon Stimulated Response Element (ISRE) Signaling of Transfected 293 Cells Following Class Π Cytokine Stimulation (Fold Induction)

<td> Ligand</td><td> ISRE-Luc.</td><td> ISRE-Luc./IL-28R</td>

<td> HuIFNa-2a (lOOng/ml)</td><td> 205</td><td> 18</td>

<td> ZcytolO (125ng/ml)</td><td> 1.3</td><td> 1</td>

<td> HuILlO (lOOng/ml)</td><td> 1</td><td> 0.5____________________</td>

Example

Signal Transduction Assays with IL-29 Cysteine mutants

Cell Transfections

To produce 293 HEK cells stably overexpressing human IL-28 receptor, 293 cells were transfected as follows: 300,000 293 cells/well (6 well plates) were plated approximately 6h prior to transfection in 2 milliliters DMEM + 10% fetal bovine serum. Per well, 2 micrograms of a pZP7 expression vector containing the cDNA of human IL-28 receptor alpha subunit (SEQ ID NO: 11) was added to 6 microliters Fugene 6 reagent (Roche Biochemicals) in a total of 100 microliters DMEM. This transfection mix was added 30 minutes later to the pre-plated 293 cells. Forty-eight hours later the transfected cells were placed under 2 microgram/milhliter puromicin selection. Puromicin resistant cells were carried as a population of cells.

The 293 HEK cells overexpressing human IL-28 receptor were transfected as follows: 700,000 293 cells/well (6 well plates) were plated approximately 18h prior to transfection in 2 milliliters DMEM + 10% fetal bovine serum. Per well, 1 microgram KZ157 containing an interferon-stimulated response element (ISRE) driving transcription of a luciferase reporter gene were added to 3 microliters Fugene 6 reagent (Roche Biochemicals) in a total of 100 microliters DMEM. This transfection mix was added 30 minutes later to the pre-plated 293HEK cells. Forty-eight hours later the transfected cells were removed from the plate using trypsin-EDTA and replated in 500 micrograms/ml G418 (Geneticin, Life Technologies). Puromycin and G418 resistant cells were carried as a population of cells.

Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: 293HEK cells overexpressing human IL-28 receptor and containing KZ157 were treated with trypsin-EDTA and replated at approximately 25,000 cells/well in 96 well microtiter plates. Approximately 18 h prior to ligand stimulation, media was changed to DMEM + 0.5%FBS.

Following an 18h incubation at 37°C in DMEM + 0.5%FBS, transfected cells were stimulated with dilutions (in DMEM + 0.5%FBS) of the different forms of E.coli-derived zcyt021 containing different cysteine binding patterns. Following a 4hour incubation at 37°C, the cells were lysed, and the relative light units (RLU) were measured on a luminometer after addition of a luciferase substrate. The results obtained are shown as the fold induction of the RLU of the experimental samples over the medium alone control (RLU of experimental samples/RLU of medium alone = fold induction).

Table 11 shows that C1-C3 form (C16-C113) of wild-type E. collderived IL-29 is better able to induce ISRE signaling than wild-type C3-C5 form (C113-C172) or a mixture of wild-type C1-C3 form and C3-C5 form (C16-C113, C113-C172), all referring to SEQ ID NO: 15.

Table 12 shows that C1-C3 (C16-C113) of wild-type E. coZi-derived IL29 and C1-C3 (C16-C113; SEQ ED NO:15) of Cysteine mutant (C172S) E. coli-dedved

IL-29 (SEQ ID NO :29) are equally able to induce ISRE signaling in 293HEK cells overexpressing human IL-28 receptor.

Table 11

ISRE Signaling by different forms of E.coli-derived IL-29 (Fold Induction)

<td> Cytokine Concentration . (ng/ml)</td><td> C1-C3 form (C16-C113)</td><td> C3-C5 form (C113-C172)</td><td> Mixture of ClC3andC3-C5</td>

Table 12

ISRE Signaling by different forms of E.coli-derived IL-29 (Fold Induction)

<td> Cytokine Concentration (ng/ml)</td><td> ׳Wild-type C1-C3</td><td> Cysteine mutant C172S C1-C3</td>

Example

Induction of IL-28 A, IL-29.1L-28B by poly I:C and viral infection

Freshly isolated human peripheral blood mononuclear cells were grown in the presence of polyinosinic acid-polycytidylic acid (poly I:C; 100 gg/ml) (SIGMA; St. Louis, MO), encephalomyocarditis virus (EMCV) with an MOI of 0.1, or in medium alone. After a 15h incubation, total RNA was isolated from cells and treated with RNase-free DNase. 100 ng total RNA was used as template for one-step RT-PCR using the Superscript One-Step RT-PCR with Platinum Taq kit and gene-specific primers as suggested by the manufacturer (Jhvitrogen).

Low to undetectable amounts of human IL-28 A, 1L-28B, and IL-29, IFN-a and IFN-β RNA were seen in untreated cells. In contrast, the amount of IL-28A, IL-29, IL-28B RNA was increased by both poly I:C treatment and viral infection, as was also seen for the type I interferons. These experiments indicate that IL-28A, IL-29, IL-28B, like type I interferons, can be induced by double-stranded RNA or viral infection.

Example

IL-28, IL-29 signaling activity compared to IFNa in HepG2 cells

A, Cell Transfections

HepG2 cells were transfected as follows: 700,000 HepG2 cells/well (6 well plates) were plated approximately 18h prior to transfection in 2 milliliters DMEM + 10% fetal bovine serum. Per well, 1 microgram pISRE-Luciferase DNA (Stratagene) and 1 microgram pIRES2-EGFP DNA (Clontech,) were added to 6 microliters Fugene

I reagent (Roche Biochemicals) in a total of 100 microliters DMEM. This transfection mix was added 30 minutes later to the pre-plated HepG2 cells. Twenty-four hours later the transfected cells were removed from the plate using trypsin-EDTA and replated at approximately 25,000 cells/well in 96 well microtiter plates. Approximately 18 h prior to ligand stimulation, media was changed to DMEM + 0.5%FBS.

B. Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: Following an 18h incubation at 37°C in DMEM + 0.5%FBS, transfected cells were stimulated

PCI7US2004/025864 with 100 ng/ml IL-28A, IL-29, IL-28B, zcyt024, zcyt025 and huIFN-a2a ligands. Following a 4־hour incubation at 37° degrees, the cells were lysed, and the relative light units (RLU) were measured on a luminometer after addition of a luciferase substrate. The results obtained are shown as the fold induction of the RLU of the experimental samples over the medium alone control (RLU of experimental samples/RLU of medium alone = fold induction). Table 13 shows that IL-28A, IL-29, IL-28B, zcyt024 and zcyt025 induce ISRE signaling in human HepG2 liver cells transfected with ISREluciferase.

Table 13: Fold Induction of Cytokine-dependent ISRE Signaling in HepG2 Cells

<td> Cytokine</td><td> Fold Induction</td>

<td> HulFN-a2a</td><td> 5.8</td>

Example

IL-29 antiviral activity compared to IFNa in HepG2 cells

An antiviral assay was adapted for EMCV (American Type Culture Collection # VR-129B, Manassas, VA) with human cells (Familletti, P., et al., Methods Enzvm. 78: 387-394, 1981). Cells were plated with cytokines and incubated 24 hours prior to challenge by EMCV at a multiplicity of infection of 0.1 to 1. The cells were analyzed for viability with a dye-uptake bioassay 24 hours after infection (Berg, K., et al., Apmis 98: 156-162, 1990 ). Target cells were given MTT and incubated at 37°C for 2 hours. A solubiliser solution was added, incubated overnight at 37°C and the optical density at 570 nm was determined. OD570 is directly proportional to antiviral activity.

The results show the antiviral activity when IL-29 and IFN on were tested with HepG2 cells: IL-29, IFN-β and IFN a-2a were added at varying concentration to HepG2 cells prior to EMCV infection and dye-uptake assay. The mean and standard‘ deviation of the OD570 from triplicate wells is plotted. OD570 is directly proportional to antiviral activity. For IL-29, the EC50 was 0.60 ng/ml; for IFNa2a, the EC50 was 0.57 ng/ml; and for IFN-β, the EC50 was 0.46ng/ml.

Example 23

TL-28RA mRNA expression in liver and lymphocyte subsets

Tn order to further examine the mRNA distribution for IL-28RA, semiquantitative RT-PCR was performed using the SDS 7900HT system (Applied Biosystems, CA). One-step RT-PCR was performed using lOOng total RNA for each 5 sample and gene-specific primers. A standard curve was generated for each, primer set using Bjab RNA and all sample values were normalized to HPRT. The normalized results are summarized in Tables 14-17. The normalized values for IFNAR2 and CRF2-4 are also shown.

Table 14: B and T cells express significant levels of IL-28RA mRNA. 10 Low levels are seen in dendritic cells and most monocytes.

Table 14

<td> Cell/Tissue</td><td> IL-28RA</td><td> IFNAR2</td><td> CRF2-4</td>

<td> Dendritic Cells unstim</td><td> .04</td><td> 5.9</td><td> 9.8</td>

<td> Dendritic Cells +IFNg</td><td> .07</td><td> 3.6</td><td> 4.3</td>

<td> Dendritic Cells</td><td> .16</td><td> 7.85</td><td> 3.9</td>

<td> CD 14+ monocytes resting</td><td> .12</td><td> 11</td><td> 15.4</td>

<td> Hu CD14+ Unact.</td><td> 4.2</td><td> TBD</td><td> TBD</td>

<td> H. Inflamed tonsil</td><td> 3</td><td> 12.4</td><td> 9.5</td>

<td> H. B-cells+PMA/Iono 4 & 24 hrs</td><td> 3.6</td><td> 1.3</td><td> 1.4</td>

<td> Hu CD19+ resting</td><td> 6.2</td><td> TBD</td><td> TBD</td>

<td> IgD+ B־cells</td><td> 6.47</td><td> 13.15</td><td> 6.42</td>

<td> IgM+ B-cells</td><td> 9.06</td><td> 15.4</td><td> 2.18</td>

<td> IgD- B-cells</td><td> 5.66</td><td> 2.86</td><td> 6.76</td>

<td> NKCells + PMA/Iono</td><td> 0</td><td> 6.7</td><td> 2.9</td>

<td> Hu CD3+ Unactivated</td><td> 2.1</td><td> TBD</td><td> TBD</td>

<td> CD4+ resting</td><td> .9</td><td> 8.5</td><td> 29.1</td>

<td> CD4+ Unstim 18 hrs</td><td> 1.6</td><td> 8.4</td><td> 13.2</td>

<td> CD3 neg resting</td><td> 1.6</td><td> 7.3</td><td> 46</td>

<td> CD3 neg unstim 18 hrs</td><td> 2.4</td><td> 13.2</td><td> 16.8</td>

<td> CD8+unstim 18 hrs</td><td> 1.8</td><td> 4.9</td><td> 13.1</td>

As shown in Table 14, normal liver tissue and liver derived cell lines display substantial levels of IL-28RA and CRF2-4 mRNA.

PCT7US2004/025864

Table 15

<td> Cell/Tissue</td><td> IL-28RA</td><td> IFNAR2</td><td> CRF2-4</td>

<td> HuH7 hepatoma - CGAT</td><td> 4.2</td><td> 7.2</td><td> 3.1</td>

<td> Liver, normal - CGAT #HXYZ020801K</td><td> 11.7</td><td> 3.2</td><td> 8.4</td>

<td> Liver, NAT - Normal adjacent tissue</td><td> 4.5</td><td> 4.9</td><td> 7.7</td>

<td> Liver, NAT - Normal adjacent tissue</td><td> 2.2</td><td> 6.3</td><td> 10.4</td>

<td> Hep SMCA hep. Artery</td><td> 0</td><td> 2.1</td><td> 7.5</td>

<td> Hep. Fibro</td><td> 0</td><td> 2.9</td><td> 6.2</td>

<td> Adenoca liver</td><td> 8.3</td><td> 4.2</td><td> 10.5</td>

<td> SK-Hep-1 adenoca. Liver</td><td> .1</td><td> 1.3</td><td> 2.5</td>

<td> AsPC-1 Hu. Pancreatic adenocarc.</td><td> .7</td><td> .8</td><td> 1.3</td>

<td> Hu. Hep. Stellate cells</td><td> .025</td><td> 4.4</td><td> 9.7</td>

As shown in Table 15, primary airway epithelial cells contain abundant levels of 1L-28RA and CRF2-4.

Table 16

<td> Cell/Tissue</td><td> IL-28RA</td><td> IFNAR2</td><td> CRF2-4</td>

<td> U87MG-glioma</td><td> 0</td><td> .66</td><td> .99</td>

<td> NHBE unstim</td><td> 1.9</td><td> 1.7</td><td> 8.8</td>

<td> NHBE + TNF-alpha</td><td> 2.2</td><td> 5.7</td><td> 4.6</td>

<td> Small Airway Epithelial Cells</td><td> 3.9</td><td> 3.3</td><td> 27.8</td>

<td> NHLF ־ Normal human lung fibroblasts</td><td> 0</td><td> nd</td><td> nd</td>

As shown in Table 16, IL-28RA is present in normal and diseased liver specimens, with increased expression in tissue from Hepatitis C and Hepatitis B infected specimens.

Table 17

<td> Cell/Tissue</td><td> IL-28RA</td><td> CRF2-4</td><td> IFNAR2</td>

<td> Liver with Coagulation Necrosis</td><td> 8.87</td><td> 15.12</td><td> 1.72</td>

<td> Liver with Autoimmune Hepatitis</td><td> 6.46</td><td> 8.90</td><td> 3.07</td>

<td> Neonatal Hepatitis</td><td> 6.29</td><td> 12.46</td><td> 6.16</td>

<td> Endstage Liver disease</td><td> 4.79</td><td> 17.05</td><td> 10.58</td>

<td> Fulminant Liver Failure</td><td> 1.90</td><td> 14.20</td><td> 7.69</td>

<td> Fulminant Liver failure</td><td> 2.52</td><td> 11.25</td><td> 8.84</td>

<td> Cirrhosis, primary biliary</td><td> 4.64</td><td> 12.03</td><td> 3.62</td>

<td> Cirrhosis Alcoholic (Laennec's) ___________</td><td> 4.17</td><td> 8.30</td><td> 4.14</td>

<td> Cirrhosis, Cryptogenic</td><td> 4.84</td><td> 7.13</td><td> 5.06</td>

<td> Hepatitis C+, with cirrhosis</td><td> 3.64</td><td> 7.99</td><td> 6.62</td>

<td> Hepatitis C+</td><td> 6.32</td><td> 11.29</td><td> 7.43</td>

<td> Fulminant hepatitis secondary to Hep A</td><td> 8.94</td><td> 21.63</td><td> 8.48</td>

<td> Hepatitis C+</td><td> 7.69</td><td> 15.88</td><td> 8.05</td>

<td> Hepatitis B+</td><td> 1.61</td><td> 12.79</td><td> 6.93</td>

<td> Nonnal Liver</td><td> 8.76</td><td> 5.42</td><td> 3.78</td>

<td> Normal Liver___________________________</td><td> 1.46</td><td> 4.13</td><td> 4.83</td>

<td> Liver NAT</td><td> 3.61</td><td> 5.43</td><td> 6.42</td>

<td> Liver NAT l</td><td> 1.97</td><td> 10.37</td><td> 6.31</td>

<td> Hu Fetal Liver</td><td> 1.07</td><td> 4.87</td><td> 3.98</td>

<td> Hepatocellular Carcinoma</td><td> 3.58</td><td> 3.80</td><td> 3.22</td>

<td> Adenocarcinoma Liver</td><td> 8.30</td><td> 10.48</td><td> 4.17</td>

<td> Hep SMCA hep. Artery</td><td> 0.00</td><td> 7.55</td><td> 2.10</td>

<td> Hep. Fibroblast</td><td> 0.00</td><td> 6.20</td><td> 2.94</td>

<td> HuH7 hepatoma</td><td> 4.20</td><td> 3.05 _</td><td> 7.24</td>

<td> HepG2 Hepatocellular carcinoma</td><td> 3.40</td><td> 5.98</td><td> 2.11</td>

<td> SK-Hep-1 adenocar. Liver</td><td> 0.03</td><td> 2.53</td><td> 1.30</td>

<td> HepG2 Unstim</td><td> 2.06</td><td> 2.98</td><td> 2.28</td>

<td> Nonnal Female Liver - degraded____________</td><td> 1.38</td><td> 6.45</td><td> 4.57</td>

<td> Normal Liver - degraded</td><td> 1.93</td><td> 4.99</td><td> 6.25</td>

<td> Normal Liver - degraded</td><td> 2.41</td><td> 2.32</td><td> 2.75</td>

<td> Disease Liver - degraded</td><td> 2.33</td><td> 3.00</td><td> 6.04</td>

<td> Primary Hepatocytes from Clonetics</td><td> 9.13</td><td> 7.97</td><td> 13.30</td>

As shown in Tables 18-22, IL-28RA is detectable in normal B cells, B lymphoma cell Enes, T ceEs, T lymphoma cell lines (Jurkat), normal and transformed 5 lymphocytes (B ceEs and T cells) and normal human monocytes.

Table 18

<td></td><td> HPRT Mean</td><td> IL-28RA Mean</td><td> IL-28RA norm</td><td> IFNAR2</td><td> IFNR2 norm</td><td> CRF2-4</td><td> CRF2-4 Norm</td>

<td> CD 14+ 24hr unstim #A38</td><td> 13.1</td><td> 68.9</td><td> 5.2</td><td> 92.3</td><td> 7.0</td><td> 199.8</td><td> 15.2</td>

<td> CD14+24hrstim#A38</td><td> 6.9</td><td> 7.6</td><td> 1.1</td><td> 219.5</td><td> 31.8</td><td> 276.6</td><td> 40.1</td>

<td> CD14+24hrunstim#A112</td><td> 17.5</td><td> 40.6</td><td> 2.3</td><td> 163.8</td><td> 9.4</td><td> 239.7</td><td> 13.7</td>

<td> CD14+24hrstim#A112</td><td> 11.8</td><td> 6.4</td><td> 0.5</td><td> 264.6</td><td> 22.4</td><td> 266.9</td><td> 22.6</td>

<td> CD 14+ 24 hr unstim #A39</td><td> 26.3</td><td> 86.8</td><td> 3.3</td><td> 297.4</td><td> 11.3</td><td> 480.6</td><td> 18.3</td>

<td> CD14+24hrstim#A39</td><td> 16.6</td><td> 12.5</td><td> 0.8</td><td> 210.0</td><td> 12.7</td><td> 406.4</td><td> 24.5</td>

<td> HL60 Resting</td><td> 161.2</td><td> 0.2</td><td> 0.0</td><td> 214.2</td><td> 1.3</td><td> 264.0</td><td> 1.6</td>

<td> U937 Resting</td><td> 246.7</td><td> 0.0</td><td> 0.0</td><td> 449.4</td><td> 1.8</td><td> 362.5</td><td> 1.5</td>

<td> Jurkat Resting</td><td> 241.7</td><td> 103.0</td><td> 0.4</td><td> 327.7</td><td> 1.4</td><td> 36.1</td><td> 0.1</td>

<td> Jurkat Activated</td><td> 130.7</td><td> 143.2</td><td> 1.1</td><td></td><td></td><td></td><td></td>

<td colspan="3"> Table 19</td>

<td></td><td> HPRTSD</td><td> IL-28RA SD</td>

<td> Mono 24hr unstim #A38</td><td> 0.6</td><td> 2.4</td>

<td> Mono 24 hr unstim #A112</td><td> 2.0</td><td> 0.7</td>

<td> Mono 24 hr unstim #A39</td><td> 0.7</td><td> 0.8</td>

<td> HL60 Resting</td><td> 19.7</td><td> 0.1</td>

<td> U937 Resting</td><td> 7.4</td><td> 0.0</td>

<td> Jurkat Resting</td><td> 3.7</td><td> 1.1</td>

<td> Jurkat Activated</td><td> 2.4</td><td> 1.8</td>

Table 20

<td></td><td> Mean Hprt</td><td> Mean IFNAR2</td><td> Mean IL28RA</td><td> MeanCRF</td>

<td> CD4/CD3+ Unstim 18 hrs</td><td> 12.9</td><td> 108.7</td><td> 20.3</td><td> 170.4</td>

<td> CD3 neg unstim 18 hrs</td><td> 15.7</td><td> 206.6</td><td> 37.5</td><td> 263.0</td>

<td> CD8+ Unstim. 18 hrs</td><td> 6.1</td><td> 29.7</td><td> 11.1</td><td> 79.9</td>

<td> 12.8.1 - NHBE Unstim</td><td> 47.4</td><td> 81.1</td><td> 76.5</td><td> 415.6</td>

<td> 12.8.2 - NHBE+TNE-alpha</td><td> 42.3</td><td> 238.8</td><td> 127.7</td><td> 193.9</td>

Table 2

<td></td><td> IL-28RA Norm</td><td> CRF Norm</td><td> IFNAR2 Norm</td><td> IL-28RA SD</td><td> CRF SD</td><td> IFNAR2 SD</td>

<td> CD4/CD3+ Unstim 18 hrs</td><td> 1.6</td><td> 13.2</td><td> 8.4</td><td> 0.2</td><td> 1.6</td><td> 1.4</td>

<td> CD3 neg unstim 18 hrs</td><td> 2.4</td><td> 16.8</td><td> 13.2</td><td> 0.4</td><td> 2.7</td><td> 2.3</td>

<td> CD8+ Unstim. 18 hrs</td><td> 1.8</td><td> 13.1</td><td> 4.9</td><td> 0.1</td><td> 1.1</td><td> 0.3</td>

<td> 12.8.1 - NHBE Unstim</td><td> 1.6</td><td> 8.8</td><td> 1.7</td><td> 0.1</td><td> 0.4</td><td> 0.1</td>

<td> 12.8.2 - NHBE+TNF-alpha</td><td> 3.0</td><td> 4.6</td><td> 5.7</td><td> 0.1</td><td> 0.1</td><td> 0.1</td>

Table 22

<td></td><td> SDHprt</td><td> SDIFNAR2</td><td> SDIL-28RA</td><td> SDCRF</td>

<td> CD4/CD3+ Unstim 18 hrs</td><td> 1.4</td><td> 13.7</td><td> 1.1</td><td> 8.5</td>

<td> CD3 neg unstim 18 hrs</td><td> 2.4</td><td> 16.2</td><td> 2.7</td><td> 12.6</td>

<td> CD8+ Unstim. 18 hrs</td><td> 0.2</td><td> 1.6</td><td> 0.5</td><td> 6.1</td>

<td> 12.8.1 - NHBE Unstim</td><td> 2.4</td><td> 5.6</td><td> 2.7</td><td> 2.8</td>

<td> 12.8.2 - NHBE+TNF-alpha</td><td> 0.5</td><td> 3.4</td><td> 3.5</td><td> 3.4</td>

Example

Mouse IL-28 Does Not Effect Daudi Cell Proliferation

Human Daudi cells were suspended in RPM1 + 10%FBS at 50,000 cells/milliliter and 5000 cells were plated per well in a 96 well plate. IL-29-CEE (IL-29 conjugated with glu tag), IFN-γ or IFN-a2a was added in 2-fold serial dilutions to each well. IL-29-CEE was used at a concentration range of from 1000 ng/ml to 0.5 ng/ml. IFN-γ was used at a concentration range from 125 ng/ml to 0.06 ng/ml. IFN-a2a was used at a concentration range of from 62 ng/ml to 0.03 ng/ml. Cells were incubated for

72 h at 37°C. After 72 h. Alamar Blue (Accumed, Chicago, IL) was added at 20 microliters/well. Plates were further incubated at 37°C., 5% CO, for 24 hours. Plates

PCT7US2004/025864 were read on the Fmax™ plate reader (Molecular Devices, Sunnyvale, CA) using the SoftMax™ Pro program, at wavelengths 544 (Excitation) and 590 (Emission). Alamar Blue gives a fluourometric readout based on the metabolic activity of cells, and is thus a direct measurement of cell proliferation in comparison to a negative control. The results indicate that IL-29-CEE, in contrast to IFN-a2a, has no significant effect on proliferation of Daudi cells.

Example

Mouse IL-28 Does Not Have Antiproliferative Effect on Mouse B cells

Mouse B cells were isolated from 2 Balb/C spleens (7 months old) by depleting CD43+ cells using MACS magnetic beads. Purified B cells were cultured in vitro with IPS, anti-IgM or anti-CD40 monoclonal antibodies. Mouse IL-28 or mouse IFNa was added to the cultures and 3H-thymidine was added at 48 hrs. and 3Hthymidine incorporation was measured after 72 hrs. culture.

IFNa at 10 ng/ml inhibited 3H-thymidine incorporation by mouseB cells stimulated with either LPS or anti-IgM. However mouse IL-28 did not inhibit 3Hthymidine incorporation at any concentration tested including 1000 ng/ml. In contrast, both mIFNa and mouse IL-28 increased 3H thymidine incorporation by mouse B cells stimulated with anti-CD40 MAb.

These data demonstrate that mouse IL-28 unlike IFNa displays no antiproliferative activity even at high concentrations. In addition, zcyto24 enhances proliferation in the presence of anti-CD40 MAbs. The results illustrate that mouse IL28 differs from IFNa in that mouse IL-28 does not display antiproliferative activity on mouse B cells, even at high concentrations. In addition, mouse IL-28 enhances proliferation in the presence of anti-CD40 monoclonal antibodies.

Example

Bone marrow expansion assay

Fresh human marrow mononuclear cells (Poietic Technologies, Gaithersburg, Md.) were adhered to plastic for 2 hrs in aMEM, 10% FBS, 50 micromolar β-mercaptoethanol, 2 ng/ml FLT3L at 37°C. Non adherent cells were then plated at 25,000 to 45,000 cells/well (96 well tissue culture plates) in aMEM, 10% FBS, 50 micromolar 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 IFNa2a, 10 ng/ml IFN-a2a or 1 ng/ml IFN-a2a. These cells were incubated with a variety of cytokines to test for expansion or differentiation of hematopoietic cells from the marrow (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 no added cytokine). After 8 to 12 days Alamar Blue (Accumed, Chicago, Hl.) was added at 20 microliters/well. Plates were further incubated at 37 °C, 5% CO, for 24 hours. Plates were read on the Fmax™ plate reader (Molecular Devices Sunnyvale, Calif.) using the SoftMax™ Pro program, at wavelengths 544 (Excitation) and 590 (Emission). Alamar Blue gives a fluourometric readout based on the metabolic activity of cells, and is thus a direct measurement of cell proliferation in comparison to a negative control.

lFN-a2a caused a significant inhibition of bone marrow expansion under all conditions tested. In contrast, IL-29 had no significant effect on expansion of bone marrow cells in the presence of IL-3, IL-4, IL-5, IL-7, IL-10, IL-12, IL-21 or no added cytokine. A small inhibition of bone marrow cell expansion was seen in the presence of IL-2 or IL-15.

Example

Inhibition of IL-28 and IL-29 signaling with soluble receptor (zcytoR19/CRF2=41

A. Signal Transduction Reporter Assay

A signal transduction reporter assay can be used to show the inhibitor properties of zcytorl9-Fc4 homodimeric and zcytorl9-Fc/CRF2-4-Fc heterodimeric soluble receptors :on zcyto20, zcyt021 and zcyt024 signaling. Human embryonal kidney (HEK) cells overexpressing the zcytorl9 receptor are transfected with a reporter plasmid containing an interferon-stimulated response element (ISRE) driving transcription of a luciferase reporter gene. Luciferase activity following stimulation of transfected cells with ligands (including zcyto20 (SEQ ID NO :2), zcyt021 (SEQ ID

NO:4), zcyt024 (SEQ ID NO:8)) reflects the interaction of the ligand with soluble receptor.

B, Cell Transfections

293 HEK cells overexpressing zcytorl9 were transfected as follows: 700,000 293 cells/well (6 well plates) were plated approximately 18h prior to transfection in 2 milliliters DMEM + 10% fetal bovine serum. Per well, 1 microgram pISRE-Luciferase DNA (Stratagene) and 1 microgram pIRES2-EGFP DNA (Clontech,) were added to 6 microliters Fugene 6 reagent (Roche Biochemicals) in a total of 100 microliters DMEM. This transfection mix was added 30 minutes later to the pre-plated 293 cells. Twenty-four hours later the transfected cells were removed from the plate using trypsin-EDTA and replated at approximately 25,000 cells/well in 96 well microtiter plates. Approximately 18 h prior to ligand stimulation, media was changed to DMEM + 0.5%FBS.

C, Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: Following an 18h incubation at 37°C in DMEM + 0.5%FBS, transfected cells were stimulated with 10 ng/ml zcyto20, zcyt021 or zcyt024 and 10 micrograms/ml of the following soluble receptors; human zcytorl9-Fc homodimer, human zcytorl9-Fc/human CRF2-4Fc heterodimer, human CRF2-4-Fc homodimer, murine zcytorl9-Ig homodimer. Following a 4-hour incubation at 37°C, the cells were lysed, and the relative light units (RLU) were measured on a luminometer after addition of a luciferase substrate. The results obtained are shown as the percent inhibition of ligand-induced signaling in the presence of soluble receptor relative to the signaling in the presence of PBS alone. Table' 23 shows that the human zcytorl9-Fc/human CRF2-4 heterodimeric soluble receptor is able to inhibit zcyto20, zcyt021 and zcyt024-induced signaling between 16 and 45% of control. The human zcytorl9-Fc homodimeric soluble receptor is also able to inhibit zcyt021-induced signaling by 45%. No significant effects were seen with huCRF2-4-Fc or muzcytorl9-Ig homodimeric soluble receptors.

PCT7US2004/025864

Table 23: Percent Inhibition of Ligand-induced Interferon Stimulated Response

<td colspan="2"> Element (ISR</td><td colspan="3"> E) Signaling by Soluble Receptors</td>

<td> Ligand</td><td> Huzcytorl9- Fc/huCRF2-4-Fc</td><td> Huzcytorl9-Fc</td><td> HuCRF2-4-Fc</td><td> Muzcytorl9-Ig</td>

<td> Zcyto20</td><td> 16%</td><td> 92%</td><td> 80%</td><td> 91%</td>

Example

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

Human immunodeficiency virus (HIV) is a pathogenic retrovirus that infects cells of the immune system. CD4 T cells and monocytes are the primary infected cell types. To test the ability of IL-28 and IL-29 to inhibit HIV replication in vitro, PBMCs from normal donors were infected with the HIV virus in the presence of IL-28, IL-29 and MetJL-29C172S-PEG.

Fresh human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood obtained from screened donors who were seronegative for HIV and HBV. Peripheral blood cells were pelleted/washed 2-3 times by low speed centrifugation and resuspended in PBS to remove contaminating platelets. The washed blood cells were diluted 1:1 with Dulbecco's phosphate buffered saline (D-PBS) and layered over 14 mL of Lymphocyte Separation Medium ((LSM; cellgro™ by Mediatech, Inc. Herndon, VA); density 1.078 +/-0.002 g/ml) in a 50 mL centrifuge tube and centrifuged for 30 minutes at 600 x G. Banded PBMCs were gently aspirated from the resulting interface and subsequently washed 2X in PBS by low speed centrifugation. After the final wash, cells were counted by trypan blue exclusion and resuspended at 1 x 107 np-Hs/mT, in RPMI 1640 supplemented with 15% Fetal Bovine Serum (FBS), 2 mM ז -glutamine, 4 [ig/mL PHA-P. The cells were allowed to incubate for 48-72 hours at 37°C. After incubation, PBMCs were centrifuged and resuspended in RPMI 1640 with 15% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 pg/mL streptomycin, 10 pg/mL gentamycin, and 20 U/mL recombinant human IL-2. PBMCs were maintained in the medium at a concentration of 1-2 x 106 cells/mL with biweekly medium changes until used in the assay protocol. Monocytes were depleted from the culture as the result of adherence to the tissue culture flask.

For the standard PBMC assay, PHA-P stimulated cells from at least two normal donors were pooled, diluted in fresh medium to a final concentration of 1 x 106 cells/mL, and plated in the interior wells of a 96 well round bottom microplate at 50 pL/well (5 x 104 cells/well). Test dilutions were prepared at a 2X concentration in microtiter tubes and 100 pL of each concentration was placed in appropriate wells in a standard format. IL-28, IL-29 and MetIL-29C172S-PEG were added at concentrations from 0-10 pg/ml, usually in 1/2 log dilutions. 50 pL of a predetermined dilution of virus stock was placed in each test well (final MOI of 0.1). Wells with only cells and virus added were used for virus control. Separate plates were prepared identically without virus for drug cytotoxicity studies using an MTS assay system. The PBMC cultures were maintained for seven days following infection, at which time cell-free supernatant samples were collected and assayed for reverse transcriptase activity and p24 antigen levels.

A decrease in reverse transcriptase activity or p24 antigen levels with IL28, IL-29 and MetIL-29C172S-PEG would be indicators of antiviral activity. Result would demonstrate that IL-28 and IL-29 may have therapeutic value in treating HIV and AIDS.

Example

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

HCV is a member of the Flaviviridae family of RNA viruses. HCV does not replicate well in either ex-vivo or in vitro cultures and therefore, there are no satisfactory systems to test 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 since it has a relatively high level of sequence identity with HCV and is a hepatotropic virus. To date, the virus can only be grown in the primary hepatocytes of certain non-human primates. This is accomplished by either isolating hepatocytes in vitro and infecting them with GBV-B, or by isolating hepatocytes from GBV-B infected marmosets and directly using them with antiviral compounds.

The effects of IL-28, IL-29 and MetIL-29C172S-PEG are assayed on GBV-B extracellular RNA production by TaqMan RT-PCR and on cytotoxicity using CellTiter96® reagent (Promega, Madison, WI) at six half-log dilutions IL-28, IL-29 or MetIL-29C172S-PEG polypeptide in triplicate. Untreated cultures serve as the cell and virus controls. Both RIBAVIRIN® (200 μg/ml at the highest test concentration) and IFN-oc (5000 lU/ml at the highest test) are included as positive control compounds. Primary hepatocyte cultures are isolated and plated out on collagen-coated plates. The next day the cultures are treated with the test samples (IL-28, IL-29, MetIL-29C172SPEG, IFNa, or RIBAVIRIN®) for 24hr before being exposed to GBV-B virions or treated directly with test samples when using in vivo infected hepatocytes. Test samples and media are added the next day, and replaced three days later. Three to four days later (at day 6-7 post test sample addition) the supernatant is collected and the cell numbers quantitated with CellTiter96®. Viral RNA is extracted from the supernatant and quantified with triplicate replicates in a quantitative TaqMan RT-PCR assay.'using an in vitro transcribed RNA containing the RT-PCR target as a standard. The average of replicate samples is computed. Inhibition of virus production is assessed by plotting the average RNA and cell number values of the triplicate samples relative to the untreated virus and cell controls. The inhibitory concentration of drug resulting in 50% inhibition of GBV-B RNA production (IC50) and the toxic concentration resulting in destruction of 50% of cell numbers relative to control values (TC50) are calculated by interpolation from graphs created with the data.

Inhibition of the GBV-B RNA production by IL-28 and 29 is an indication of the antiviral properties of IL-28 and IL-29 on this Hepatitis C-like virus on hepatocytes, the primary organ of infection of Hepatitis C, and positive results suggest that IL-28 or IL-29 may be useful in treating HCV infections in humans.

Example

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 of humans belonging to the Hepadnaviridae family of viruses. To test the antiviral activities of IL-28 and IL-29 against HBV, IL-28, IL-29 and MetlL-29C172SWO 2005/023862 PCT/US2004/025864

PEG were tested against HBV in an in vitro infection system using a variant of the human liver line HepG2. IL-28, IL-29 and MefIL-29C172S-PEG inhibited viral replication in this system, suggesting therapeutic value in treating HBV in humans.

WT10 cells are a derivative of the human liver cell line HepG2 2.2.15. WT10 cells are stably transfected with the HBV genome, enabling stable expression of HBV transcripts in the cell line (Fu and Cheng, Antimicrobial Agents Chemothen 44(12):3402-3407, 2000). In the WT10 assay the drug in question and a 3TC control will be assayed at five concentrations each, diluted in a half-log series. The endpoints are TaqMan PCR for extracellular HBV DNA (IC50) and cell numbers using CellTiter96 reagent (TC50). The assay is similar to that described 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 plated in 96-well microtiter plates. After 16-24 hours the confluent monolayer of HepG2-2.2.15 cells is washed and the medium is replaced with complete medium containing varying concentrations of a test samples in triplicate. 3TC is used as the positive control, while media alone is added to cells as a negative control (virus control, VC). Three days later the culture medium is replaced with fresh medium containing the appropriately diluted test samples. Six days following the initial addition of the test compound, the cell culture supernatant is collected, treated with pronase and DNAse, and used in a real-time quantitative TaqMan PCR assay. The PCR-amplified HBV DNA is detected in real-time by monitoring, increases in fluorescence signals that result from the exonucleolytic degradation of a quenched fluorescent probe molecule that hybridizes to the amplified HBV DNA. For each PCR amplification, a standard curve is simultaneously generated using dilutions of purified HBV DNA. Antiviral activity is calculated from the reduction in HBV DNA levels (IC50). A dye uptake assay is then employed to measure cell viability which is used to calculate toxicity (TC50). The therapeutic index (ΊΊ) is calculated as TC50/IC50

IL-28, IL-29 and MetEL-29C172S-PEG inhibited HepB viral replication in WT10 cells with an IC50 < 0.032ug/ml. This demonstrates antiviral activity of IL-28 and IL-29 against HBV grown in liver cell lines, providing evidence of therapeutic value for treating HBV in human patients.

Example

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 the bovine viral diarrhea virus (BVDV) and yellow fever virus (YFV). HCV does not replicate well in either ex vivo or in vitro cultures and therefore there are no systems to test anti-HCV activity in vitro. The BVDV and YFV assays are used as surrogate viruses for HCV to test the antiviral activities against the Flavivirida family of viruses.

The antiviral effects of IL-28, IL-29 and MetlL-29C172S-PEG were assessed in inhibition of cytopathic effect assays (CPE). The assay measured cell death using Madin-Darby bovine kidney cells (MDBK) after infection with cytopathic BVDV virus and the inhibition of cell death by addition of IL-28, IL-29 and MetIL-29C172SPEG. The MDBK cells were propagated in Dulbecco’s modified essential medium (DMEM) containing phenol red with 10% horse serum, 1% glutamine . and ’1% penicillin-streptomycin. CPE inhibition assays were performed in DMEM without phenol red with 2% FBS, 1% glutamine and 1% Pen-Strep. On the day preceding the assays, cells were trypsinized (1% trypsin-EDTA), washed, counted and plated out at 104 cells/well in a 96-well flat-bottom BioCoat® plates (Fisher Scientific, Pittsburgh, PA) in a volume of 100 μΐ/well. The next day, the medium was removed and a pretitered aliquot of virus was added to the cells. The amount of virus was the maximum dilution that would yield complete cell killing (>80%) at the time of maximal CPE development (day ר for BVDV). Cell viability was determined using a CellTiter96® reagent (Promega) according to the manufacturer’s protocol, using a Vmax plate reader (Molecular Devices, Sunnyvale, CA). Test samples were tested at six concentrations each, diluted in assay medium in a half-log series. IFNa and RIBAVIRIN® were used as positive controls. Test sample were added at the time of viral infection. The average background and sample color-corrected data for percent CPE reduction and percent cell viability at each concentration were determined relative to controls and the IC50 calculated relative to the TC50.

IL-28, IL-29 and MetIL-29C172S-PEG inhibited cell death induced by BVDV in MDBK bovine kidney cells. IL-28 inhibited cell death with an IC50 of 0.02 pg/ml, IL-29 inhibited cell death with an IC50 of 0.19 gg/ml, and MetIL-29C172S-PEG inhibited cell death with an IC50 of 0.45 gg/ml. This demonstrated that IL-28 and IL-29 have antiviral activity against the Flavivirida family of viruses.

Example 32

Induction of Interferon Stimulated Genes bv IL-28 and IL-29

A, Human Peripheral Blood Mononuclear Cells

Freshly isolated human peripheral blood mononuclear cells were grown in the presence of IL-29 (20 ng/mL), IFNa2a (2 ng/ml) (PBL Biomedical Labs, Piscataway, NJ), or in medium alone. Cells were incubated for 6, 24, 48, or 72 hours, and then total RNA was isolated and treated with RNase-free DNase. 100 ng total RNA was used as a template for One-Step Semi-Quantitative RT-PCR® using Taqman One-Step RT-PCR Master Mix® Reagents and gene specific primers as suggested by the manufacturer: (Applied Biosystems, Branchburg, NJ) Results were normalized to HPRT and are shown as the fold induction over the medium alone control for each time-point. Table 24 shows that IL-29 induces Interferon Stimulated Gene Expression in human peripheral blood mononuclear cells at all time-points tested.

Table 24

<td></td><td> MxA Fold induction</td><td> Pkr Fold Induction</td><td> OAS Fold Induction</td>

<td> 6 hrIFNa2a</td><td> 17.2</td><td> 9.6</td><td> 16.2</td>

B. Activated Human T Cells

Human T cells were isolated by negative selection from freshly harvested peripheral blood mononuclear cells using the Pan T-cell Isolation® kit according to manufacturer’s instructions (Miltenyi, Auburn, CA). T cells were then activated and expanded for 5 days with plate-bound anti-CD3, soluble apti-CD28 (0.5ug/ml), (Pharmingen, San Diego, CA) and Interleukin 2 (IL-2; 100 U/ml) (R&D Systems, Minneapolis, MN), washed and then expanded for a further 5 days with IL-2.

Following activation and expansion, cells were stimulated with IL-28A (20 ng/ml), IL29 (20 ng/ml), or medium alone for 3, 6, or 18 hours. Total RNA was isolated and treated with. RNase-Free DNase. One-Step Semi-Quantitative RT-PCR® was performed as described in the example above. Results were normalized to HPRT and are shown as the fold induction over the medium alone control for each time-point.

Table 25 shows that IL-28 and IL-29 induce Interferon Stimulated Gene expression in activated human T cells at all time-points tested.

PCT7US2004/025864

Table 25

<td></td><td> MxA Fold Induction</td><td> Pkr Fold Induction</td><td> OAS Fold Induction</td>

<td> Donor #1 3 hr IL28</td><td> 5.2</td><td> 2.8</td><td> 4.8</td>

<td> Donor #1 3 hr IL29</td><td> 5.0</td><td> 3.5</td><td> 6.0</td>

<td> Donor #1 6 hr 1L28</td><td> 5.5</td><td> 2.2</td><td> 3.0</td>

<td> Donor #16 hrIL29</td><td> 6.4</td><td> 2.2</td><td> 3.7</td>

<td> Donor #118 hr IL28</td><td> 4.6</td><td> 4.8</td><td> 4.0</td>

<td> Donor #1 18 hr IL29</td><td> 5.0</td><td> 3.8</td><td> 4.1</td>

<td> Donor #2 3 hr IL28</td><td> 5.7</td><td> 2.2</td><td> 3.5</td>

<td> Donor #2 3 hr IL29</td><td> 6.2</td><td> 2.8</td><td> 4.7</td>

<td> Donor #2 6 hr IL28</td><td> 7.3</td><td> 1.9</td><td> 4.4</td>

<td> Donor #2 6 hr IL29</td><td> 8.7</td><td> 2.6</td><td> 4.9</td>

<td> Donor #2 18 hr IL28</td><td> 4.7</td><td> 2.3</td><td> 3.6</td>

<td> Donor #2 18 hr IL29</td><td> 4.9</td><td> 2.1</td><td> 3.8</td>

C. Primary Human Hepatocytes

Freshly isolated human hepatocytes from two separate donors (Cambrex, 5 Baltimore, MD and CellzDirect, Tucson, AZ) were stimulated with IL-28A (50 ng/ml), IL-29 (50 ng/ml), IFNa2a (50 ng/ml), or medium alone for 24 hours. Following stimulation, total RNA was isolated and treated with RNase-Free DNase. One-step semi-quantitative RT-PCR was performed as described previously in the example above. Results were normalized to HPRT and are shown as the fold induction over the 10 medium alone control for each time-point. Table 26 shows that IL-28 and IL-29 induce Interferon Stimulated Gene expression in primary human hepatocytes following 24-hour stimulation.

Table 26

<td> —----</td><td> MxAFold Induction</td><td> Pkr Fold Induction</td><td> OAS Fold Induction</td>

<td> Donor #1IL28</td><td> 31.4</td><td> 6.4</td><td> 30.4</td>

<td> Donor #1IL29</td><td> 31.8</td><td> 5.2</td><td> 27.8</td>

<td> Donor #1 IFN-a2a</td><td> 63.4</td><td> 8.2</td><td> 66.7</td>

<td> Donor #2IL28</td><td> 41.7</td><td> 4.2</td><td> 24.3</td>

<td> Donor #2IL29</td><td> 44.8</td><td> 5.2</td><td> 25.2</td>

<td> Donor #2 IFN-a2a</td><td> 53.2</td><td> 4.8</td><td> 38.3</td>

D. HepG2 andHuH7: Human Liver Hepatoma Cell Lines

HepG2 and HuH7 cells (ATCC NOS. 8065, Manassas, VA) were 5 stimulated with IL-28A (10 ng/ml), IL-29 (10 ng/ml), IFNa2a (10 ng/ml), IFNB (1 ng/ml) (PBL Biomedical, Piscataway, NJ), or medium alone for 24 or 48 hours. In a separate culture, HepG2 cells were stimulated as described above with 20 ng/ml of MetIL-29C172S-PEG or MetIL-29-PEG. Total RNA was isolated and treated with RNase-Free DNase. 100 ng Total RNA was used as a template for one-step semi10 quantitative RT-PCR as described previously. Results were normalized to HPRT and are shown as the fold induction over the medium alone control for each time-point. Table 27 shows that IL-28, and IL-29 induce ISG expression in HepG2 and HuH7 liver hepatoma cell lines after 24 and 48 hours.

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Table 27

<td></td><td> MxA Fold Induction</td><td> Pkr Fold Induction</td><td> OAS Fold Induction</td>

<td> HepG2 48hrIFNp</td><td> 15.9</td><td> 1.8</td><td> 3.3</td>

<td> HuH7 24hrIFNP</td><td> 279.8</td><td> 5.6</td><td> 151.8</td>

Table 28

<td></td><td> MxA Fold Induction</td><td> OAS Fold Induction</td><td> Pkr Fold Induction</td>

<td> MetIL-29-PEG</td><td> 36.7</td><td> 6.9</td><td> 2.2</td>

<td> MetIL-29C172S-PEG</td><td> 46.1</td><td> 8.9</td><td> 2.8</td>

Data shown is for 20 ng/ml metH^/PEG and metIL-29C172S-PEG versions of: IL-29 after culture for 24 hours.

Data shown is normalized to HPRT and shown as fold induction over unstimulated cells.

Example

Antiviral Activity of IL-28 and IL-29 in HCV Replicon System

The ability of antiviral drugs to inhibit HCV replication can be tested in vitro with the HCV replicon system. The replicon system consists of the Huh7 human hepatoma cell line that has been transfected with subgenomic RNA replicons that direct constitutive replication of HCV genomic RNAs (Blight, KJ. et al. Science 290:197215 1974, 2000). Treatment of replicon clones with IFNa at 10 lU/ml reduces the amount of HCV RNA by 85% compared to untreated control cell lines. The ability of IL-28A and IL-29 to reduce the amount of HCV RNA produced by the replicon clones in 72 hours indicates the antiviral state conferred upon Huh7 cells by IL-28A/IL-29 treatment is effective in inhibiting HCV replicon replication, and thereby, very likely effective in inhibiting HCV replication.

The ability of IL-28A and IL-29 to inhibit HCV replication as determined by Bayer Branched chain DNA kit, is be done under the following conditions:

1. IL28 alone at increasing concentrations (6)* up to 1.0 μg/nϊl

2. π .29 alnne at increasing concentrations (6)* up to 1.0 μg/ml

3. PEGIL29 alone at increasing concentrations (6)* up to 1.0 pg/ml

4. IFNa2A alone at 0.3,1.0, and 3.0 lU/ml

5. Ribavirin alone.

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

The cells are stained after 72 hours with Alomar Blue to assess viablility.

*The concentrations for conditions 1-3 are:

1.0 gg/ml, 0.32 pg/ml, 0.10 pg/ml, 0.032 pg/ml, 0.010 μg/ml, 0.0032 μ^πύ.

The replicon clone (BB7) is treated IX per day for 3 consecutive days with the doses listed above. Total HCV RNA is measured after 72 hours.

Example

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

Two methods are used to assay in vitro antiviral activity of IL-28 and IL29 against a panel of pathogenic viruses including, among others, adenovirus, parainfluenza virus, respiratory syncytial virus, rhino virus, coxsackie virus, influenza virus, vaccinia virus, west nile virus, dengue virus, Venezuelan equine encephalitis virus, pichinde virus and polio virus. These two methods are inhibition of virusinduced cytopathic effect (CPE) determined by visual (microscopic) examination of the cells and increase in neutral red (NR) dye uptake into cells. In the CPE inhibition method, seven concentrations of test drug (loglO dilutions, such as 1000, 100, 10, 1, 0.1, 0.01, 0.001 ng/ml) are evaluated against each virus in 96-well flat-bottomed

PCT7US2004/025864 microplates containing host cells. The compounds are added 24 hours prior to virus, which is used at a concentration of approximately 5 to 100 cell culture infectious doses per well, depending upon the virus, which equates to a multiplicity of infection (MOI) of 0.01 to 0.0001 infectious particles per cell. The tests are read after incubation at 37°C for a specified time sufficient to allow adequate viral cytopathic effect to develop. In the NR uptake assay, dye (0.34% concentration in medium) is added to the same set of plates used to obtain the visual scores. After 2 h, the color intensity of the dye absorbed by and subsequently eluted from the cells is determined using a microplate autoreader. Antiviral activity is expressed as the 50% effective (virus-inhibitory) concentration (EC50) determined by plotting compound concentration versus percent inhibition on semilogarithmic graph paper. The EC5O/IC5O data in some cases may be determined by appropriate regression analysis software. In general, the EC50s determined by NR assay are two-to fourfold higher than those obtained by the CPE method.

PCI7US2004/025864

Table 29: Visual Assay

<td> : Virus</td><td> Cell line</td><td> Drug .</td><td> ' . EC50 Visual</td><td> IC50 Visual</td><td> SI Visual '(IC50Z . EC50) .</td>

<td> Adenovirus</td><td> A549</td><td> IL-28A</td><td> >10 μΕ/ml</td><td> >10 με/ml</td><td> 0</td>

<td> Adenovirus</td><td> A549</td><td> IL-29</td><td> >10 μΕ/ml</td><td> >10 με/ml</td><td> 0</td>

<td> Adenovirus</td><td> A549</td><td> MetIL-29 C172S-PEG</td><td> >10 gg/ml</td><td> >10 pig/ml</td><td> 0</td>

<td> Parainfluenza virus</td><td> MA-104 ii</td><td> IL-28A</td><td> >10 gg/ml</td><td> >10 μβ/ιηΐ</td><td> 0</td>

<td> Parainfluenza virus</td><td> MA-104</td><td> IL-29</td><td> >10 μg/ml</td><td> >10 μg/nנl</td><td> 0</td>

<td> Parainfluenza virus</td><td> MA-104</td><td> MetIL-29 C172S-PEG</td><td> >10 μΕ/ml</td><td> >10 }Xg/iril</td><td> 0</td>

<td> Respiratory syncytial virus</td><td> MA-104</td><td> IL-28A</td><td> >10 μg/ml</td><td> >10 jxg/ml</td><td> 0</td>

<td> Respiratory syncytial virus</td><td> MA-104</td><td> IL-29</td><td> >10 μg/ml</td><td> >10 μβ/ιηΐ</td><td> 0</td>

<td> Respiratory syncytial virus</td><td> MA-104</td><td> MetIL-29 C172S-PEG</td><td> >10 με/ml</td><td> >10 μg/ml</td><td> 0</td>

<td> Rhino 2</td><td> KB</td><td> 1L-28A</td><td> >10 μβ/πύ</td><td> >10 μΕ/ml</td><td> 0</td>

<td> Rhino 2</td><td> KB</td><td> IL-29</td><td> >10 μΕ/ml</td><td> >10 με/ml</td><td> 0</td>

<td> Rhino 2</td><td> KB</td><td> MetIL-29 C172S-PEG</td><td> >10 μg/ml</td><td> >10 μg/ml</td><td> 0</td>

<td> Rhino 9</td><td> HeLa</td><td> IL-28A</td><td> >10 μg/ml</td><td> >10 μΕ/ml</td><td> 0</td>

<td> Rhino 9</td><td> HeLa</td><td> IL-29</td><td> >10 μg/ml</td><td> >10 μΕ/ml</td><td> 0</td>

<td> Rhino 9</td><td> HeLa</td><td> MetIL-29 C172S-PEG</td><td> >10 μg/nנl</td><td> >10 μΕ/ml</td><td> 0</td>

<td> Coxsackie B4 virus</td><td> KB</td><td> IL-28A</td><td> >10 pg/ml</td><td> >10 μΕ/ml</td><td> 0</td>

<td> Coxsackie B4 virus</td><td> KB</td><td> IL-29</td><td> >10 με/πιΐ</td><td> >10 μΕ/ml</td><td> 0</td>

<td> Coxsackie B4 virus</td><td> KB 1'</td><td> MetIL-29 C172S-PEG</td><td> >10 μβ/πύ</td><td> >10 μΕ/ml</td><td> 0</td>

PCI7US2004/025864

<td> Influenza (type A[H3N2])</td><td> MadenDarby Canine Kidney</td><td> IL-28A</td><td> >10 pg/ml</td><td> >10 pg/ml</td><td> 0</td>

<td> Influenza (type A[H3N2])</td><td> MadenDarby Canine Kidney</td><td> IL-29</td><td> >10 pg/ml</td><td> >10 pg/ml</td><td> 0</td>

<td> Influenza (type A[H3N2])</td><td> MadenDarby Canine Kidney</td><td> MetIL-29 C172S-PEG</td><td> >10 pg/ml</td><td> >10 pg/ml</td><td> 0</td>

<td> Influenza (type A[H3N2])</td><td> Vero</td><td> IL-28A</td><td> 0.1 pg/ml</td><td> >10 pg/ml</td><td> >100</td>

<td> Influenza (type: ΑΓΗ3Ν2]) י</td><td> Vero</td><td> IL-29</td><td> >10 gg/ml</td><td> >10 pg/ml</td><td> 0</td>

<td> Influenza (type ΑΙΉ3Ν2])</td><td> Vero</td><td> MetIL-29 C172S-PEG</td><td> 0.045 pg/ml</td><td> >10 pg/ml</td><td> >222</td>

<td> Vaccinia virus</td><td> Vero</td><td> IL-28A</td><td> >10 pg/ml</td><td> >10 pg/ml</td><td> 0</td>

<td> Vaccinia virus</td><td> Vero</td><td> IL-29</td><td> >10 pg/ml</td><td> >10 pg/ml</td><td> 0</td>

<td> Vaccinia virus</td><td> Vero</td><td> MetIL-29 C172S-PEG</td><td> >10 pg/ml</td><td> >10 pg/ml</td><td> 0</td>

<td> West Nile virus</td><td> Vero</td><td> IL-28A</td><td> 0.00001 pg/ml</td><td> >10 pg/ml</td><td> >1,000,0 00</td>

<td> West Nile virus</td><td> Vero</td><td> IL-29</td><td> 0.000032 _</td><td> >10 pg/ml</td><td> >300,000</td>

<td> West Nile virus</td><td> Vero</td><td> MetIL-29 C172S-PEG</td><td> 0.001 pg/ml</td><td> >10 pg/ml</td><td> >10,000</td>

<td> Dengue virus</td><td> Vero</td><td> IL-28A</td><td> 0.01 pg/ml</td><td> >10 pg/ml</td><td> >1000</td>

<td> Dengue virus</td><td> ,Vero</td><td> IL-29</td><td> 0.032 pg/ml</td><td> >10 pg/ml</td><td> >312</td>

<td> Dengue virus</td><td> Vero</td><td> MetIL-29 C172S-PEG</td><td> 0.0075 pg/ml</td><td> >10 pg/ml</td><td> >1330</td>

PCI7US2004/025864

<td> Venezuelan equine encephalitis virus</td><td> Vero</td><td> IL-28A</td><td> 0.01 μβ/πύ</td><td> >10 μβ/ml</td><td> >1000</td>

<td> Venezuelan equine encephalitis virus</td><td> Vero</td><td> IL-29</td><td> 0.012 μβ/ml</td><td> >10 μβ/ml</td><td> >833</td>

<td> Venezuelan equine encephalitis virus</td><td> Vero</td><td> MetIL-29 C172S-PEG</td><td> 0.0065 μβ/ml</td><td> >10 μβ/ιηΐ</td><td> >1538</td>

<td> Pichinde virus</td><td> BSC-1</td><td> IL-28A</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

<td> Pichinde virus</td><td> BSC-1</td><td> IL-29</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

<td> Pichinde virus</td><td> BSC-1</td><td> MetIL-29 C172S-PEG</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

<td> Polio virus</td><td> Vero</td><td> IL-28A</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

<td> Polio virus</td><td> : Vero</td><td> IL-29</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

<td> Polio virus</td><td> Vero</td><td> MetIL-29 C172S-PEG</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

Table 30:

<td> Adenovirus</td><td> A549</td><td> IL-28A</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

<td> Adenovirus</td><td> A549</td><td> IL-29</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

<td> Adenovirus</td><td> A549</td><td> MetIL-29 C172S-PEG</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

<td> Parainfluenza virus</td><td> MA-104</td><td> IL-28A</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

<td> Parainfluenza virus</td><td> MA-104</td><td> IL-29</td><td> >10 μβ/ml.</td><td> >10 μβ/ml</td><td> 0</td>

<td> Parainfluenza virus</td><td> MA-104</td><td> MetIL-29 C172S-PEG</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

<td> Respiratory syncytial virus</td><td> MA-104</td><td> IL-28A</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

<td> Respiratory syncytial virus</td><td> MA-104</td><td> IL-29</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

<td> Respiratory syncytial virus</td><td> MA-104</td><td> MetIL-29 C172S-PEG</td><td> 5.47 μβ/ml</td><td> >10 μβ/ml</td><td> >2</td>

<td> Rhino 2 ,</td><td> KB</td><td> IL-28A</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

<td> Rhino 2</td><td> KB</td><td> IL-29</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

<td> Rhino 2</td><td> KB</td><td> MetIL-29 C172S-PEG</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

<td> Rhino 9</td><td> HeLa</td><td> IL-28A</td><td> 1.726 μβ/ml</td><td> >10 μβ/ml</td><td> >6</td>

<td> Rhino 9</td><td> HeLa</td><td> IL-29</td><td> 0.982 μβ/ml</td><td> >10 μβ/ml</td><td> >10</td>

<td> Rhino 9 1</td><td> HeLa</td><td> MetIL-29 C172S-PEG</td><td> 2.051 μβ/ml</td><td> >10 μβ/ml</td><td> >5</td>

<td> Coxsackie B4</td><td> KB</td><td> IL-28A</td><td> >10 μβ/ml</td><td> >10 μβ/ml</td><td> 0</td>

<td> virus</td><td></td><td></td><td></td><td></td><td></td>

<td> Coxsackie B4 virus</td><td> KB</td><td> IL-29</td><td> >10 pg/ml</td><td> >10 pig/x□!</td><td> 0</td>

<td> Coxsackie B4 virus</td><td> KB</td><td> MetIL-29 C172S-PEG</td><td> >10 gg/ml</td><td> >10 μg/nנl</td><td> 0</td>

<td> Influenza (type A [H3N2])</td><td> MadenDarby Canine Kidney</td><td> IL-28A</td><td> >10 pg/ml</td><td> >10 μg/nנl</td><td> 0</td>

<td> Influenza (type A [H3N2])</td><td> MadenDarby Canine Kidney</td><td> IL-29</td><td> >10 gg/ml</td><td> >10 μg/ml</td><td> 0</td>

<td> Influenza (type A [H3N2])</td><td> MadenDarby Canine Kidney</td><td> MetIL-29 C172S-PEG</td><td> >10 μg/πנl</td><td> >10 μg/nבl</td><td> 0</td>

<td> Influenza (type A [H3N2])</td><td> Vero</td><td> IL-28A</td><td> 0.25 gg/ml</td><td> >10 μg/π11</td><td> >40</td>

<td> Influenza (type A [H3N2])</td><td> Vero</td><td> IL-29</td><td> 2 gg/ml</td><td> >10 μg/Inl</td><td> >5</td>

<td> Influenza (type A [H3N2])</td><td> Vero</td><td> MetIL-29 C172S-PEG</td><td> 1.4 gg/ml</td><td> >10 μg/ml</td><td> >7</td>

<td> Vaccinia virus</td><td> Vero</td><td> IL-28A</td><td> >10 ug/ml</td><td> >10 μg/π11</td><td> 0</td>

<td> Vaccinia virus</td><td> Vero</td><td> IL-29</td><td> >10 ug/ml</td><td> >10 μg/ml</td><td> 0</td>

<td> Vaccinia virus</td><td> Vero</td><td> MeflL-29 C172S-PEG</td><td> >10 gg/ml</td><td> >10 μg/ml</td><td> 0</td>

<td> West Nile virus</td><td> Vero</td><td> IL-28A</td><td> 0.0001 ug/ml</td><td> >10 μβ/ιηΐ</td><td> >100,000</td>

<td> West Nile virus</td><td> Vero</td><td> IL-29</td><td> 0.00025 μg/π11</td><td> >10 μκ/ml</td><td> >40,000</td>

<td> West Nile virus</td><td> Vero</td><td> MetIL-29 C172S-PEG</td><td> 0.00037 μg/πנl</td><td> >10 μg/ml</td><td> >27,000</td>

<td> Dengue virus</td><td> Vero</td><td> IL-28A</td><td> 0.1 μg/ml</td><td> >10 μβ/ιηΐ</td><td> >100</td>

<td> Dengue virus</td><td> Vero</td><td> IL-29</td><td> 0.05 μg/ml</td><td> >10 μg/πנl</td><td> >200</td>

<td> Dengue virus</td><td> Vero</td><td> MetIL-29 C172S-PEG</td><td> 0.06 μg/ml</td><td> >10 μg/π11</td><td> >166</td>

<td> Venezuelan 1 equine encephalitis virus</td><td> Vero</td><td> IL-28A</td><td> 0.035 μg/ml</td><td> >10 μg/πנl</td><td> >286</td>

<td> Venezuelan equine encephalitis virus</td><td> Vero</td><td> IL-29</td><td> 0.05 μg/π11</td><td> >10 μg/ml</td><td> >200</td>

<td> Venezuelan equine encephalitis virus</td><td> Vero</td><td> MetIL-29 C172S-PEG</td><td> 0.02 μg/π11</td><td> >10 μg/ml</td><td> >500</td>

<td> Pichinde virus</td><td> BSC-1</td><td> IL-28A</td><td> >10 μg/ml</td><td> >10 μ8/π11</td><td> 0</td>

<td> Pichinde virus</td><td> BSC-1</td><td> IL-29</td><td> >10 μg/Inl</td><td> >10 μg/ml</td><td> 0</td>

<td> Pichinde virus</td><td> BSC-1</td><td> MetIL-29 C172S-PEG</td><td> >10 μg/ml</td><td> >10 μg/π11</td><td> 0</td>

<td> Polio virus</td><td> . Vero</td><td> IL-28A</td><td> >1.672 μg/ml</td><td> >10 μg/Iπl</td><td> >6</td>

<td> Polio virus</td><td> Vero</td><td> IL-29</td><td> >10 μg/π11</td><td> >10 μβ/πιΐ</td><td> 0</td>

<td> Polio virus</td><td> Vero</td><td> MetIL-29 C172S-PEG</td><td> >10 μg/ml</td><td> >10 μg/πגl</td><td> 0</td>

PCI7US2004/025864

Example

TL-28. IL-29, metIL-29-PEG and metIL-29C172S-PEG Stimulate ISG induction in the Mouse Liver Cell line AML-12

Interferon stimulated genes (ISGs) are genes that are induced by type I interferons (IFNs) and also by the IL-28 and IL-29 family molecules, suggesting that IFN and IL-28 and IL-29 induce similar pathways leading to antiviral activity. Human type I IFNs (IFNal-4 and ΙΕΝβ) have little or no activity on mouse cells, which is thought to be caused by lack of species cross-reactivity. To test if human IL-28 and IL29 have effects on mouse cells, ISG induction by human IL-28 and IL-29 was evaluated by real-time PCR on the mouse liver derived cell line AML-12.

AML-12 cells were plated in 6-well plates in complete DMEM media at a concentration of 2 x 106 cells/well. Twenty-four hours after plating cells,, human IL28 and IL-29 were added to the culture at a concentration of 20 ng/ml. As a control, cells were either stimulated with mouse IFN□ (positive control) or unstimulated (negative). Cells were harvested at 8, 24, 48 and 72 hours after addition of CHOderived human IL-28A (SEQ ID NO:2) or IL-29 (SEQ ID NO:4) . RNA was isolated from cell pellets using RNAEasy-kit® (Qiagen, Valencia, CA). RNA was treated with DNase (Millipore, Billerica, MA) to clean RNA of any contaminating DNA. cDNA was generated using Perkin-Elmer RT mix. ISG gene induction was evaluated by real-time PCR using primers and probes specific for mouse OAS, Pkr and Mxl. To obtain quantitative data, HPRT real-time PCR was duplexed with ISG PCR. A standard curve was obtained using known amounts of RNA from IFN-stimulated mouse PBLs. All data are shown as expression relative to internal HPRT expression.

Human JL-28A and IL-29 stimulated ISG induction in the mouse hepatocyte cell line AML-12 and demonstrated that unlike type I IFNs, the IL-28/29 family proteins showed cross-species reactivity.

Table 31

<td> Stimulation</td><td> OAS</td><td> PkR</td><td> Mxl</td>

<td> Human IL-28</td><td> 0.04</td><td> 0.02</td><td> 0.06</td>

<td> Human IL-29</td><td> 0.04</td><td> 0.02</td><td> 0.07</td>

<td> Mouse IL-28</td><td> 0.04</td><td> 0.02</td><td> 0.08</td>

<td> Mouse IFNa</td><td> 0.02</td><td> 0.02</td><td> 0.01</td>

All data shown were expressed as fold relative to HPRT gene expression ng of OAS mRNA = normalized value of OAS mRNA amount relative to internal ng of HPRT mRNA housekeeping gene, HPRT

As an example, the data for the 48 hour time point is shown.

Table 32

<td> ruvu-׳xx׳ o</td><td> Mxl Fold Induction</td><td> OAS Fold Induction</td><td> Pkr Fold Induction</td>

<td> MetIL-29-PEG</td><td> 728</td><td> 614</td><td> 8</td>

<td> MetIL-29C172S-PEG</td><td> 761</td><td> 657</td><td> 8</td>

Cells were stimulated with 20 ng/ml or hours.

Data shown is normalized to HPRT and shown as fold induction over unstimulated cells.

Example

ISGs are Efficiently Induced in Spleens of Transgenic Mice Expressing Human IL-29 Transgenic (Tg) mice were generated expressing human IL-29 under the control of the Eu-lck promoter. To study if human IL-29 has biological activity in vivo in mice, expression of ISGs was analyzed by real-time PCR in the spleens of Eu-lck IL29 transgenic mice.

Transgenic mice (C3H/C57BL/6) were generated using a construct that expressed the human IL-29 gene under the control of the Eu-lck promoter. This promoter is active in T cells and B cells. Transgenic mice and their non-transgenic littermates (n=2/gp) were sacrificed at about 10 weeks of age. Spleens of mice were isolated. RNA was isolated from cell pellets using RNAEasy-kit® (Qiagen). RNA was treated with DNase to clean RNA of any contaminating DNA. cDNA was generated using Perkin-Rimer RT® mix. ISG gene induction was evaluated by real-time PCR using primers and probes (5’ FAM, 3’ NFQ) specific for mouse OAS, Pkr and Mxl. To obtain quantitative data, HPRT real-time PCR was duplexed with ISG PCR.

Furthermore, a standard curve was obtained using known amounts of IFN stimulated mouse PBLs. All data are shown as expression relative to internal HPRT expression.

Spleens isolated from IL-29 Tg mice showed high induction of ISGs OAS, Pkr and Mxl compared to their non-Tg littermate controls suggesting that human IL-29 is biologically active in vivo in mice.

Table 33

All data shown are fold expression relative to HPRT gene expression.

The average expression in two mice is shown

Example

Human IL-28 and IL-29 Protein Induce ISG Gene Expression In Liver, Spleen and Blood of Mice

To determine whether human IL-28 and IL-29 induce interferon stimulated genes in vivo, CHO-derived human IL-28A and IL-29 protein were injected into mice. In addition, E. coli derived IL-29 was also tested in in vivo assays as described above using MetlL-29C172S-PEG and MetIL-29-PEG. At various׳ time points and at different doses, ISG gene induction was measured in the blood, spleen and livers of the mice.

C57BL/6 mice were injected i.p or i.v with a range of doses (10 pg - 250 pg) of CHO-derived human 1L-28A and IL-29 or MetlL-29C172S-PEG and MetIL29C16-C113-PEG. Mice were sacrificed at various time points (lhr - 48hr). Spleens and livers were isolated from mice, and RNA was isolated. RNA was also isolated from the blood cells. The cells were pelleted and RNA isolated from pellets using RNAEasy®-kit (Qiagen). RNA was treated with DNase (Amicon) to rid RNA of any contaminating DNA. cDNA was generated using Perkin-Elmer RT mix (Perkin-Elmer). ISG gene induction was measured by real-time PCR using primers and probes specific for mouse OAS, Pkr and Mxl. To obtain quantitative data, HPRT real-time PCR was duplexed with ISG PCR. A standard curve was calculated using known amounts of IFN-stimulated mouse PBLs. All data are shown as expression relative to internal HPRT expression.

Human IL-29 induced ISG gene expression (OAS, Pkr, Mxl) in the livers, spleen and blood of mice in a dose dependent manner. Expression of ISGs peaked between 1-6 hours after injection and showed sustained expression above control mice upto 48 hours. In this experiment, human IL-28A did not induce ISG gene expression.

Table 34

<td> Injection</td><td> OAS- Ihr</td><td> OAS-6hr</td><td> OAS-24hr</td><td> OAS-48hr</td>

<td> None - liver</td><td> 1.6</td><td> 1.6</td><td> 1.6</td><td> 1.6</td>

<td> IL-29 liver</td><td> 2.5</td><td> 4</td><td> 2.5</td><td> 2.8</td>

<td> None - spleen</td><td> 1.8</td><td> 1.8</td><td> 1.8</td><td> 1.8</td>

<td> IL-29 - spleen</td><td> 4</td><td> 6</td><td> 3.2</td><td> 3.2</td>

<td> None - blood</td><td> 5</td><td> 5</td><td> 5</td><td> 5</td>

<td> IL-29 blood</td><td> 12</td><td> 18</td><td> 11</td><td> 10</td>

Results shown are fold expression relative to HPRT gene expression. A sample data set for IL-29 induced OAS in liver at a single injection of 250 pg i.v. is shown. The data shown is the average expression from 5 different animals/group.,

Table 35

<td> Injection</td><td> OAS (24hr)</td>

Table 36

MetIL-29-PEG ________ MetIL-29C172S-PEG_________Naive

<td> tvfxl</td><td> 537.51</td><td> 124.99</td><td> 33.58</td><td> 35.82</td><td> 27.89</td><td> 29.34</td><td> 16.61</td><td> 0.00</td><td> 10.98</td>

Mice were injected with 100 pg of proteins i.v. Data shown is fold expression over HPRT expression from livers of mice. Similar data was obtained from blood and spleens of mice.

Example

IL-28 and IL-29 Induce ISG Protein In Mice

To analyze of the effect of human IL-28 and IL-29 on induction of ISG protein (OAS), serum and plasma from IL-28 and IL-29 treated mice were tested for

OAS activity.

C57BL/6 mice were injected i.v with PBS or a range of concentrations (10 pg-250 pg) of human IL-28 or IL-29. Serum and plasma were isolated from mice at varying time points, and OAS activity was measured using the OAS radioimmunoassay (RIA) kit from Eiken Chemicals (Tokyo, Japan).

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

Table 37

<td> Injection</td><td> OAS-lhr</td><td> OAS-6hr</td><td> OAS-24hr</td><td> OAS-48hr</td>

OAS activity is shown at pmol/dL of plasma for a single concentration (250 pg) of human IL-29.

Example

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

To test the antiviral activities of IL-28 and IL-29 against viruses that infect the liver, the test samples were tested in mice against infectious adenoviral vectors expressing an internal green fluorescent protein (GFP) gene. When injected intravenously, these viruses primarily target the liver for gene expression. The adenoviruses are replication deficient, but cause liver damage due to inflammatory cell infiltrate that can be monitored by measurement of serum levels of liver enzymes like AST and ALT, or by direct examination of liver pathology.

C57B1/6 mice were given once daily intraperitoneal injections of 50 pg mouse IL28־ (zcyt024 as shown in SEQ ID NO:8) or metIL-29C172S- PEG for 3 days. Control animals were injected with PBS. One hour following the 3rd dose, mice were given a single bolus intravenous tail vein injection of the adenoviral vector, AdGFP (1 X 109 plaque-forming units (pfu)). Following this, every other day mice were given an additional dose of PBS, mouse IL-28 or metIL-29C172S- PEG for 4 more doses (total of 7 doses). One hour following the final dose of PBS, mouse IL-28 or metIL

29C172S- PEG mice were terminally bleed and sacrificed. The serum and liver tissue were analyzed. Serum was analyzed for AST and ALT liver enzymes. Liver was isolated and analyzed for GFP expression and histology. For histology, liver specimens were fixed in formalin and then embedded in paraffin followed by H&E staining. Sections of liver that had been blinded to treat were examined with a light microscope. Changes were noted and scored on a scale designed to measure liver pathology and inflammation.

Mouse IL-28 and IL-29 inhibited adenoviral infection and gene expression as measured by liver fluorescence. PBS-treated mice (n=8) had an average relative liver fluorescence of 52.4 (arbitrary units). In contrast, IL-28-treated mice (n=8) had a relative liver fluorescence of 34.5, and IL-29-treated mice (n=8) had a relative liver fluorescence of 38.9. A reduction in adenoviral infection and gene expression led to a reduced liver pathology as measured by serum ALT and AST levels and histology. PBS-treated mice (n=8) had an average serum AST of .234,' U/L (units/liter) and serum ALT of 250 U/L. In contrast, IL-28-treated mice (n=8) had an average serum AST of 193 U/L and serum ALT of 216 U/L, and IL-29-treated mice (n=8) had an average serum AST of 162 U/L and serum ALT of 184 U/L. In addition, the liver histology indicated that mice given either mouse IL-28 or IL-29 had. lower liver and inflammation scores than the PBS-treated group. The livers from the IL-29 group also had less proliferation of sinusoidal cells, fewer mitotic figures and fewer changes in the hepatocytes (e.g. vacuolation, presence of multiple nuclei, hepatocyte enlargement) than in the PBS treatment group. These data demonstrate that mouse IL28 and IL-29 have antiviral properties against a liver-trophic virus.

Example

LCMV Models

Lymphocytic choriomeningitis virus (LCMV) infections in mice mice are an excellent model of acture and chronic infection. These models are used to evaluate the effect of cytokines on the antiviral immune response and the effects IL-28 and IL-29 have viral load and the antiviral immune response. The two models used are: LCMV Armstrong (acute) infection and LCMV Clone 13 (chronic) infection. (See,

e.g, Wherry et al, J. Virol. 77:4911-4927,2003; Blattman et al. Nature Med. 9(5):540547, 2003; Hoffman et al, J, Immunol 170:1339-1353, 2003.) There are three stages of CD8 T cell development in response to virus: 1) expansion, 2) contraction, and 3) memory (acute model). IL-28 or IL-29 is injected during each stage for both acute and chronic models. In the chronic model, IL-28 or IL-29 is injected 60 days after infection to assess the effect of IL-28 or IL-29 on persistent viral load. For both acute and chronic models, IL-28 or IL-29 is injected, and the viral load in blood, spleen and liver is examined. Other paramenter that can be examined include: tetramer staining by flow to count the number of LCMV-specific CD8+ T cells; the ability of tetramer+ cells to produce cytokines when stimulated with their cognate LCMV antigen; and the ability of LCMV-specific CD8+ T cells to proliferate in response to their cognate LCMV antigen. LCMV-specific T cells are phenotyped by flow cytometry to assess the cells activation and differentiation state. Also, the ability of LCMV-specific CTL to lyse target cells bearing their cognate LCMV antigen is examined. The number and function of LCMV-specific CD4+ T cells is also assessed.

A reduction in viral load after treatment with IL-28 or IL-29 is determined. A 50% reduction in viral load in any organ, especially liver, would be significant. For IL-28 or IL-29 treated mice, a 20% increase in the percentage of tetramer positive T cells that proliferate, make cytokine, or display a mature phenotype relative to untreated mice would also be considered significant.

IL-28 or IL-29 injection leading to a reduction in viral load is due to more effective control of viral infection especially in the chronic model where untreated the viral titers remain elevated for an extended period of time. A two fold reduction, in viral titer relative to untreated mice is considered significant.

Example

Influenza Model of Acute Viral Infection

A, Preliminary Experiment to test antiviral activity

To determine the antiviral activity of IL-28 or IL-29 on acute infection by Influenza virus, an in vivo study using influenza infected c57Bl/6 mice is performed using the following protocol:

Animals: 6 weeks-old female. BALB/c mice (Charles River) with 148 mice, 30 per group.

Groups:

(1) Absolute control (not infected) to run in parallel for antibody titre and histopathology (2 animals per group) (2) Vehicle (i.p.) saline (3) Amantadine (positive control) 10 mg/day during 5 days (per os) starting 2 hours before infection (4) IL-28 or IL-29 treated (5 pg, i.p. starting 2 hours after infection) (5) IL-28 or IL-29 (25 pg, i.p. starting 2 hours after infection) (6) IL-28 or IL-29 (125 pg, i.p. starting 2 hours after infection)

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

For viral load (10 at LD50)

For immunology workout (LD30)

Day 0 - 9 - daily injections of IL-28 or IL-29 (i.p.)

Body weight and general appearance recorded (3 times/week)

Day 3 - sacrifice of 8 animals per group

Viral load in right lung (TCID50)

Histopathology in left lung

Blood sample for antibody titration

Day 10 - sacrifice of all surviving, animals collecting blood samples for antibody titration, isolating lung lymphocytes (4 pools of 3) for direct CTL assay ( in all 5 groups), and quantitative immunophenotyping for the following markers: CD3/CD4, CD3/CD8, CD3/CD8/CDllb, CD8/CD44/CD62L, CD3/DX5, GR-1/F480, and CD19.

Study N0.2

Efficacy study of IL-28 or IL-29 in C57B1/6 mice infected with mouseadapted virus is done using 8 weeks-old female C57B1/6 mice (Charles River).

Group 1: Vehicle (i.p.)

Group 2: Positive control: Anti-influenza neutralizing antibody (goat anti-influenza A/USSR (H1N1) (Chemicon International, Temecula, CA); 40 μg/mouse at 2 h and 4 h post infection (10 μΐ intranasal)

Group 3: IL-28 or IL-29 (5 pg, i.p.)

Group 4: IL-28 or IL-29 (25 pg, i.p.)

Group 5: IL-28 or IL-29 (125 pg, i.p.) . . Following-life observations and immunological workouts are prepared:

Day 0 - all animals infected with Influenza virus (dose determined in experiment 2)

Day 0 — 9 — daily injections of IL-28 or IL-29 (i.p.)

Body weight and general appearance recorded every other day

Day 10 - sacrifice of surviving animals and perform viral assay to determine viral load in lung.

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

Tetramer staining: The number of CD8+ T cells binding MHC Class I tp-tramp.TR containing influenza A nucleoprotein (NP) epitope are assessed using complexes of MHC class I with viral peptides: FLU-NP366-374/Db (ASNENMETM), (LMCV peptide/Db ).

Quantitative immunophenotyping of the following: CD8, tetramer, intracellular IFNy, NK1.1, CD8, tetramer, CD62L, CD44, CD3(+ or -), NK1.1(+), intracellular ΙΡΝγ, CD4, CD8, NK1.1, DX5, CD3 (+ or -), NK1.1, DX5, tetramer, Single colour samples for cytometer adjustment.

Survival/Re-challenge Study

Day 30: Survival study with mice are treated for 9 days with different doses of IL-28 or IL-29 or with positive anti-influenza antibody control. Body weight and antibody production in individual serum samples (Total, IgGl, IgG2a, IgG2b) are measured.

Re-challenge study:

Day 0: Both groups will be infected with A/PR virus (1LD30).

Group 6 will not be treated.

Group Ί will be treated for 9 days with 125 μg of IL-28 or IL-29.

Day 30: Survival study

Body weight and antibody production in individual serum samples (Total, IgGl, IgG2a, IgG2b) are measured.

Day 60: Re-challenge study

Survivors in each group will be divided into 2 subgroups

Group 6A and 7A will be re-challenge with A/PR virus (1 LD30) Group 6B and 7B will be re-challenge with A/PR virus (1 LD30).

Both groups will be followed up and day of sacrifice will be determined. Body weight and antibody production in individual serum samples (Total, IgGl, IgG2a, IgG2b) are measured.

Example

IL-28 and IL-29 have Antiviral Activity Against Hepatitis B virus (HBV) in vivo

A transgenic mouse model (Guidotti et al., J. Virology 69:6158-6169, 1995) supports the1 replication of high levels of infectious HBV and has been used as a chemotherapeutic model for HBV infection. Transgenic mice are treated with antiviral drugs and the levels of HBV DNA and RNA are measured in the transgenic mouse liver and serum following treatment. HBV protein levels can also be measured in the transgenic mouse serum following treatment. This model has been used to evaluate the effectiveness of lamivudine and IFN-a in reducing HBV viral titers.

PCT7US2004/025864

HBV TG mice (male) are given intraperitoneal injections of 2.5, 25 or 250 micrograms IL-28 or IL-29 every other day for 14 days (total of 8 doses). Mice are bled for serum collection on day of treatment (day 0) and day 7. One hour following the final dose of IL-29 mice undergo a terminal bleed and are sacrificed. Serum and liver are analyzed for liver HBV DNA, liver HBV RNA, serum HBV DNA, liver HBc, serum Hbe and serum HBs.

Reduction in liver HBV DNA, liver HBV RNA, serum HBV DNA, liver HBc, serum Hbe or serum HBs in response to IL-28 or IL-29 reflects antiviral activity of these compounds against HBV.

Example

IL-28 and IL-29 inhibit human herpesvirus-8 (HHV-8) replication in BCBL-1 cells

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

In the HHV-8 assay the test compound and a ganciclovir control, were assayed at five concentrations, each, diluted in a half-log series. The endpoints were TaqMan PCR for extracellular HHV-8 DNA (IC50) and cell numbers using CellTiter96® reagent (TC50; Promega, Madison, WI). Briefly, BCBL-1 cells were plated in 96-well microtiter plates. After 16-24 hours the cells were washed and the medium was replaced with complete medium containing various concentrations of the test compound in triplicate. Ganciclovir was the positive control, while media alone was a negative control (virus control, VC). Three days later the culture medium was replaced with fresh medium containing the appropriately diluted test compound. Six days following the initial administration of the test compound, the cell culture supernatant was collected, treated with pronase and DNAse and then used in a real-time quantitative TaqMan PCR assay. The PCR-amplified HHV-8 DNA was detected in real-time by monitoring increases in fluorescence signals that result from the exonucleolytic degradation of a quenched fluorescent probe molecule that hybridizes to the amplified HHV-8 DNA. For each PCR amplification, a standard curve was simultaneously generated using dilutions of purified HHV-8 DNA. Antiviral activity was calculated from the reduction in HHV-8 DNA levels (IC50). A novel dye uptake

PCT7US2004/025864 assay was then employed to measure cell viability which was used to calculate toxicity (TC50). The therapeutic index (ΤΓ) is calculated as TC50/IC50.

IL-28 and IL-29 inhibit HHV-8 viral replication in BCBL-1 cells. IL28A had an IC50 of 1 pg/ml and a TC50 of >10 pg/ml (ΊΙ >10). IL-29 had an IC50 of 6.5 pg/ml and a TC50 of >10 pg/ml (Π >1.85). MetIL-29C172S-PEG had an IC50 of 0.14 pg/ml and a TC50 of >10 pg/ml (Π >100).

Example

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

The antiviral activities of IL-28 and IL-29 are tested against EBV in an in vitro infection system in a B-lymphoid cell line, P3HR-1. In the EBV assay the test compound and a control are assayed at five concentrations each, diluted in a half-log series. The endpoints are TaqMan PCR for extracellular EBV DNA (IC50) and cell numbers using CellTiter96® reagent (TC50; Promega). Briefly, P3HR-1 cells are plated in 96-well microtiter plates. After 16-24 hours the cells are washed and the medium is replaced with complete medium containing various concentrations of the test compound in triplicate. In addition to a positive control, media alone is added to cells as a negative control (virus control, VC). Three days later the culture medium is replaced with fresh medium containing the appropriately diluted test compound. Six days following the initial administration of the test compound, the cell culture supernatant is collected, treated with pronase and DNAse and then used in a real-time quantitative TaqMan PCR assay. The PCR-amplified EBV DNA is detected in real-time by monitoring increases in fluorescence signals that result from the exonucleolytic degradation of a quenched fluorescent probe molecule that hybridizes to the amplified EBV DNA. For each PCR amplification, a standard curve was simultaneously generated using dilutions of purified EBV DNA. Antiviral activity is calculated from the reduction in EBV DNA levels (IC50). A novel dye uptake assay was then employed to measure cell viability which was used to calculate toxicity (TC50). The therapeutic index (ΊΊ) is calculated as TC50/IC50.

Example 45

IL-28 and IL-29 antiviral activity against Herpes Simplex Virus-2 (HSV-2־)

The antiviral activities of IL-28 and IL-29 were tested against HSV-2 in an in vitro infection system in Vero cells. The antiviral effects of IL-28 and IL-29 were assessed in inhibition of cytopathic effect assays (CPE). The assay involves the killing of Vero cells by the cytopathic HSV-2 virus and the inhibition of cell killing by IL-28 and IL-29. The Vero cells are propagated in Dulbecco’s modified essential medium (DMEM) containing phenol red with 10% horse serum, 1% glutamine and 1% penicillin-streptomycin, while the CPE inhibition assays are performed in DMEM without phenol red with 2% FBS, 1% glutamine and 1% Pen-Strep. On the day preceding the assays, cells were trypsinized (1% trypsin-EDTA), washed, counted and plated out at 104 cells/well in a 96-well flat-bottom BioCoat® plates (Fisher Scientific, Pittsburgh, PA) in a volume of 100 μΐ/well. The next morning, the medium was removed and a pre-titered aliquot of virus was added to the cells. The amount of virus used is the maximum dilution that would yield complete cell killing (>80%) at the time of maximal CPE development. Cell viability is determined using a CellTiter 96® reagent (Promega) according to the manufacturer’s protocol, using a Vmax plate reader (Molecular Devices, Sunnyvale, CA). Compounds are tested at six concentrations each, diluted in assay medium in a half-log series. Acyclovir was used as a positive control. Compounds are added at the time of viral infection. The average background and drug color-corrected data for percent CPE reduction and percent cell viability at each concentration are determined relative to controls and the IC50 calculated relative to the TC50.

IL-28A, IL-29 and MetIL-29C172S-PEG did not inhibit cell death (IC50 of >10ug/ml) in this assay. There was also no antiviral activity of IFN□ in the assay.

The complete disclosure of all patents, patent applications, and publications, and; electronically available material (e.g., GenBank amino acid and nucleotide sequence submissions) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

SEQUENCE LISTING <110> ZymoGenetics, Inc.

<120> HOMOGENEOUS PREPARATIONS OF IL-28 AND IL-29 <130> 03-10PC <150> US 60/493,194 <151> 2003-08-07 .

<150> US 60/551,841 <151> 2004-03-10 <150> US 60/559,142 <151> 2004-04-02 <160> 161 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 734 <212> DNA <213> Homo sapiens <220>

<221> sig_peptide <222> (53) . . . (127) <221> mat_peptide <222> (128)..(655) <221> CDS <222> (53)..(655) <400> 1 ' ' tcraataacag cctcagagtg tttcttctgc tgacaaagac cagagatcag ga atg aaa 58 . . Met Lys

155 160 165 agt ggg gac ctg tgt gtc tga ccctcccacc agtcatgcaa cctgagattt 685 Ser Gly Asp Leu Cys Vai *

170 175 tatttataaa ttagccactt gtcttaattt attgccaccc agtcgctat 734

<td> <213></td><td> Homo sapiens</td>

<td> <221></td><td> SIGNAL</td>

170 .175 <210> 3 <211* 856 <212> DNA <213> Homo sapiens <220>

<221> sig_peptide <222> (98) . . . (154) <221> mat_peptide <222> (155) . . . (700) <221> CDS <222> (98) . . . (700) <400> 3 aattaccttt tcactttaca cacatcatct tggattgccc attttgcgtg gctaaaaagc 60 agagccatgc cgctggggaa gcagttgcga tttagcc atg get gca get tgg acc 115 a Met Ala Ala Ala Trp Thr gtg gtg etg gtg act ttg gtg eta Vai Vai Leu Vai Thr Leu Vai Leu -10

ecc act tcc aag ccc acc aca act Pro Thr Ser Lys Pro Thr Thr Thr 5 10 ttc aaa tet etg tea cca cag gag

Phe Lys Ser Leu Ser Pro Gin Glu

25 gac gcc ttg gaa gag tea etc aag

Asp Ala Leu Glu׳ Glu Ser Leu Lys 40 cct gtc ttc ccc ggg aat tgg gac

Pro Vai Phe Pro. Gly Asn Trp Asp 55:

ege cct gtg gee,ttg gag get gag

Arg Pro Vai Ala;Leu Glu Ala Glu

7075 gag gcc get get ggc cca gcc etg Glu Ala Ala Ala Gly Pro Ala Leu 8590 cac acc etg cac cac ate etc. tec

His Thr Leu His ,His lie LeuSer

100 . . . ,.105 cag ccc aca gca ggg ccc aggccc

Gin Pro Thr Ala Gly Pro ArgPro cac egg etc cag gag gcc cccaaa

His Arg Leu Gin Glu Ala ProLys .135 gca tet gtc acc ttc aac etcttc

Ala Ser Vai Thr Phe . Asn LeuPhe ., .. . ' 150 . . -.155 tat gtg gcc gat ggg aac ctg tgt ctg aga acg tea ace cac cct gag691

Tyr Vai Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr His Pro Glu

165 170175 tcc acc tga caccccacac cttatttatg cgctgagccc tactccttcc740

Ser Thr * 180 ttaatttatt tcctctcacc ctttatttat gaagetgaag ccctgactga gacatagggc 800 tgagtttatt gttttacttt tatacattat gcacaaataa acaacaagga attgga856

<td> <210> <211> <212> <213></td><td> 4 200 PRT Homo</td><td> sapiens</td>

<td> <221></td><td colspan="2"> SIGNAL</td>

175 180 <210>

׳ ׳ 734 <211>

<212> DNA‘ <213> Homo sapiens <220>

<221> sig peptide <222> (53) . . . (127) <221> mat_peptide <222> (128)..(655) <221> CDS <222> (53) <400> 5 tgggtgacag cctcagagtg tttcttctgc tgacaaagac cagagatcag ga atg aaa Met Lys -25 eta gac atg acc ggg gac tgc atg cca gtg etg gtg etg atg gee gca106

Leu. Asp Met' Thr Gly Asp Cys Met Pro Vai Leu Vai Leu Met Ala Ala

10- 15-סבgtg etg acc gtg act gga gca gtt cct gtc gee agg etc ege ggg get

Vai Leu Thr Vai Thr Gly Ala Vai Pro Vai Ala Arg Leu Arg GlyAla etc ccg gat gca agg ggc tgc cac ata gee cag ttc aag tee etg tet202

Leu Pro Asp Ala Arg Gly Cys His He Ala Gin Phe Lys Ser LeuSer

15 2025 cca cag gag etg cag gee ttt aag agg gee aaa gat gee tta gaa gag250 pro Gin Glu Leu Gin Ala Phe Lys Arg Ala Lys Asp Ala Leu GluGlu

3540 teg ett etg etg aag gac tgc aag tgc ege tee.ege etc ttc ccc agg298

Ser Leu Leu Leu Lys Asp Cys Lys Cys Arg Ser Arg Leu Phe ProArg

5055 acc tgg gac etg agg cag etg cag gtg agg gag ege ccc gtg get ttg346

Thr Trp Asp Leu Arg Gin Leu Gin Vai Arg Glu Arg Pro Vai AlaLeu

6570 gag get gag etg gee etg acg etg aag gtt etg gag gee acc get gac3;94

Glu Ala Glu. Leu Ala Leu Thr Leu Lys Vai Leu Glu Ala Thr AlaAsp

8085 act gac cca gee etg ggg gat gtc ttg gac cag ccc ett cac acc etg442.

Thr Asp Pro Ala Leu Gly Asp Vai Leu Asp Gin Pro Leu His ThrLeu

95 100105 cac cat ate etc tee cag etc egg gee tgt ate cag cct cag ccc acg490

His His He Leu Ser Gin Leu Arg Ala Cys He Gin Pro Gin ProThr

110 115 .120 gca ggg ccc agg acc egg ggc ege etc cac cat tgg etg cac egg etc538

Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu His ArgLeu

125 130135 cag gag gee cca aaa aag gag tee cct ggc tgc etc gag gee tet gtc586 (רדוד Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala SerVai

140 145150 acc ttc aac etc ttc ege etc etc acg ega gac etg aat tgt gtt gee634

Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys VaiAla !55 160165 age ggg gac etg tgt gtc Ser Gly Asp Leu Cys Vai 170 175 tga cccttccgcc agtcatgcaa cctgagattt *

tatttataaa ttagccactt ggcttaattt attgccaccc agtegetat <210> 6 <211> 200 <212> PRT <213> Homo sapiens <220>

<221>

<222>

SIGNAL (1) . - - (25)

Sit°Lys Leu Asp Met Thr Gly Asp Cys Met Pro Vai Leu Vai Leu Met

10־ 15־ 20־25Ala Ala Vai Leu Thr Vai Thr Gly Ala Vai Pro Vai Ala Arg Leu Arg -5 15

Gly Ala Leu Pro Asp Ala Arg Gly Cys His lie Ala Gin Phe Lys Ser 10 15

Leu Ser Pro Gin Glu Leu Gin Ala Phe Lys Arg Ala Lys Asp Ala Leu 25 2035

Glu Glu Ser Leu Leu Leu Lys Asp Cys Lys Cys Arg Ser Arg LeuPhe

45 5055

Pro Arg Thr Trp Asp Leu Arg Gin Leu Gin Vai Arg Glu Arg ProVai 6570

Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Vai Leu Glu Ala Thr 75 8085

Ala Asp Thr Asp Pro Ala Leu Gly Asp Vai Leu Asp Gin Pro Leu His 90 95100

Thr Leu His His He Leu Ser Gin Leu Arg Ala Cys He Gin Pro Gin 105 HO

Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp LeuHis

120 125 130135

Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu GluAla a140 145.150

Ser Vai Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys 155 160165

Vai Ala Ser Gly Asp Leu Cys Vai 170175 .

<210> 7 <211> 633 .

<212> DNA <213> Mus musculus <220>

<221> sig_peptide <222> (22) . -(105) ' <221> mat_peptide <222> (106)..(630) <221> CDS <222> (22)..(630) <400> 7 ' ' ' ' '' tcacagaccc cggagagcaa c atg aag cca gaa aca get ggg ggc cac atg51

Met Lys Pro Glu Thr Ala Gly Gly HisMet _ . -25 .

etc etc etg etg ttg׳cct etg etg etg gee gca gtg etg aca aga ace99

Leu Leu Leu Leu Leu Pro Leu Leu Leu Ala Ala.Vai Leu Thr ArgThr

. 5 . ' 10 .. .־... 15־ ' caa get gac cct gtc ccc agg gcc acc.agg etc cca gtg gaa gca aag14

Gin Ala Asp Pro Vai Pro Arg Ala Thr Arg Leu Pro Vai Glu AlaLys

5 . . .10 .

gattgc cac att get cag ttc aag.tct etg tcc cca aaa gag etg cag19

Asn Cys His He Ala Gin Phe Lys Ser Leu Ser Pro Lys Glu LeuGin . ' . 20 .. . 25 .30 gcc ttc aaa aag gcc aag gat gcc ate gag aag agg etg ett gag aag24

Ala Phe Lys Lys Ala Lys Asp Ala He Glu Lys Arg Leu Leu GluLys

... 40 . :. .. 45

tga cct <210> 8.

<211> 202 <212> PRT <213> Mus musculus <220>..

<221> SIGNAL :.

<222> (1)..(28) <400> 8 .' '' .

Met Lys Pro Glu Thr Ala Gly Gly His Met Leu Leu Leu Leu Leu Pro

15־ 20־ 25-.

Leu Leu Leu Ala Ala Vai Leu Thr Arg Thr Gin Ala Asp Pro Vai Pro

5־ ct Ala Tb־r zx־rg Lgu Pxo Vai Gin Ala Lys Asp Cys His Ils Ala Gin 5 10 1520

Phe Lys Ser Leu Ser Pro Lys Glu Leu Gin Ala Phe Lys Lys AlaLys

30 .35

Asp Ala lie Glu Lys Arg Leu Leu Glu Lys Asp Leu Arg Cys SerSer

45 .50

His Leu Phe Pro Arg Ala Trp Asp Leu Lys Gin Leu Gin Vai Gin Glu 55 6065.

־ atq־ pro Lys Ala Leu Gin Ala Glu Vai Ala Leu Thr Leu Lys Vai Trp 70 75 .80

Ash Met Thr Asp Ser Ala Leu Ala Thr lie Leu Gly Gin Pro Leu

00-^ . ־ 95 9085

His Thr'Leu Ser His lie His Ser Gin.Leu Gin Thr Cys. Thr Gin Leu 105 . . ־ . . HO

Gin Ala Thr. Ala Glu Pro Arg Ser Pro Ser Arg Arg Leu Ser Arg Trp.

120125

Leu His Arg Leu Gin Glu Ala Gin Ser Lys 135 140 u 7.

Glu Ala Ser Vai Thr Ser Asn Leu Phe Arg

150155

Lys Cys Vai Ala Asn Gly Asp Gin Cys Vai

165170

Glu Thr Pro Gly Cys Leu 145

Leu Leu Thr Arg Asp Leu <210> 9 <211> 632 <212> DNA <213> Mus musculus <220>

<221> sig_peptide <222> (22). . .(105) <221> mat_peptide <222> (106)..(630) <221> CDS <222> (22).-.(630) <400> 9 tcacagaccc cggagagcaa c atg aag

Met Lys cca gaa aca get ggg ggc cac atg

Pro Glu Thr Ala Gly Gly His Met

20־ 25etg gcc gca gtg etg aca aga acc

Leu Ala Ala Vai Leu Thr Arg Thr 5־ 10acc agg etc cca gtg gaa gca aag

Thr Arg Leu Pro Vai Glu Ala Lys 10 tet etg tcc cca aaa gag etg cag

Ser Leu Ser Pro Lys Glu Leu Gin

2530 ate gag aag agg etg ett gagaag

He Glu Lys Arg Leu Leu GluLys ate tcc agg gcc tgg gac etg aag

He Ser Arg Ala Trp Asp Leu Lys aag gee ttg cag get gag gtg gcc

Lys Ala Leu Gin Ala Glu Vai Ala 75 ata aat gac tea gcc etg acc acc

He Asn Asp Ser Ala Leu Thr Thr 90 etg age cac att cac tcc cag etg

Leu Ser His He His Ser Gin Leu 105 '. 11° aca gca gag ccc aag ccc ccg agt Thr Ala Glu Pro Lys Pro Pro Ser 120 125 agg etc cag gag gee cag age aag.

.387 etc etc etg etg ttg cct etg etg Leu Leu Leu Leu Leu Pro Leu Leu -15 .

caa get gac cct gtc ccc agg gee

Gin Ala Asp Pro Vai Pro Arg Ala 1 5 gat tgc cac att get cag ttc aag

Asp Cys His He Ala Gin Phe Lys

20 gec ttc aaa aag gee aag ggt gee

Ala Phe Lys Lys Ala Lys Gly Ala gac atg agg tgc agt tcc cac etc

Asp Met Arg Cys Ser Ser His Leu cag etg cag gtc caa gag ege ccc Gin Leu Gin Vai Gin Glu Arg Pro 65 70 etg acc etg aag gtc tgg gag aac Leu Thr Leu Lys Vai Trp Glu Asn 80 85 ate ctg.ggc cag cct ett cat aca He Leu Gly Gin Pro Leu His Thr

. 100 - : . . . . י 95 cag acc tgt aca cag ett cag gcc Gin Thr Cys Thr Gin Leu Gin Ala 115 .

ege ege etc tcc ege tgg etg cac

<210> 10 <211> 202 <212> PRT <213> Mus musculus <220>

<221> SIGNAL <222> (1) . . . (28) ״ ״ 10 <400>

Met Lys Pro Glu Thr Ala Gly Gly His Met Leu Leu Leu Leu Leu Pro

15־ 20־25Leu Leu'Leu Ala Ala Vai Leu Thr Arg Thr Gin Ala Asp Pro Vai Pro ?

5־10Arg Ala Thr Arg Leu Pro Vai Glu Ala Lys Asp Cys His lie AlaGin

10 1520

Phe Lys Ser Leu Ser Pro Lys Glu Leu Gin Ala Phe Lys Lys AlaLys

3035

Gly Ala lie Glu Lys Arg Leu Leu Glu Lys Asp Met Arg Cys SerSer

4550

His Leu He Ser Arg Ala Trp Asp Leu Lys Gin Leu Gin Vai GinGlu , 60 . . '65

Arg Pro Lys Ala Leu Gin Ala Glu Vai Ala Leu Thr Leu Lys VaiTrp

7580

Glu Asn lie Asn Asp Ser Ala Leu Thr Thr He Leu Gly Gin Pro Leu

90 95100

His Thr Leu Ser His lie His Ser Gin Leu Gin Thr Cys Thr Gin Leu

HO115

Gin Ala Thr Ala Glu Pro Lys Pro Pro Ser Arg Arg Leu Ser Arg Trp 120 125130

Leu His Arg Leu Gin Glu Ala Gin Ser Lys.Glu Thr Pro Gly Cys Leu 135 140145.

Glu Asp Ser Vai Thr Ser Asn Leu Phe Gin Leu Leu Leu Arg Asp Leu 150 155160

Lys Cys Vai Ala Ser Gly Asp Gin Cys Vai 165 170 ... .' . ' <210> 11 <211> 520 <212> PRT :

<213> Homo sapiens י ' 11' <400>

Met Ala Gly Pro Glu Arg Trp Gly Pro Leu Leu Leu Cys Leu Leu Gin . ; 5 10 15

Ala Ala Pro Gly Arg Pro Arg Leu Ala Pro Pro Gin'Asn Vai Thr. Leu

25 30 . .

Leu Ser Gin Ash, Phe Ser Vai Tyr Leu. Thr Trp Leu Pro Gly Leu Gly . ' '. - 40 . . . /:. . .. 45

Asn Pro Gin Asp Vai Thr Tyr Phe Vai Ala Tyr Gin Ser Ser Pro Thr 50 5560

Arg Arg Arg Trp Arg Glu Vai Glu Glu Cys Ala Gly Thr Lys Glu Leu 65 . 70 7580

Leu Cys Ser Met Met Cys Leu Lys Lys Gin Asp Leu Tyr Asn Lys Phe 85 9095

Lys Gly Arg Vai, Arg Thr Vai Ser Pro Ser Ser Lys Ser Pro Trp Vai 100 105110

Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Vai Glu Pro Ala Pro Pro 115 120125

Vai Leu Vai: Leu Thr Gin Thr Glu Glu lie Leu Ser Ala Asn Ala Thr 130 135140

Tyr Gin Leu Pro-Pro Cys Met Pro Pro Leu Asp Leu Lys Tyr GluVai

145 150 155160

Ala Phe Trp Lys Glu Gly Ala Gly Asn Lys Thr Leu Phe Pro VaiThr '165 . 170175

Pro His Gly Gin Pro Vai Gin Tie Thr Leu Gin Pro Ala Ala Ser Glu 180 185190

His His Cys Leu Ser Ala Arg Thr He Tyr Thr Phe Ser Vai Pro Lys 195 ’ 200205

Tyr Ser Lys Phe Ser Lys Pro Thr Cys Phe Leu Leu Glu Vai Pro Glu

220 215 ׳210

Ala Asn Trp Ala Phe Leu Vai Leu Pro Ser Leu Leu lie Leu LeuLeu

225 230 235240

Vai He Ala Ala Gly Gly Vai He Trp Lys Thr Leu Met Gly AsnPro

245 250255

Trp Phe Gin Arg Ala Lys Met Pro Arg Ala Leu Asp Phe Ser Gly His 260 265270

Thr His Pro Vai Ala Thr Phe Gin Pro Ser Arg Pro Glu Ser Vai Asn 275 280285

Asp Leu Phe Leu Cys Pro Gin Lys Glu Leu Thr Arg Gly Vai Arg Pro 290 295300

Thr Pro Arg Vai Arg Ala Pro Ala Thr Gin Gin Thr Arg Trp LysLys

305 ! 310' 315320

Asp Leu Ala Glu Asp Glu Glu Glu Glu Asp Glu Glu Asp Thr' GluAsp

325 330335

Gly Vai Ser Phe Gin Pro Tyr He Glu Pro Pro Ser Phe Leu Gly Gin 340 345350'

Glu His Gin Ala Pro Gly His Ser Glu Ala Gly Gly Vai Asp Ser Gly 355 360365

Arg Pro Arg Ala Pro Leu Vai Pro Ser Glu Gly Ser Ser Ala Trp Asp 370 375.380

Ser Ser Asp Arg Ser Trp Ala Ser Thr Vai Asp Ser Ser Trp Asp Arg

335 390 395400

Ala Gly Ser Ser Gly Tyr Leu Ala Glu Lys Gly Pro Gly Gin Gly Pro

405 410415

Gly Gly Asp Gly His Gin Glu Ser Leu Pro Pro Pro Glu Phe Ser Lys 420 425430

Asp Ser Gly Phe Leu Glu Glu Leu Pro Glu Asp Asn Leu Ser Ser Trp 435 440445

Ala Thr Trp Gly Thr Leu Pro Pro Glu Pro Asn Leu Vai Pro Gly Gly 450 - 455460

Pro Pro Vai Ser Leu Gin Thr Leu Thr Phe Cys Trp Glu Ser Ser' Pro

465 470 475480

Glu Glu Glu Glu Glu Ala Arg Glu Ser Glu He Glu Asp Ser Asp Ala

485 490495

Gly Ser Trp Gly Ala Glu Ser Thr Gin Arg Thr Glu Asp Arg Gly Arg 500 ! .505510

Thr Leu Gly His Tyr .Met Ala Arg.

515. . . , 520 .

<210> 12 <211> 531 :.

<212> DNA <213> Artificial Sequence' <22 0>

<223> mature protein of SEQ ID NO: 1, with 3' Met added <221> CDS <222> (1) . . -:(531) <400 12 atg gtt cot gtc gcc agg etc cac

Met Vai Pro Vai Ala Arg Leu His 5 tgc cac ata gcc cag ttc aag tcc

Cys His lie Ala Gin Phe Lys Ser 20 ggg get etc ccg gat gca agg ggc 48

Gly Ala Leu Pro Asp Ala Arg Gly etg tet cca cag gag etg cag gcc96

Leu Ser Pro Gin Glu Leu Gin Ala

2530 ttt aag agg gcc aaa gat

Phe Lys Arg Ala Lys Asp 35 tgc agg tgc cac tcc ege

Cys Arg Cys His Ser Arg 50 etg cag gtg agg gag ege Leu Gin Vai Arg Glu Arg 65 70 acg etg aag gtt etg gag Thr Leu Lys Vai Leu Glu 85 gac gtc ttg gac cag ccc

Asp Vai Leu Asp Gin Pro 100 ttc egg gcc tgt ate cag

Phe Arg Ala Cys lie Gin 115 ggc ege etc cac cat tgg

Gly Arg Leu His His Trp 130 gag tcc cct ggc tgc etc Glu Ser Pro Gly Cys Leu 145. 150 etc etc acg ega gac etg Leu Leu Thr Arg Asp Leu .; 165 gcc tta gaa gag teg Ala Leu Glu Glu Ser 40 etc ttc ccc agg ace

Leu Phe Pro Arg Thr 55 ccc atg get ttg gag

Pro Met Ala Leu Glu gcc acc get gac act Ala Thr Ala Asp Thr 90 ett cac acc etg cac

Leu His Thr Leu His 105 cct cag ccc acg gca

Pro Gin Pro Thr Ala 120 etg tac egg etc cag Leu Tyr Arg Leu Gin 135 gag gcc tet gtc acc Glu Ala Ser Vai Thr 155 aat tgt gtt gcc agt Asn Cys Vai Ala Ser 170 ett etg etg aag gac Leu Leu Leu Lys Asp 45 tgg gac etg agg cag

Trp Asp Leu Arg Gin 60 get gag etg gcc etg

Ala Glu Leu Ala Leu gac cca gee etg gtg Asp Pro Ala Leu Vai 95 cat ate etc tcc cag

His He Leu Ser Gin

110 ggg ccc agg acc egg

Gly Pro Arg Thr Arg . 125.

gag gcc cca aaa aag Glu Ala Pro Lys Lys 140 ttc aac etc ttc ege

Phe Asn Leu Phe Arg ggg gac etg tgt gtc Gly Asp Leu Cys Vai 175

336.

<210> 13 <211> 176 <212> PRT <213> Artificial Sequence <223> mature protein of SEQ ID NO: 1, with 3' Met.added׳ <400> 13. .... ; 7. .

165 '170 175

<td> <213></td><td> Artificial Sequence</td>

<td> <220> <223></td><td> mature protein of SEQ ID NO: 3, . with 3' Met added</td>

<221> CDS <222> (1)..(549) <400> 14

115 ; . 120125 cac cac tgg ctg cac egg etc cag gag gcc ccc aaa aag gag teeget

His His Trp Leu His Arg Leu Gin Glu Ala pro Lys Lys Glu SerAla

130 135140 ggc tgc ctg gag gca tet gtc acc ttc aac etc ttc ege etc etcacg

Gly Cys Leu Glu Ala Ser Vai Thr Phe Asn Leu Phe Arg Leu LeuThr

145 150 155160 ega gac etc aaa tat gtg gcc gat ggg aac ctg tgt ctg aga acgtea

Arg Asp Leu Lys Tyr Vai Ala Asp Gly Asn Leu Cys Leu Arg ThrSer

165 170175 acc cac cct gag tee acc tga caccccacac cttatttatg cgctgagccc

Thr His Pro Glu Ser Thr * 180 tactccttcc ttaatttatt tcctctcacc ctttatttat ga <210> 15 <211> 182 <212> PRT <213> Artificial Sequence

י- י_י. .<220>

<223> mature protein of SEQ ID NO: 3, with 3' Met added <400> 15

Met Gly Pro Vai Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys 1 5 10 .15

His lie Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe 20 2530

Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 4045

Ser Cys Ser Ser Pro Vai Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 5560

Gin Vai Arg Glu Arg Pro Vai Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 7580

Leu Lys Vai Let! Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Vai Leu 85 90 '95

Asp Gin Pro Leu His Thr Leu His His lie Leu Ser Gin Leu Gin Ala 100 105HO

Cys lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120

His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Ala 130 135 1140

Gly Cys Leu Glu Ala Ser Vai Thr Phe Asn Leu Phe Arg Leu LeuThr

145 150 155160

Arq Asp Leu Lys Tyr Vai Ala Asp Gly Asn Leu Cys Leu Arg ThrSer . 165 170 :175

Thr His Pro, Glu Ser Thr . 180' <210> 16 <211> 531 ״r ; <212> DNA . .

<213> Artificial Sequence . . י. <220>

<223> mature protein of SEQ ID NO:' 5, with 3’ Met added <221> CDS : 'יי <222> (1)-- - (531) .־.. .'. .

<400> 16 ata gtt cat gtc gcc agg etc ege ggg get etc ccg gat gca agg ggc

Met Vai Pro Vai Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly

5 10 tgc cac.ata gcc cag ttc aag tee etg tet cca cag gag etg caggee

Cys His lie Ala Gin Phe Lys Ser Leu Ser Pro Gin Glu Leu GinAla

2530 ttt aag agg gcc aaa gat gcc tta gaa gag teg ett etg etg aag gac Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp

4045

׳384

׳432 tga * <210> 17 <211> 176 <212> PRT :

<213> Artificial Sequence <223> mature protein of SEQ ID NO: 5, with 3' Met added <400> 17 ' ' _

Met Vai Pro Vai: Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly y 5 10 15 . ׳

Cvs His He Ala Gin Phe Lys Ser Leu Ser Pro Gin Glu Leu Gin Ala 20 25 30

Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp.

45 ־ . . . . . 40 ׳ 35

Cys Lys Cys Arg ,.Ser Arg’ Leu' Phe Pro Arg Thr Trp Asp Leu Arg Gin

5560

Leu Gin Vai Arg Glu Arg Pro Vai Ala Leu Glu Ala Glu Leu AlaLeu

70 7580

Thr Leu Lys Vai Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala LeuGly

9095

Asp Vai Leu Asp' Gin Pro Leu His Thr Leu His His He Leu Ser Gin 100' 105110

Leu Arg Ala Cys lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Thr Arg 115 120125

Gly Arg Leu His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys 130 135140

Glu Ser Pro Gly Cys Leu Glu Ala Ser Vai.Thr Phe Asn Leu Phe Arg 145 150 155, 160

Leu Leu Thr Arg Asp Leu Asn Cys Vai Ala Ser Gly Asp Leu Cys Vai 165 170175 <210> 18 <211> 528 <212> DNA <213> Artificial Sequence <220> .

<223> IL-28A mutant C48S <221> CDS <222> (1)..(528) <400> 18

His lie. Ala Gin Phe Lys Ser Leu Ser Pro Gin Glu Leu Gin Ala Phe 20 25 30 aag agg gcc aaa gat gcc tta gaa gag teg ett etg etg aag gac tcc 144

Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Ser 35 40 45

־ 528 <210> 19 <211> 175 <212> PRT <213> Artificial Sequence <220>

<223> IL-28A mutant C48S <400> 19

Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly Cys 1 5 1015

His He Ala Gin ,Phe Lys Ser Leu Ser Pro Gin Glu Leu Gin Ala Phe 20 : 2530

Lvs Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Ser 35 4045

Arg Cys His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gin Leu 50 5550

Gin Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala LeuThr

70 7580

Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu ValAsp

9095

Val Leu Asp Gin Tro Leu His Thr Leu His His He Leu Ser Gin Phe 100 105HO

Arg Ala Cys lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Thr Arg Gly 115 ' 120125

Arg Leu His His Trp Leu Tyr Arg Leu Gin Glu Ala Pro Lys LysGlu : 130 . 135140

Ser Pro Gly Cys Leu Glu. Ala Ser Val Thr Phe Asn Leu Phe ArgLeu

145 150 155160

Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165 170175 <210> 20.

<211> 531 <212> DNA.

<213> Artificial Sequence <220>

<223> met IL-28A‘ mutant C49S <221> CDS <222> (1). - (531) <400> 20 i '

tcc agg tgc cac tcc cgc etc ttc ccc agg acc tgg gac etg agg cag192

Ser Arg Cys His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu ArgGin

55.60 etg cag gtg agg gag cgc ccc atg get ttg gag get gag etg gee etg240

Leu Gin Vai Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu AlaLeu . 70 7580 acg etg aag gtt etg gag gee acc get gac act gac cca gee etg gtg288

Thr Leu Lys Vai Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala LeuVai

9095 gac gtc ttg gac cag ccc ett cac acc etg cac cat ate etc tee cag336

Asp Vai Leu Asp Gin Pro Leu His Thr Leu His His He Leu SerGin

100 105110 ttc egg gee tgt ate cag cct cag ccc acg gca ggg ccc agg acc egg384

Phe Arg Ala Cys He Gin Pro Gin Pro Thr Ala Gly Pro Arg ThrArg

115 120125 ggc cgc etc cac cat tgg etg tac egg etc cag gag gee cca aaa aag432

Gly Arg Leu His His Trp Leu Tyr Arg Leu Gin Glu Ala Pro LysLys

130 135140 gag tcc cct ggc tgc etc gag gee tet gtc acc ttc aac etc ttc cgc480

Glu Ser Pro Gly Cys Leu Glu Ala Ser Vai Thr Phe Asn Leu PheArg

145 150 155 etc etc acg ega gac etg aat tgt gtt gee agt ggg gac etg tgt gtc5:28

Leu Leu Thr Arg. Asp Leu Asn Cys Vai Ala Ser Gly Asp Leu CysVai

165 170. 175 <210> 21 <211> 176 <212> PRT <213> Artificial Sequence <220>

<223> met IL-28A mutant C49S <400> 21

Met Vai Pro Vai Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly 1 5 1015

Cys His lie Ala Gin Phe Lys Ser Leu Ser Pro Gin Glu Leu Gin Ala 20 2530

Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp 35 4045

Ser Arg Cys His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gin 50 5560

Leu Gin Vai Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu AlaLeu

70 75 .80

Thr Leu Lys Vai Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala LeuVai

9095

Asp Vai Leu Asp Gin Pro Leu His Thr Leu His His lie Leu Ser Gin 100'. - 105 . . 110 '

Phe Arg Ala Cys He Gin Pro Gin Pro Thr Ala Gly Pro Arg Thr Arg 115 120 . 125 . .

Gly Arg Leu His His Trp Leu Tyr Arg Leu Gin Glu Ala Pro Lys Lys

. 140 135 ׳ 130

Glu Ser Pro Gly Cys Leu Glu Ala Ser Vai Thr Phe Asn Leu Phe Arg 145 /. .150 . . . 155 '.160

Leu Leu Thr Arg Asp Leu Asn

Cys Vai Ala Ser Gly Asp Leu Cys Vai

170 175 <210> 22 <211> 528 <212> DNA <213> Artificial Sequence <220>

<223> IL-28A mutant C50S <221> CDS <222> (1). . . (528) <400> 22

<210> 23 . . .

<211> 175 <212> PRT ' ' <213> Artificial Sequence <220>

<223> IL-28A mutant C50S <400> 23

<td> <213></td><td> Artificial Sequence</td>

<td> <220> <223></td><td> met IL-28A mutant C5IS</td>

tga <210> 25 .

<211> 176 <212> PRT <213> Artificial Sequence <220> .

<223> met IL-28A mutant C51S <400> 25

<210> 26 <211> 546 <212> DNA .

<213> Artificial Sequence <220>

<223> IL-29 mutant C171S <221> CDS ; .

<222> (1) . . . (546) <400> 26 ggt ccg gtt ccg acc tct aaa cca acc acc act ggt aaa ggt tgccac

Gly Pro Vai Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly CysHis . 5 1015 ate ggt cgt ttc aaa tct etg tct ccg cag gaa etg get tct ttcaaa

He Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser PheLys

2530 aaa get cgt gac get.etg gaa gaa tct etg aaa etg aaa aac tggtct

Lys Ala Arg׳ Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn TrpSer

4045 tgc tct tct ccg gtt. ttc ccg ggt aac tgg gat etg cgt etg etgcag

Cys Ser Ser Pro Vai Phe Pro Gly Asn Trp Asp Leu Arg Leu LeuGin

5560 gtt cgt gaa cgt ccg gtt get etg gaa get gaa etg get etg accetg

Vai Arg Glu Arg Pro Vai Ala Leu Glu Ala Glu Leu Ala Leu Thr.Leu

70 7580 aaa gtt etg gaa get get gca ggt cct get etg gaa gat gtt etggat

Lys Vai Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Vai LeuAsp

9095 cag ccg etg cac act etg cac cac ate etg tct cag etg cag gettgc

Gin Pro Leu His Thr Leu His His He Leu Ser Gin Leu Gin AlaCys

100 105'110 att caa ccg caa ccg acc get ggt ccg cgt ccg cgt ggt cgt etgcac lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg LeuHis

115 120125 cac tgg etg cat cgt etg cag gaa get ccg aaa aaa gaa tct getggt

His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser AlaGly

130 135140 tgc etg gaa get tct gtt acc ttc aac etg ttc cgt etg etg acccgt

Cys Leu Glu Ala Ser Vai Thr Phe Asn Leu Phe Arg Leu Leu ThrArg

145 .150 15516,0 gat etg aaa tac gtt get gat ggt aac etg tct etg cgt acc tctacc

Asp Leu Lys Tyr Vai Ala Asp Gly Asn Leu Ser Leu Arg Thr SerThr ' 165 170175 cat ccg gaa tct acc taa

His Pro Glu Ser Thr *

180'

432:

<210> 27 <211> 181 <212> PRT .

<213> Artificial Sequence <220>

<223> IL-29 mutant C171S <400> 27

Gly Pro Vai Pro :Thr Ser Lys Pro

5.:. י 1

He Gly Arg Phe Lys Ser Leu Ser

: 20 ־

Lys Ala Arg Asp Ala Leu Glu Glu

Thr Thr Thr Gly Lys Gly Cys His

Pro Gin Glu Leu Ala Ser Phe Lys

Ser Leu Lys Leu Lys Asn Trp Ser

Cys Ser Ser Pro Val Phe 50

Val Arg Glu Arg Pro Val 65 70

Lys Val Leu Glu Ala Ala 85

Gin Pro Leu His Thr Leu 100

He Gin Pro Gin Pro Thr 115

His Trp Leu His Arg Leu 130

Cys Leu Glu Ala Ser Val 145 I50

Asp Leu Lys Tyr Val Ala 165

His Pro Glu Ser Thr .

Pro Gly Asn Trp Asp Leu Arg Leu Leu Gin 55

Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu 75B0

Ala Gly Pro Ala Leu Glu Asp Val Leu Asp 9095

His His He Leu Ser Gin Leu Gin Ala Cys 105HO

Ala Gly Pro Arg Pro Arg Gly Arg Leu His 120125

Gin Glu Ala Pro Lys Lys Glu Ser Ala Gly

135 I40 τ־Ητ Ph.B Asn Lsu Ptis Aiircj Lbu. Lbu Tnx Axg״

155160

Asp Gly Asn Leu Ser Leu Arg Thr Ser Thr 170175 <210> 28 .

<211> 549 <212> DNA <213> Artificial Sequence <220>

<223> met IL-29 mutant C172S <221> CDS <222> (1) . . . (549) <400> 28 atg ggt ccg gtt ccg acc tct aaacca

Met Gly Pro Val Pro Thr Ser LysPro.

. 1 5 cac ate ggt cgt ttc aaa tct etgtct

His He Gly Arg Phe Lys Ser LeuSer aaa aaa get cgt gac get etg gaagaa

Lys Lys Ala Arg Asp Ala Leu GluGlu ;40 tct tgc tct tct ccg gtt ttc ccgggt

Ser Cys Ser Ser Pro Val Phe ProGly cag gtt cgt gaa cgt ccg gtt getetg

Gin Val Arg Glu Arg Pro Val AlaLeu

70 . etg aaa gtt etg gaa get get gcaggt

Leu Lys Val Leu Glu Ala Ala AlaGly gat'cag ccg etg cac act etg cac cac ' Asp Gin Pro Leu His Thr Leu.HisHis

100 .

tgc att caa ccg caa ccg acc getggt

Cys He Gin Pro Gin Pro Thr AlaGly

115120 cac cac tgg etg cat cgt etg cag gaa acc acc act ggt aaa ggt tgc 48

Thr Thr Thr Gly Lys Gly Cys

15 ccg cag gaa etg get tct ttc 96

Pro Gin Glu Leu Ala Ser Phe tct etg aaa etg aaa aac tgg 144

Ser Leu Lys Leu Lys Asn Trp .45 aac tgg gat etg cgt etg etg 192

Asn Trp Asp Leu Arg Leu Leu gaa get gaa etg get etg acc 240

Glu Ala Glu Leu Ala Leu Thr

80 cct get etg gaa gat gtt etg 288

Pro Ala Leu Glu Asp Val Leu

95 ate etg tct cag etg cag get 336 lie Leu Ser Gin Leu Gin Ala ccg cgt ccg cgt ggt cgt etg 384

Pro Arg Pro Arg Gly Arg Leu '125 '.

get ccg aaa. aaa gaa tct get 432

<210> 29 <211> 182 <212> PRT <213> Artificial Sequence <220>

<223> met IL-29 mutant C172S

Met°Gly9pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys GlyCys

5 10 LS1.

His He Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala SerPhe

2530

Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 . 4045

Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 5580

Gin Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala LeuThr

70 7580

Leu Lys Val Leu Glu Ala Ala Ala , Gly Pro Ala Leu Glu Asp ValLeu

9095

Asn Gin Pro Leu His Thr Leu His His lie Leu Ser Gin Leu Gin Ala 100 105HO

Cys lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 : 120125

His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Ala 130 135140

G1V Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu LeuThr

145 ' 150 155160

Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Ser Leu Arg ThrSer

165 170175 .

Thr His Pro Glu Ser Thr 180 <210> 30 <211> 525 <212> DNA <213> Artificial Sequence .

<220>

<223> degenerate sequence of SEQ ID NO: 18 <221> misc_feature . .: , <222> (1)' - . - (525) <223> n = A,T,C or G <400> 30 gtnccngtng cnmgnytnca yggngcnytn ccngaygcnm. gnggntgyca yathgcncar 60 ttyaarwsny tnwsnccnca rgarytncar gcnttyaarm gngcnaarga ygcnytngar 12 garwsnytny ytnmgncary ytnaargtny ccnytncaya acngcnggnc ccnaaraarg ytnacnmgng tnytnaarga tncargtnmg tngargcnac cnytncayca cnmgnacnmg arwsnccngg ayytnaaytg ywsnmgntgy ngarmgnccn ngcngayacn yathytnwsn nggnmgnytn ntgyytngar ygtngcnwsn caywsnmgny atggcnytng gayccngcny carttymgng caycaytggy gcnwsngtna ggngayytnt tnttyccnmg nacntgggay 180 argcngaryt ngcnytnacn 240 tngtngaygt nytngaycar 300 cntgyathca rccncarccn 360 tntaymgnyt ncargargcn 420 cnttyaayyt nttymgnytn 480 gygtn 525 <210> 31 <211> 525 <212> DNA <213> Artificial Sequence <223> degenerate sequence of SEQ ID NO: 20 <221> misc_feature <222> (1) . . . (525) <223> n = A,T,C or G <400> 31 gtnccngtng ttyaarwsny garwsnytny ytnmgncary ytnaargtny ccnytncaya acngcnggnc ccnaaraarg ytnacnmgng cnmgnytnca tnwsnccnca tnytnaarga tncargtnmg tngargcnac cnytncayca cnmgnacnmg arwsnccngg ayytnaaytg yggngcnytn rgarytncar ywsnmgntgy ngarmgnccn ngcngayacn yathytnwsn nggnmgnytn ntgyytngar ygtngcnwsn ccngaygcnm gnggntgyca yathgcncar 60 gcnttyaarm gngcnaarga ygcnytngar 120 caywsnmgny tnttyccnmg nacntgggay 180 atggcnytng argcngaryt ngcnytnacn 240 gayccngcny tngtngaygt nytngaycar 300: carttymgng cntgyathca rccncarccn 360 caycaytggy tntaymgnyt ncargargcn 420 gcnwsngtna cnttyaayyt nttymgnytn 480 ; ggngayytnt gygtn 525 <210> 32 <211> 525 <212> DNA <213> Artificial Sequence <223> degenerate .sequence of SEQ ID NO: 22 <221> misc_feature <222> (1) . . . (525) <223> n = A,T,C or G gtnccngtng cnmgnytnca yggngcnytn ccngaygcnm gnggntgyca yathgcncar 60 ttyaarwsny tnwsnccnca rgarytncar gcnttyaarm gngcnaarga ygcnytngar 120 garwsnytny tnytnaarga ywsnmgntgy caywsnmgny tnttyccnmg nacntgggay 180 ytnmgncary tncargtnmg ngarmgnccn atggcnytng argcngaryt ngcnytnacn 240 ytnaargtny tngargcnac ngcngayacn gayccngcny tngtngaygt nytngaycar 300 ccnytncaya cnytncayca yathytnwsn carttymgng cntgyathca rccncarccn 360 acngcnggnc cnmgnacnmg nggnmgnytn caycaytggy tntaymgnyt ncargargcn 420 ccnaaraarg arwsnccngg ntgyytngar gcnwsngtna cnttyaayyt nttymgnytn 48 ytnacnmgng ayytnaaytg ygtngcnwsn ggngayytnt gygtn 52b <210> 33 <211> 525 <212> DNA <213> Artificial Sequence ' <220> '. '.. ” <223> degenerate sequence of SEQ ID NO: 24 <221> misc_feature <222> (1) - . -(525) <223> n = A,T,C or G .

<400> 33 gtnccngtng ttyaarwsny garwsnytny ytnmgncary ytnaargtny ccnytncaya acngcnggnc ccnaaraarg ytnacnmgng cnmgnytnca tnwsnccnca tnytnaarga tncargtnmg tngargcnac cnytncayca cnmgnacnmg arwsnccngg ayytnaaytg yggngcnytn rgarytncar ywsnmgntgy ngarmgnccn ngcngayacn yathytnwsn nggnmgnytn ntgyytngar ygtngcnwsn ccngaygcnm. gnggntgyca yathgcncar gcnttyaarm caywsnmgny atggcnytng gayccngcny carttymgng caycaytggy gcnwsngtna ggngayytnt gngcnaarga ygcnytngar tnttyccnmg nacntgggay argcngaryt ngcnytnacn tngtngaygt cntgyathca tntaymgnyt cnttyaayyt gygtn nytngaycar rccncarccn ncargargcn nttymgnytn

300 360 420 480 <210>

<211>

<212>

<213>

DNA

Artificial Sequence <220>

<223>

degenerate sequence of SEQ

ID NO: 26 <221>

<222>

<223>

misc_feature (1) ..(525) n = A,T,C or G <400> 34 gtnccngtng cnmgnytnca ttyaarwsny tnwsnccnca garwsnytny tnytnaarga ytnmgncary tncargtnmg ytnaargtny tngargcnac ccnytncaya cnytncayca acngcnggnc cnmgnacnmg ccnaaraarg arwsnccngg ytnacnmgng ayytnaaytg yggngcnytn ccngaygcnm rgarytncar gcnttyaarm ywsnmgntgy caywsnmgny ngarmgnccn atggcnytng ngcngayacn gayccngcny yathytnwsn carttymgng nggnmgnytn caycaytggy ntgyytngar gcnwsngtna ygtngcnwsn ggngayytnt gnggntgyca yathgcncar 60 gngcnaarga ygcnytngar 120. tnttyccnmg nacntgggay 180 argcngaryt ngcnytnacn 240 tngtngaygt nytngaycar 3:00'. cntgyathca rccncarccn 360 tntaymgnyt ncargargcn 420 cnttyaayyt nttymgnytn 480 gygtn 525.

<210> 35 <211> 525 <212> DNA <213> Artificial Sequence <223> degenerate sequence of SEQ ID NO: 28 <221> misc_feature <222> (1). . (525) <223> n = A,T,C׳or G <400> 35 gtnccngtng ttyaarwsny garwsnytny ytnmgncary ytnaargtny ccnytncaya acngcnggnc ccnaaraarg ytnacnmgng cnmgnytnca tnwsnccnca tnytnaarga tncargtnmg tngargcnac cnytncayca cnmgnacnmg arwsnccngg. ayytnaaytg yggngcnytn rgarytncar ywsnmgntgy ngarmgnccn ngcngayacn yathytnwsn nggnmgnytn ntgyytngar ygtngcnwsn ccngaygcnm gcnttyaarm caywsnmgny atggcnytng gayccngcny carttymgng caycaytggy gcnwsngtna ggngayytnt gnggntgyca yathgcncar 60 gngcnaarga ygcnytngar 120 tnttyccnmg nacntgggay 1&0 argcngaryt ngcnytnacn 240 tngtngaygt nytngaycar 300 cntgyathca rccncarccn 360 tntaymgnyt ncargargcn 420 cnttyaayyt nttymgnytn 480 gygtn 525 <210> 36 <211> 175 <212> PRT <213> Artificial Sequence <22 0>

<223> IL-28A mutant C48X <221> VARIANT .׳ <222> (48) . - - (48) <223> Xaa = Ser, Ala, Thr, Vai or Asn

Vai Pro Vai Ala Arg‘ Leu His Gly Ala Leu Pro Asp Ala Arg Gly Cys

5. 10

His He Ala Gin Phe Lys Ser Leu Ser Pro Gin Glu Leu Gin Ala Phe

2530

Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Xaa 35 4045

Arg Cys His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gin Leu 50 5560 gin val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala LeuThr

70 7580

Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu ValAsp 9099

Val Leu Asp Gin Pro Leu His Thr Leu His His He Leu Ser Gin Phe 100 105110

Ara Ala Cys He Gin Pro Gin Pro Thr Ala Gly Pro Arg Thr Arg Gly 115 120125

Arg Leu His His Trp Leu Tyr Arg Leu Gin Glu Ala Pro Lys Lys Glu !30 135140

Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu 145 150 155I

Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val .165 170175 <210> 37 <211> 176 <212> PRT <213> Artificial Sequence <220>

<223> met IL-28A mutant C49X <221> VARIANT <222> (49)..(49) <223> Xaa = Ser, Ala, Thr, Val or Asn

Met Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly 1 5 1015

Cys His He Ala Gin Phe Lys Ser Leu Ser Pro Gin Glu Leu Gin Ala 20 25

Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp 35 4045

Xaa Arg Cys His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gin 50 5560

Leu Gin Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu 65 70 75 80

Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Val 85 90

Aso Val Leu Asp Gin Pro Leu His Thr Leu His His He Leu Ser Gin 100 : . 105HO

Phe Arg Ala Cys lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Thr Arg

125 120 ׳115

Gly Arg Leu His His Trp.Leu Tyr Arg Leu Gin Glu Ala Pro Lys Lys 130 135140

Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg

160 .155 ' 150 י.'.. 145

Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165 - -. 170 I75' <210> 38 <211> 175 <212> PRT <213> Artificial Sequence <220>

<223> IL-28A mutant C50X <221> VARIANT <222> (50) . . (50) <223> Xaa = Ser, Ala, Thr, Vai or Asn <400> 38

Vai Pro Vai Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly Cys 1 5 10,15

His He Ala Gin Phe Lys Ser Leu Ser Pro Gin Glu Leu Gin Ala Phe 20 2530

Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys 35 4045

Arg Xaa His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gin Leu 50 5560

Gin Vai Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala LeuThr

70 7580

Leu Lys Vai Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu VaiAsp

90.95

Vai Leu Asp Gin Pro Leu His Thr Leu His His He Leu Ser Gin Phe 100 105HO

Arg Ala Cys He Gin Pro Gin Pro Thr Ala Gly Pro Arg Thr Arg Gly 115 120125

Arg Leu His His Trp Leu Tyr Arg Leu Gin Glu Ala Pro Lys Lys Glu 130 135140

Ser Pro Gly Cys Leu Glu Ala Ser Vai Thr Phe Asn Leu Phe Arg Leu 145 150 155160

Leu Thr Arg Asp Leu Asn Cys Vai Ala Ser Gly Asp Leu Cys Vai

165 170175 <210> 39 <211> 176 <212> PRT <213> Artificial Sequence <220>

<223> met IL-28A mutant C51X <221> VARIANT .

<222> (51)..(51) <223> Xaa = Ser, Ala, Thr, Vai or Asn . י 39 <400>

Met Vai Pro Vai Ala Arg Leu His. Gly Ala Leu Pro Asp Ala Arg. Gly 1 5 1015

Cys His He Ala Gin Phe Lys Ser Leu Ser Pro Gin Glu Leu Gin Ala 20 2530

Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp 35 40 .45

Cys Arg Xaa.His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gin 50 5560

Leu Gin Vai Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu AlaLeu

70 7580

Thr Leu Lys Vai Leu Glu Ala Thr Ala AspThr Asp Pro Ala LeuVai . 85 . . 90 ..95 .:

Asp Vai Leu Asp Gin Pro Leu His .Thr Leu His His He Leu SerGin ־

100 .. 105 : . ' ' 110.

Phe Arg Ala Cys lie Gin Pro Gin Pro Thr Ala Gly Pro Arg. Thr Arg . . 115 . 120 . . 125

Gly Arg Leu His His Trp Leu . .Tyr Arg Leu Gin Glu Ala Pro Lys Lys

130 135I

Glu Ser Pro Gly Cys Leu Glu Ala Ser Vai Thr Phe Asn Leu PheArg

145 150 155160

Leu Leu Thr Arg Asp Leu Asn Cys Vai Ala Ser Gly Asp Leu CysVai

165 170175 <210> 40 <211> 181 <212> PRT <213> Artificial Sequence <220>

<223> IL-29 mutant C171X <221> VARIANT <222> (171)..(171) <223> Xaa = Ser, Ala, Thr, Vai or Asn <400> 40'

Gly Pro Vai Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His

15 10 5 ׳ . .1 lie Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe Lys 20 2530

Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser 35 40

Cys Ser Ser Pro Vai Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gin 5055

Vai Arg Glu Arg Pro Vai Ala Leu Glu Ala Glu Leu Ala Leu ThrLeu

70 7580

Lvs Vai Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Vai LeuAsp

90. 95

Gin Pro Leu His Thr Leu His His He Leu Ser Gin Leu Gin Ala Cys 100 105HO lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His

125 י 120115.

His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Ala Gly 130 135140

Cvs Leu Glu Ala Ser Vai Thr Phe Asn Leu Phe Arg Leu Leu ThrArg

145 .150 155160

Asp Leu Lys Tyr Vai Ala Asp Gly Asn Leu Xaa Leu Arg Thr SerThr

165 . 170175

His Pro Glu Ser Thr 180 י 41 <210>

<211> 182 <212> PRT ' <213> Artificial Sequence ' <220> ' <223> met IL-29 mutant C172X . .

<221> VARIANT ' <222> (172)..(172) <223> Xaa = Ser,. Ala, Thr, Vai or Asn .

<400>-41'

Met Gly Pro Vai Pro Thr Ser Lys Pro Thr Thr.Thr Gly Lys Gly Cys 1. 5 .1015

His He Gly Arg' Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe 20 , ' 25 ' 30

Lys Lys Ala Arg Asp Ala׳ Leu Glu Glu.Ser Leu Lys Leu Lys AsnTrp

... . ’ 35 . . . 40 .45

Ser Cys Ser Ser Pro Vai Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu ’50 5560 gin val Arg Glu. Arg Pro Vai Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75.80

Leu Lys Vai Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Vai Leu 85 90

Asn Gin Pro Leu His Thr Leu His His He Leu Ser Gin Leu Gin Ala 100 105HO

Cvs He Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120125

His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Ala 130 135140

Gly Cys Leu Glu Ala Ser Vai Thr Phe Asn Leu Phe Arg Leu LeuThr

145 150 155150

Arg Asp Leu Lys Tyr Vai Ala Asp Gly Asn Leu Xaa Leu Arg ThrSer

165 170I

Thr His Pro Glu Ser Thr 180 <210> 42 <211> 49 <212> DNA <213> Artificial Sequence .

<220>

<223> oligonucleotide primer ZC40923 <400> 42 tccagggaat tcatataggc cggccaccat gaaactagac atgactggg 4.9 <210> 43 <211> 74 <212> DNA <213> Artificial Sequence <220>

<223> oligonucleotide primer ZC43152 <400> 43 cn ggggtgggta caaccccaga gctgttttaa ggcgcgcctc tagactattt ttagacacac 60 aggtccccac tggc 74 <210> 44 <211> 50 <212> DNA <213> Artificial Sequence <220>

<223> oligonucleotide primer ZC29740 <400> 44 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa□ט <210> 45 .

<211> 42' <212> DNA .

. <213> Artificial Sequence .

<220> . .' <223> oligonucleotide primer ZC29741 <400> 45 ';

tctgatttaa tctgtatcag gctgaaaatc ttatctcatc eg.

י,';.’ 46 <210>

.. .-. .׳־; '. >>. .' '. 62 <211>

<212> DNA <213> Artificial Sequence <220>

<223> oligonucleotide primer ZC29736 <400> 46 . , gtggaattgt gagcggataa caatttcaca cagaattcat taaagaggag aaattaactc 60 cc <210> 47 <211> 63 <212> DNA <213> Artificial Sequence <223> oligonucleotide primer ZC29738 gctgaaaatc ttatctcatc cgccaaaaca cccgggagtt aatttctcct ctttaatgaa 60 ttc 6 <210> 48 <211> 78 <212> DNA <213> Artificial Sequence <220>

<223> oligonucleotide primer ZC44566 <400> 48 י ' cn tcttccagag cgtcacgagc ttttttgaaa gaagccagtt cctgcggaga cagagatttg 6u aaacgaccga tgtggcaa <210> 49 <211> 84 .

<212> DNA <213> Artificial Sequence <223> oligonucleotide primer ZC44565 <400> 49 tcgtgacgct ctggaagaat ctctgaaact gaaaaactgg tcttgctctt ctccggtttt 60 cccgggtaac tgggatctgc gtct 84 <210> 50 <211> 71 <212> DNA <213> Artificial Sequence <220>

<223> oligonucleotide primer ZC44564

י.' / 50 <400>

aacagaagct tccaggcaac cagcagattc ttttttcgga gcttcctgca gacgatgcag 60 ccagtggtgca.

<210> 51 .' . ׳,. ״י.,..

<211> 73 ' : ,/ . : ./. / ,'י.

<212> DNA .י .־ <213> Artificial Sequence .

; ־ ' <220>

<223> oligonucleotide primer ZC44563 , . . . / aactggctct gaccctgaaa gttctggaag ctgctgcagg tcctgctctg gaagatgttc 60 tggatcagcc get ־ 52 <210>

<211> 74 <212> DNA <213> Artificial Sequence <220>

<223> oligonucleotide primer ZC44562 <400> 52 tcagggtcag agccagttca gcttccagag caaccggacg ttcacgaacc tgcagcagac 60 gcagatccca gtta <210> 53 <211> 76 <212> DNA <213> Artificial Sequence <223> oligonucleotide primer ZC44561 teagetgeag gettgeatte aaccgcaacc gaccgctggt ccgcgtccgc gtggtcgtct 60, gcaccactgg etgeat 76 <210> 54 <211> 60 <212> DNA <213> Artificial Sequence <220>

<223> oligonucleotide primer ZC44560 <400> 54 ’ cn atgcaagcct gcagctgaga caggatgtgg tgcagagtgt gcagcggctg atccagaaca 60 <210> 55 .

<211> 62 .

<212> DNA <213> Artificial Sequence <220>

<223> oligonucleotide primer ZC44559 <400> 55 atgggtccgg ttccgacctc taaaccaacc accactggta aaggttgcca catcggtcgt 60 <210> 56 ...,.

<211> 65 .

<212> DNA <213> Artificial Sequence י' ' ' . <220>

<223> oligonucleotide primer ZC44558 . ' י.’ ..:.ץ..' 56 <400>

ttaggtagat tccggatggg tagaggtacg caggc’acagg ttaccatcag caacgtattt 60 cagat - ' 65 : ,. ' ' . '־ <210> 57 ./.:'/. ?..T'.'י y.! .

<211> 69 ,./. / \ .. .:λ. .־.''λ '7:. .,J.: / . ..7 <212> DNA <213> Artificial Sequence <220>

<223> oligonucleotide primer ZC44557 <400> 57 tgcctggaag cttctgttac cttcaacctg ttccgtctgc tgacccgtga tctgaaatac 60 gttgctgat 59 <210> 58 <211> 41 <212> DNA <213> Artificial Sequence <220>

<223> oligonucleotide primer ZC44340 <400> 58 cgttgctgat ggtaacctgt ctctgcgtac ctctacccat c <210> 59 , <211> 41 :

<212> DNA <213> Artificial Sequence <220>

<223> oligonucleotide primer ZC44341 <400> 59 gatgggtaga ggtacgcaga gacaggttac catcagcaac g <210> 60 ' <211>,68. <212>. DNA . <213> Artificial Sequence <220> <223> oligonucleotide primer ZC41212 <400> 60 ctagaaataa ttttgtttaa ctttaagaag gagatatata tatgggccct gtccccactt 60 ccaagccc . 68 <210> 61 <211> 67 .

<212> DNA <213> Artificial Sequence ' ־ <220>' <223> oligonucleotide primer ZC41041 <400> 61 rn tctgtatcag gctgaaaatc ttatctcatc cgccaaaaca ttaggtggac tcagggtggg 60 ttgacgt 67 <210> 62 <211> 65 - .'.. ' <212> DNA <213> Artificial Sequence ' ' . . ' ' י <220> ־ <223> oligonucleotide primer ZC43431 <400> 62 ' ' V ctagaaataa ttttgtttaa ctttaagaag gagatatata tatggttcct gtcgccaggc. 60.

<210> 63 <211> 67 <212> DNA.

<213> Artificial Sequence .

<220>

<223> oligonucleotide primer ZC43437 <400> 63 4. ״- cn taatctgtat caggctgaaa atcttatctc atccgccaaa acatcagaca cacaggtccc 60 cactggc 67 <210> 64 <211> 39 <212> DNA <213> Artificial Sequence <220>

<223> oligonucleotide primer ZC44327.

<400> 64 gtggccgatg ggaacctgtc cctgagaacg tcaacccac <210> 65 <211> 39 .

<212> DNA <213> Artificial Sequence <220>

<223> oligonucleotide primer ZC44328 <400> 65 gtgggttgac gttctcaggg acaggttccc atcggccac

<td> <210> <211> <212> <213></td><td> 66 83 DNA . Artificial Sequence'</td>

<td> <220> <223></td><td> oligonucleotide primer ZC45399</td>

<400> 66 tcaggtccca ggtcctgggg aagaggcggg agtggcacct ggagtccttc agcagaagcg 60 actcttctaa ggcatctttg gcc . 83 <210> 67 <211> 531 <212> DNA <213> Artificial Sequence <220> ' <223> zcyto20 mature start from pYEL7b <221> CDS . ־.: '. ' (531) - . . (1) <222>

<400> 67 atg gtt cct gtc gcc agg etc cac ggg get etc ccg gat gca agg ggc48

Met Vai Pro Vai Ala Arg Leu His Gly Ala Leu Pro Asp Ala ArgGly

15 10 . ׳ 51 tgc‘cac ata gcc cag ttc aag tcc etg tct cca cag gag'ctg cag gcc96

Cys His lie Ala Gin Phe'Lys .Ser Leu Ser Pro Gin Glu Leu Gin Ala .

ttt aag agg gcc aaa gat

Phe Lys Arg Ala Lys Asp 35 tgc agg tgc cac tcc cgc

Cys Arg Cys His Ser Arg 50 etg cag gtg agg gag cgc Leu Gin Val Arg Glu Arg 65 70 acg etg aag gtt etg gag Thr Leu Lys Val Leu Glu 85 gac gtc ttg gac cag ccc

Asp Val Leu Asp Gin Pro 100 ttc egg gee tgt ate cag

Phe Arg Ala Cys He Gin 115 ggc cgc etc cac cat tgg

Gly Arg Leu His His Trp 130 gag tee cct ggc tgc etc Glu Ser Pro Gly Cys Leu 145 150 etc etc acg ega gac etg Leu Leu Thr Arg Asp Leu 165 tga gcc tta gaa gag teg ett

Ala Leu Glu Glu Ser Leu 40 etc ttc ccc agg acc tgg

Leu Phe Pro Arg Thr Trp

50 ccc atg get ttg gag get Pro Met Ala Leu Glu Ala 75 gcc acc get gac act gac

Ala Thr Ala Asp Thr Asp 90 ett cac acc etg cac cat

Leu His Thr Leu His His 105 cct cag ccc acg gca ggg

Pro Gin Pro Thr Ala Gly 120 etg tac egg etc cag gag Leu Tyr Arg Leu Gin Glu 135 140 gag gcc tet gtc acc ttc Glu Ala Ser Val Thr Phe .155 aat tgt gtt gcc agt ggg Asn Cys Val Ala Ser Gly 170 etg etg aag gac 144

Leu Leu Lys Asp 45 gac etg agg cag 192

Asp Leu Arg Gin gag etg gcc etg 240

Glu Leu Ala Leu 80 cca gcc etg gtg 288

Pro Ala Leu Val ate etc tcc cag 336 lie Leu Ser Gin

׳ 110 ccc agg acc egg 384

Pro Arg Thr Arg gcc cca aaa aag 432

Ala Pro Lys Lys aac etc ttc cgc 480

Asn Leu Phe Arg gac etg tgt gtc 528

Asp Leu Cys Val .

<210> 68 <211> 83 <212> DNA <213> Artificial Sequence / <220>.

<223>’ oligonucleotide primer ZC45398 <400> 68 ׳', l. cn ggccaaagat geettagaag agtegettet gctgaaggac tccaggtgcc actcccgcct 60 cttccccagg acctgggacc tga . .

<210> 69 <211> 83 .' '.'.?.

<212> DNA <213> Artificial Sequence ' 'י ־'/ ' י./'' . <220>

<223> oligonucleotide primer ZC45397 ״ ' . 69 <400>

.gctgcctcag gtcccaggtc ctggggaaga ggcgggagtg ggacctgcag tccttcagca 60 gaagcgactc ttetaaggea tet : ..83 .. י,-־ <210> 70 <211> 83 <212> DNA <213> Artificial Sequence <220>

<223> oligonucleotide primer ZC45396 <400> 70 agatgcctta gaagagtcgc ttctgctgaa ggactgca.gg tcccactccc gcctcttccc 60 caggacctgg gacctgaggc age 83 <210> 71 <211> '1013 <212> DNA <213> Homo sapiens <220>

<221> CDS <222> (14)..(991) <400> 71 t ccagcgtccg tcc.atg geg tgg age ett ggg age tgg ctg ggt ggc tgc Met Ala Trp Ser Leu Gly Ser Trp Leu Gly Gly Cys , 5 10

<td> acc Thr</td><td> ccg Pro</td><td> cct Pro</td><td colspan="2"> ggg cag ggg Gly Gin Gly</td><td> ccc Pro</td><td> caa age Gin Ser</td><td colspan="3"> tag getetgagaa ggaaacacac ★</td><td> 1011</td>

320 325 tc . 1013 <210> 72 <211> 49 <212> DNA <213> Artificial Sequence <220>

<223> oligonucleotide primer ZC40922 <400> 72 tccagggaat tcatataggc cggccaccat ggctgcagct tggaccgtg 49 .'.־.. 73 <210>

<211> 71 <212> DNA' <213> Artificial׳. Sequence <220> 'י'.''. ' ' י י <223> oligonucleotide primer ZC43153־ <400> 73 .

ggggtgggta caaccccaga gctgttttaa ggcgcgcctc tagactattt ttaggtggac 60 tcagggtggg t ... Ϊ . ..,.'. J 71 <ט21>

<211> 546 <212> DNA <213> Artificial Sequence . .

<220>

<223> IL29 mutant C15X, Asnl69 <221> CDS <222> (1)..(546) <221> variation <222> (44)..(45) <223> n = A, G, T, or C <400> 74 ggc cct gtc ccc act tcc aag ccc acc aca act ggg aag ggc dnn cac48

Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly XaaHis

5 1015 att ggc agg ttc aaa tct ctg tea cca cag.gag eta geg age ttc aag96

He Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser PheLys

2530 aag gcc agg gac gcc ttg gaa gag tea etc aag ctg aaa aac tgg agt144

Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn TrpSer 35 40. 45 tgc age tct cct gtc ttc ccc ggg aat tgg gac ctg agg ett etc cag 192 -׳

Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu LeuGin

5560 gtg agg. gag ege cct gtg gcc ttg gag get gag ctg gcc ctg acg ctg240

Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu ThrLeu

70 7580 aag gtc ctg gag gcc get get ggc cca gcc ctg gag gac gtc eta gac288

Lys Val heu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val LeuAsp 85 9095 .

cag ccc ett cac acc ctg cac cac ate etc tee cag etc cag gcc tgt336

Gin Pro Leu His Thr Leu His His He Leu Ser Gin Leu Gin AlaCys

100 105110 ate cag cct cag ccc aca gca ggg ccc agg ccc egg ggc ege etc cac384 lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg LeuHis

115 120.!25 cac tgg ctg cac egg etc cag gag gee ccc aaa aag gag tee get ggc432

His Trp.Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser AlaGly

130 135 . .140 tgc ctg gag gca tct gtc acc ttc aac etc ttc ege etc etc acg ega480

Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu ThrArg 145 . 150 . 155 ,. .160 gac etc aaa tat gtg gcc gat ggg aay ctg tgt ctg aga acg tea acc . 528

Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr SerThr . 165 , . 170 .175 cac cct gag tee acc tga . ':. ' ? 546 .

His Pro Glu Ser Thr * ׳ .-/ י-.'׳. י' .'.׳..'

180 . . <. ..' . /'/ <210> 75 <211> 181 <212> PRT <213> Artificial Sequence <220>

<221> VARIANT <222> (15)..(15) <223> Xaa = Ser, Ala, Thr, Val, or Asn <223> IL29 mutant C15X, Asnl69 <400> 75

<210> 76 <211> 549 <212> DNA <213> Artificial Sequence <220>

<223> Met IL29 mutant C16X, Asnl70 <221> CDS <222> (1)..(549) <221> variation <222> (47)..(48) <223> n. = A, T, G, or C י־.'' 76 <400>

atg ggc cct gtc ccc act tcc aag ccc acc aca act ggg aag ggc dun Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa

15 10 י. 51 ' cac att ggc agg ttc aaa tct etg tea cca cag gag eta geg age ttc His He Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe

2530 aag aag gcc agg gac gcc ttg. gaa gag tea etc aag etg aaa aac tgg . Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp

י...... 45 . ׳ ' י 40 , י' 35 /

165 170 175

<210> 77 <211> 182 <212> PRT <213> Artificial Sequence <220>

<221> VARIANT <222> (16)..(16) <223> Xaa = Ser, Ala, Thr, Val, or Asn <223> Met IL29 mutant C16X, Asnl70 <400> 77

His His

Gly Cys 145

Arg Asp

Thr His

Trp Leu

Leu Glu

Leu Lys

Pro Glu

His Arg

Ala Ser

Tyr Val 165

Ser Thr

Leu Gin 135

Val Thr

Ala Asp

Glu Ala

Phe Asn

Gly Asn

Pro Lys

Leu Phe 155

Leu Cys

Lys Glu

Arg Leu

Leu Arg

Ser Ala

Leu Thr

160 Thr Ser 175 <210> 78 <211> 546 <212> DNA <213> Artificial Sequence <220>

<td> <223</td><td> > IL29 mutant Cl5X, Aspl69</td>

<td colspan="2"> <221> variation</td>

<td> att</td><td> ggc agg ttc aaa tet etg tea cca caggag eta geg age ttc aag 96</td>

145 . 150 155 . 160 gac etc aaa tat gtg Asp Leu Lys Tyr Val 165 gcc gat ggg gay ctg tgt Ala Asp Gly Asp Leu Cys 170 ctg aga acg tea acc Leu Arg Thr Ser Thr 175 cac cct gag tcc acc tga His. Pro Glu Ser Thr * 180 <210> 79 <211> 181 <212> PRT <213> Artificial Sequence <220>

<223> IL29 mutant C15X, Aspl69 <221> VARIANT <222> (15) . . (15) <223> Xaa = Ser, Ala, Thr, Val, or Asn

Gly°Pro9Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys 1 510 lie Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala

2025

Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys

3540

Cvs Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg 50 5560

Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala

7075

Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp 85 .90

Gin Pro Leu His Thr Leu His His lie Leu Ser Gin Leu . 100 lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly

115 120125

His Trp Leu His Arg Leu Gin Glu Ala Pro Lys LysGlu

130 I35 ' 140_

Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe ArgLeu !45 150155

Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg

165170

His Pro Glu Ser Thr

Gly Xaa His

Ser Phe Lys

Asn Trp Ser

Leu Leu Gin

Leu Thr Leu

Val Leu Asp

Gin Ala Cys no

Arg Leu His

Ser Ala Gly

Leu Thr Arg . 160

Thr Ser Thr <210> 80 <211> 549 ' <212> DNA . <213> Artificial Sequence <220>

<223> Met IL29 mutant C16X, Aspl70 <221> CDS <222> (1) . - - (549).

<221> variation <222> (47) . . - (48) <223> n = A, T, G, or C <400> 80 ata qgc cct gtc ccc act tcc aag ccc acc acaact ggg aagggc dnn .8

Met Gly Pro Val Pro.Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa γη

ctg gcc ctg acg 240

Leu Ala Leu Thr gtg gcc ttg gag get gag Vai Ala Leu Glu Ala Glu 75 cag gtg agg gag cgc cct

Gin Vai Arg Glu Arg Pro 70

acc cac cct gag tcc acc tga Thr His Pro Glu Ser Thr * 180 <210> 81 <211> 182 <212> PRT <213> Artificial Sequence <220> _ <223> Met IL29 mutant C16X, Aspl70 <221> VARIANT <222> (16) - . (16) <223> Xaa = Ser, Ala, Thr, Vai, <400> .81

Met Gly Pro Vai Pro Thr Ser Lys 1 5

His He Gly Arg Phe Lys Ser Leu 20 .

Lys Lys Ala Arg Asp Ala Leu Glu . 7S .: 40 or Asn

Pro Thr Thr Thr Gly Lys Gly Xaa 10 15

Ser’Pro Gin Glu Leu Ala Ser. Phe 25 . . 30.

Glu Ser Leu Lys Leu Lys Asn Trp.

Ser Cys Ser Ser Pro Vai Phe Pro Gly Asn Trp Asp 5055

Gin Vai Arg Glu Arg Pro Vai Ala Leu Glu Ala Glu 65 7075

Leu Lys Vai Leu Glu Ala Ala Ala Gly Pro Ala Leu .90

Asp Gin.Pro Leu His Thr Leu His His He Leu Ser 100

Cys He Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro 115120

His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys 130I

Gly Cys Leu Glu Ala Ser Vai Thr Phe Asn Leu Phe

145 150I

Arg Asp Leu Lys Tyr Vai Ala Asp Gly Asp Leu Cys

165' 170

Thr His Pro Glu Ser Thr

Leu Arg Leu Leu

Leu Ala Leu Thr 80

Glu Asp Vai Leu 95

Gin Leu Gin Ala

Arg Gly Arg Leu

Lys Glu Ser Ala

Arg Leu Leu Thr

Leu Arg Thr Ser 175.

<210> 82 <211> 546 <212> DNA <213> Artificial Sequence <223> IL29 mutant Aspl69, C171X <221>.CDS <222> (1).--(546) <221> variation <222> (512) . .(513) <223> n = A, T, G, or C <400> 82 ggc cct gtc ccc act Gly Pro Vai Pro Thr

5 ־ . ' 1 att ggc agg ttc aaa He Gly Arg Phe Lys 20 .

tcc aag ccc acc aca

Ser Lys Pro Thr Thr tet etg tea cca cag

Ser Leu Ser Pro Gin act ggg aag ggc tgc cac

Thr Gly Lys Gly Cys His gag eta geg age ttc aag

Glu Leu Ala Ser Phe Lys aag gcc agg gac gcc ttg gaa gag

Lys Ala Arg Asp Ala Leu Glu Glu '

tgc age tet cct gtc ttc ccc ggg

Cys Ser Ser Pro Vai Phe Pro Gly

5055 gtg agg qag ege cct gtg gccttg

Vai Arg Glu Arg Pro Vai Ala Leu .

aag gtc etg gag gcc get getggc

Lys Vai Leu Glu: Ala Ala AlaGly cag ccc ett cac acc etg caccac

Gin Pro Leu His Thr Leu HisHis ' 100 :

ate cag . cct cag ccc aca.’gca ggg lie Gin Pro Gin. Pro'.Thr Ala Gly tea etc aag etg aaa aac tgg agt

Ser Leu Lys Leu Lys Asn Trp Ser . 45 aat tgg gac etg agg ett etc cag

Asn Trp Asp Leu Arg Leu Leu Gin . 60 ..

gag get gag etg gcc etg acg etg

Glu Ala Glu Leu Ala Leu Thr Leu

75.80 cca gcc etg gag gac gtc eta gac Pro Ala Leu Glu Asp Vai Leu Asp 90 .:95 ate'etc tcc cag etc cag gcc tgt lie Leu Ser Gin Leu Gin Ala Cys

־ ' 11° :. .105 ccc agg ccc egg ggc ege etc cac . Pro Arg Pro Arg Gly Arg Leu His

<210> 83 <211> 181 <212> PRT <213> Artificial Sequence <223> IL29 mutant Aspl69, C171X <221> VARIANT <222> (171). .(171) <223> Xaa = Ser, Ala, Thr, Vai, or Asn <400> 83

Gly Pro Vai Pro Thr Ser Lys Pro 1 5

He Gly Arg ,Phe Lys Ser Leu Ser 20

Lys Ala Arg Asp Ala Leu Glu Glu 35

Cvs Ser Ser Pro Vai Phe Pro Gly . 5055

Vai Arg Glu Arg Pro Vai Ala Leu

ד7065

Lys Vai Leu Glu Ala Ala Ala Gly 85 , Gin Pro Leu His Thr Leu His His . 100 lie Gin Pro Gin Pro Thr Ala Gly 115I

His Trp Leu His Arg Leu Gin Glu 130 ..135

Cys Leu Glu Ala Ser Vai' Thr Phe

150.145 י

Asp Leu Lys Tyr Vai Ala Asp Gly . 165

His Pro Glu Ser Thr 180

Thr Thr Thr Gly Lys Gly Cys His

Pro Gin Glu Leu Ala Ser Phe Lys 25 30

Ser Leu Lys Leu Lys Asn Trp Ser

Asn Trp Asp Leu Arg Leu Leu Gin 60

Glu Ala Glu Leu Ala Leu Thr Leu 7580

Pro Ala Leu Glu Asp Vai Leu Asp

9095 lie Leu Ser Gin Leu Gin Ala Cys 105HO

Pro Arg Pro Arg Gly Arg Leu His

125

Ala Pro Lys Lys Glu Ser Ala Gly 140

Asn Leu Phe Arg Leu Leu Thr Arg

18° י 155

Asp Leu Xaa Leu Arg Thr Ser Thr 170 I75 <210> 84 <211> 549 <212> DNA <213> Artificial Sequence., <220> : .

<223> Met IL29 mutant Aspl70,. C172X <221> CDS <222> (1)..(549) <221> variation <222> (515)..(516) <223> n = A, T, G, or C <400> 84 atg ggc cot gtc ccc act tcc aag ccc acc aca act ggg aag ggc tgc-ט

Met Gly Pro Vai Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys GlyCys

5 1015 cac att ggc agg ttc aaa tct ctg tea cca cag gag eta geg age ttc96

His He Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala SerPhe

2530 aag aag gee agg gac gcc ttg gaa gag tea etc aag ctg aaa aac tgg144

Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys AsnTrp . 4045 agt tgc age tct cct gtc ttc ccc ggg aat tgg gac ctg agg ett etc192

Ser Cys Ser Ser Pro Vai Phe Pro Gly Asn Trp Asp Leu Arg LeuLeu

5560 cag gtg agg gag ege cct gtg gcc ttg gag get gag ctg gcc ctg acg . 240

Gin Vai Arg Glu Arg Pro Vai Ala Leu Glu Ala Glu Leu Ala LeuThr

70 7580 ctg aag gtc ctg gag gcc get get ggc cca gcc ctg gag gac gtc eta288

Leu Lys Vai Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Vai Leu

9095 gac cag ccc ett cac acc ctg cac cac ate etc tec cag etc cag gcc33S

Asp Gin Pro Leu His Thr Leu His His He Leu Ser Gin Leu Gin Ala

100 105110 tgt ate cag cct cag ccc aca gca ggg ccc agg ccc egg ggc ege etc384

Cys He Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly ArgLeu

115 120125 cac cac tgg ctg cac egg etc cag gag gcc ccc aaa aag gag tec get432

His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu SerAla

130 135140 ggc tgc ctg gag gca tct gtc acc ttc aac etc ttc ege etc etc acg480

Gly Cys Leu Glu Ala Ser Vai Thr Phe Asn Leu Phe Arg Leu LeuThr

145 150 155160 ega gac etc aaa tat gtg gcc gat ggg gay ctg dnn ctg aga acg tea528

Arc! Asp Leu Lys Tyr Vai Ala Asp Gly Asp Leu Xaa Leu Arg Thr Ser

'. 165. 170 . ,. 175 ' acc cac cct gag tec acc tga Thr His Pto Glu Ser Thr *

180 .

<210> 85 <211> 182 <212> PRT <213> Artificial Sequence <220>

<223> Met IL29 mutant Aspl70, C172X <221> VARIANT <222> (172)..(172) <223> Xaa = Ser, Ala, Thr, Vai, <400> 85

Met Gly Pro Vai Pro Thr Ser Lys 1 5

His He Gly Arg Phe Lys Ser Leu

Lys Lys Ala Arg Asp Ala Leu Glu

3540

Ser Cys Ser Ser Pro Vai Phe Pro 5055

Gin Vai Arg Glu Arg Pro Vai Ala

6570

Leu Lys Vai Leu Glu Ala Ala Ala

Asp Gin Pro Leu His Thr Leu His 100

Cvs He Gin Pro Gin Pro Thr Ala

115 Λ120

His His Trp Leu His Arg Leu Gin 130135.

Gly Cys Leu Glu Ala Ser Vai Thr

145I

Arg Asp Leu Lys Tyr Vai Ala Asp

Thr His Pro Glu Ser Thr or Asn

Pro Thr Thr Thr Gly Lys Gly Cys

Ser Pro Gin Glu Leu Ala Ser Phe

Glu Ser Leu Lys Leu Lys Asn Trp

Gly Asn Trp Asp Leu Arg Leu Leu 60

Leu Glu Ala Glu Leu Ala Leu Thr

7580

Gly Pro Ala Leu Glu Asp Vai Leu

9095

His He Leu Ser Gin Leu Gin Ala

105. 11°

Gly Pro Arg Pro Arg Gly Arg Leu

Glu Ala Pro Lys Lys Glu Ser Ala

Phe Asn Leu .Phe Arg Leu Leu Thr

155 160

Gly Asp Leu Xaa Leu Arg Thr Ser 170 175 <210> 86 <211> 546 <212> DNA <213> Artificial Sequence <220> ’ <223> IL29 mutant T10P, Asnl69, C171X <221> CDS <222> (1). -.(546) <221> variation <222> (512) .. (513) <223> n = A, T, G, or C <400> 86 ' ' ' Λα gac cct.gtc ccc act tcc aag ccc acc ccn act ggg aag ggc tgc cac 4B Gly Pro Vai Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Cys His att ggc agg ttc aaa tct ctg tea cca cag gag eta geg age ttc aag96 lie Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser PheLys

2530 aag gcc'agg gac gcc ttg gaa gag tea etc aag ctg aaa aac tgg agt144

Lys. Ala Arg Asp AlaLeu Glu Glu Ser Leu Lys Leu Lys Asn TrpSer

40 ..? 45 tgc age tct cct gtc ttc ccc ggg aat tgg gac ctg agg ett etc cag192

Cys Ser Ser Pro Vai Phe Pro Gly Asn Trp Asp Leu Arg Leu LeuGin :

60 55 ״ 50.

gtg agg gag ege cct gtg gcc. ttg gag get. gag ctg gcc ctg acg ctg240

Vai Arg .Glu Arg Pro Vai Ala Leu Glu Ala Glu Leu Ala Leu ThrLeu

70 ..'. ' .75 . ' . ;' . . ; 80

<td> <213></td><td> Artificial</td><td> Sequence</td>

<td> <223></td><td> IL29 mutant</td><td> T10P, Asnl69, C1.71X</td>

<221> VARIANT <222> (171)..(171) <223> Xaa = Ser, Ala, Thr, Vai, or Asn <400> 87

180'. \ <21O>.8B <211> 549 <212> DNA .

<213> Artificial Sequence <220 <223>

Met IL29 mutant T11P, Asnl70, C172X <221> CDS <222> .(1) . . . (549) <221> variation <222> (515)..(516) <223> n = A, T, G, or C <400> 88 atg ggc cct gtc ccc act tcc aag ccc

Met Gly Pro Vai Pro Thr Ser Lys Pro 15 cac att ggc agg ttc aaa tct etg tea

His He. Gly Arg Phe Lys Ser Leu Ser 2025 aag aag gcc agg gac gcc ttg gaa gag Lys Lys Ala Arg Asp Ala Leu Glu Glu 35 agt tgc age tct cct gtc ttc ccc.ggg

Ser Cys Ser Ser Pro Vai Phe Pro Gly 5055 cag gtg agg gag ege cct gtg gcc ttg

Gin Vai Arg Glu Arg Pro Vai Ala Leu 65 etg aag gtc etg gag gcc get get ggc

Leu Lys Vai Leu Glu Ala Ala Ala Gly 85 gac cag ccc ctt cac acc etg cac cac

Asp Gin Pro Leu His Thr Leu His His 100105 tgt ate cag cct cag ccc aca gca ggg Cys He Gin Pro Gin Pro Thr Ala Gly . 115 .120 cac cac tgg etg cac egg etc cag gag

His His Trp Leu His Arg Leu Gin Glu 130. 135 ggc tgc etg gag gca tct gtc acc ttc Gly Cys Leu Glu Ala Ser Vai Thr Phe 1'45 . 150 ega gac etc aaa tat gtg gcc gat ggg

Arg Asp Leu Lys.Tyr Vai Ala Asp Gly 165 acc cac cct gag tee acc tga

Thr His Pro Glu .Ser Thr *

'י 180' acc ccn act ggg aag ggc tgc 48 Thr Pro Thr Gly Lys Gly Cys

15 cca cag gag eta geg age ttc 96 Pro Gin Glu Leu Ala Ser Phe tea etc aag etg aaa aac tgg 144

Ser Leu Lys Leu Lys Asn Trp aat tgg gac etg agg ctt etc 192. Asn Trp Asp Leu Arg Leu Leu gag get gag etg gcc etg acg 240' Glu Ala Glu Leu Ala Leu Thr

80 cca gcc etg gag gac gtc eta 288 Pro Ala Leu Glu Asp Vai Leu

95 ate etc tec cag etc cag gcc 336 lie Leu Ser Gin Leu Gin Ala ccc agg ccc egg ggc ege etc 384 Pro Arg Pro Arg Gly Arg Leu gee ccc aaa aag gag tec get 432 Ala Pro Lys Lys Glu Ser Ala aac etc ttc ege.etc etc acg . 480

Asn Leu Phe Arg Leu Leu Thr : 155 160 aac etg dnn etg aga acg tea 528 Asn Leu Xaa Leu Arg Thr Ser 170 175 <210> 89 <211> 182 <212> PRT <213> Artificial Sequence <223> Met IL29 mutant Τ11Ρ, Asnl70, C172X <221> VARIANT <222> (172)..(172) <223> Xaa = Ser, Ala, Thr, Val, or Asn

Met°Gly9pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Cys T_ 5ד

His lie Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe 20 25

Lvs Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 4045

Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 5560

Gin Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala LeuThr

70 75

Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp ValLeu

9095 .

Asp Gin Pro Leu His Thr Leu His His He Leu Ser Gin Leu Gin Ala 100 10511°

Cvs lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg . Gly Arg Leu 115 I20 '125

His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Ala 130 135140

Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu LeuThr

145 150 155 x.n

Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg ThrSer

165 170. 175

Thr His Pro Glu Ser Thr 180 <210> 90 <211> 546 <212> DNA <213> Artificial Sequence <220>

<223> IL29 mutant T10P, C15X, Asnl69 <221> CDS <222> (1)- . -(546) <221> variation <222> 30, 44, 45 <223> n = A, T, G, or C <400> 90 ggc cct gtc ccc act tcc aag cccacc

Gly Pro Val Pro. Thr Ser Lys ProThr ; ' ..,? 5 ' . .. ..

att ggc agg ttc.aaa tet etg teacca lie Gly Arg Phe Lys Ser Leu SerPro

25 י..'.20 aag gcc agg gac gcc ttg gaa gag tea Lys Ala Arg Asp Ala. Leu Glu, Glu Ser, ccn act ggg aag ggc dim cac 48 .

Pro'Thr Gly Lys Gly Xaa His . . . 15 /':

cag gag eta geg age ttc aag ' 96

Gin Glu Leu Ala Ser Phe Lys . .30 י .

etc.aag etg aaa aac tgg agt 144 ׳ Leu Lys Leu Lys Asn Trp Ser • 35 40 45

<210> 91 <211> 181 <212> PRT <213> Artificial Sequence

220>

223> IL29 mutant T10P, C15X, Asnl69 <221> VARIANT <222> (15)..(15) <223> Xaa = Ser, Ala, Thr, Vai, <400> 91

Gly Pro Vai Pro Thr Ser Lys Pro ' 5 ' :

He Gly Arg Phe :Lys Ser Leu Ser 20

Lys .Ala Arg Asp Ala Leu Glu Glu

40 י . ..-. . 35 •

Cys Ser Ser Pro Vai Phe Pro Gly .:.י ... j 55 .

Vai Arg Glu Arg Pro Vai Ala Leu .' .-: . .. 70 : :.

Lys Vai Leu Glu Ala Ala Ala Gly 85

Gin Pro Leu?His Thr Leu His His

... 100 . -.-' :.

or Asn'

Thr Pro Thr Gly Lys Gly Xaa His 10 .15

Pro Gin Glu Leu Ala Ser Phe Lys

2530

Ser Leu Lys Leu Lys Asn Trp Ser 45

Asn Trp Asp Leu Arg Leu Leu Gin 60

Glu Ala Glu Leu Ala Leu Thr Leu 75 ..80 '

Pro Ala Leu Glu Asp Vai Leu Asp 9095

He Leu Ser Gin Leu Gin Ala Cys

105.-:.HO:

He Gin

His Trp

Cys Leu 145

Asp Leu

Pro׳ Gin 115

Leu His

Glu Ala

Lys

His Pro Glu

Ser

Pro

Arg

Ser

Vai 165 Thr

Thr

Leu

Vai 150 Ala

Ala Gly

Gin Glu 135 Thr

Asp

Pro

Ala

Arg

Pro

Lys

Arg

Phe

Asn

Gly

Asn

Leu Phe

Leu Cys 170

Lys 140

Arg Leu

Gly Arg 125 Glu Ser

Leu Arg

Leu

Thr

Leu

Ala

Thr

Ser

His

Gly

Arg 160 Thr <210>

<211>

<212>

<213>

DNA

Artificial Sequence <220>

<223>

Met IL29 mutant T11P

C16X, Asnl70 <221>

<222>

CDS (1) .. . (549) <221>

<223>

variation 33, 47, 48 . n = A, T

G, or C <400> ggc Gly atg

Met 1 cct Pro gtc

Vai ccc

Pro act tcc

Ser aag

Lys ccc

Pro acc

Thr ccn

Pro act

Thr ggg

Gly aag

Lys ggc

Gly dim

Xaa

״ '8־4 cac

His att

He ggc

Gly ttc agg

Arg Phe aaa

Lys tct

Ser ctg

Leu

ttg

Leu gtg

Vai ttc

Phe get

Ala gaa

Glu Glu 40 ccc

Pro gcc

Ala get

Ala gac . Asp cag

Gin ccc

Pro ett

Leu acc cac

His Thr ctg

Leu cac

His tgt

Cys ate lie cct cag cag

Gin Pro.Gin ccc

Pro aca

Thr gca

Ala cac.

His cac His 1 130'.

tgg

Trp ctg

Leu cac egg etc

His

Arg Leu

135 - ׳ • cag

Gin ggc tgc ctg

Gly Cys Leu gag

Glu tct gtc gca

Ala.Ser Vai acc ttc

Thr Phe gag ttg

Leu ggg

Gly tea

Ser gag Glu ggc

Gly ggg Gly cac

His

145 - 150 . 155 . *bu cga gac etc aaa tat gtg gcc gat ggg aay etg tgt etg aga acg tea528

Arg Asp Leu Lys Tyr Vai Ala Asp Gly Asn Leu Cys Leu Arg ThrSer

165 170175 acc cac cct gag tcc acc tga549

Thr His Pro Glu Ser Thr * <210> 93 <211> 182 <212> PRT <213> Artificial Sequence <220>

<223> Met IL29 mutant T11P, C16X, Asnl70 <221> VARIANT <222> (16) . . . (16) <223> Xaa = Ser, Ala, Thr, Vai, or Asn י 93 <400>

Met Gly Pro Vai Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Xaa 1 5 10 15.

His lie Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe 20 2530

Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 4045

Ser Cys Ser Ser Pro Vai Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 5560

Gin Vai Arg Glu Arg Pro Vai Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 7580

Leu Lys Vai Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Vai Leu 85 9095

Asp Gin Pro Leu His Thr Leu His His He Leu Ser Gin Leu Gin Ala 100 10511°

Cys lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120125

His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys LysיGlu Ser Ala 130 135140

Glv Cvs Leu Glu Ala Ser Vai Thr Phe Asn Leu Phe Arg Leu Leu Thr . !45 150 155 .160

Ara Asp Leu Lys Tyr Vai Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser 165 170 .175

Thr His Pro Glu. Ser Thr

י'.? ' 'י.'180

<td> <213></td><td> Artificial Sequence</td>

<td> <220> <223></td><td> IL29 mutant T10P, Aspl69, C171X</td>

<td> <221></td><td> variation . .</td>

aac cct gtc ccc act tcc aag ccc acc ccn act ggg aag ggc tgc cac-8

Glv Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly CysHis

5 1015 att ggc agg ttc aaa tct ctg tea cca cag gag eta geg age ttc aag96

He Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser PheLys

2530 aag gcc agg gac gcc ttg gaa gag tea etc aag ctg aaa aac tgg agt144

Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn TrpSer . 4045 tgc age tct cct gtc ttc ccc ggg aat tgg gac ctg agg ett etc cag192

Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu LeuGin

5560 gtg agg gag ege cct gtg gcc ttg gag get gag ctg gee ctg acg ctg240

Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu ThrLeu

70 7580 aag gtc ctg gag gcc get get ggc cca gcc ctg gag gac gtc eta gac288

Lvs Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Gia Asp Val LeuAsp

90. 95 cag ccc ett cac acc ctg cac cac ate etc tcc cag etc cag gee tgt336 (ח וי pro Leu His Thr Leu His His He Leu Ser Gin Leu Gin AlaCys

100 105HO ate cag cct cag. ccc aca gca ggg ccc agg ccc egg ggc ege etc cac 3'.84.

lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg LeuHis

125 ״120115 cac tgg ctg cac egg etc cag gag gcc ccc aaa aag gag tee get ggc432

His Trp Leu His. Arg Leu Gin Glu. Ala Pro Lys Lys Glu Ser Ala Gly

130 135 .140 tgc ctg gag gca tct gtc acc ttc aac etc ttc ege etc . etc acg ega480.

Cvs Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg

145 150 155160 gac etc aaa tat gtg gcc gat ggg gay ctg dnn ctg aga acg tea acc528

Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Xaa Leu Arg Thr Ser Thr

165 170175

546 cac cct gag tcc acc tga

His Pro Glu Ser Thr . * <210> 95 .

<211> 181 <212> PRT' .

<213> Artificial Sequence

'. ’ :י '<220>

<223> IL29 mutant T10P, Aspl69, C171X <221> VARIANT'י .

<222> (171) . - - (171) .' ~ .

<223> Xaa = Ser, Ala, Thr, Val, or Asn .

.״ ״ ' . 95 <400>

Glv Pro Val Pro Thr Ser Lys'Pro Thr Pro Thr Gly Lys Gly Cys His 1 : 5 . - 10 15.

lie Glv Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe Lys

A; 20 25 / .a30 , ./; .־ :. .

Lys Ala Arg Asp Ala Leu Glu Glu 3540

Cys Ser Ser Pro Val Phe Pro Gly 5055

Val Arg Glu Arg Pro Val Ala Leu 6570

Lys Val Leu Glu Ala Ala Ala Gly 85

Gin Pro Leu His Thr Leu His His 100

He Gin Pro Gin Pro Thr Ala Gly . 115120

His Trp Leu His Arg Leu Gin Glu

130135

Cys Leu Glu Ala Ser Val Thr Phe

145.150

Asp Leu Lys Tyr Val Ala Asp Gly

His Pro Glu Ser Thr

Ser Leu Lys Leu Lys Asn Trp Ser 45

Asn Trp Asp Leu Arg Leu Leu Gin 60

Glu Ala Glu Leu Ala Leu Thr Leu 7580

Pro Ala Leu Glu Asp Val Leu Asp 9095 lie Leu Ser Gin Leu Gin Ala Cys 105HO

Pro Arg Pro Arg Gly Arg Leu His 125

Ala Pro Lys Lys Glu Ser Ala Gly 140

Asn Leu Phe Arg Leu Leu Thr Arg 155 160

Asp Leu Xaa Leu Arg Thr Ser Thr 170 175 <210> 96 <211> 549' <212> DNA <213> Artificial Sequence <220>

<223> Met IL29 mutant T11P, Aspl70, C172X <221> CDS <222> (1)..(549) <221> variation <222> 33, 515, 516 <223> n = A, T, G, or C <400> 96' atg ggc cct gtc ccc act tcc aag ccc acc ccn act ggg aag ggc tgc48

Met Gly Pro Val Pro' Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Cys

5 10 15.

cac att ggc agg ttc aaa tct ctg tea cca cag gag eta geg age ttc96־

His He Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala SerPhe

2530 aag aag gcc agg gac gcc ttg gaa gag tea etc aag ctg aaa aac tgg144

Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys AsnTrp

4045 agt tgc age tct cct gtc ttc ccc ggg aat tgg gac ctg agg ett etc192

Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg LeuLeu

5560 cag gtg agg gag ege cct gtg gcc ttg gag get gag ctg gcc ctg acg240

Gin Val -Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala LeuThr ' 65 . 70 . 75 ..80 ctg aag gtc ctg 1.gag gcc get get ggc cca gcc ctg gag gac gtc eta288 .

Leu Lys Val Leu Glu Ala Ala Ala.Gly Pro Ala Leu Glu Asp ValLeu . 85 . . 9095 gac cag ccc ett cac acc ctg cac cac ate etc tcc cag etc cag gcc336

Asp Gin Pro Leu His Thr Leu His His lie Leu Ser Gin Leu GinAla .

. /.:. 100 J..:.110 105 .־. _.

tgt ate cag cct cag ccc aca gca ggg ccc agg ccc egg ggc ege etc384

Cys He Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu

115 120 .125 cac cac tgg etg cac egg etc cag gag gee ccc aaa aag gag tee get432

His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Ala

130 . 135I ggc tgc etg gag gca tet gtc acc ttc aac etc ttc ege etc etc acg480

Gly Cys Leu Glu Ala Ser Vai Thr Phe Asn Leu Phe Arg Leu Leu. Thr

145 150. 155160 ega gac etc aaa tat gtg gcc gat ggg gay etg .dim etg aga acg tea528

Arg Asp Leu Lys Tyr Vai Ala Asp Gly Asp Leu Xaa Leu Arg Thr Ser

165 170175 acc cac cct gag tcc acc tga Thr His Pro Glu Ser Thr * 180 <210> 97 <211> 182 <212> PRT <213> Artificial Sequence <220>

<223> Met IL29 mutant T11P, Aspl7.0(, C172X <221> VARIANT <222> (172)..(172) <223> Xaa = Ser, Ala, Thr, Vai, or Asn <400> 97

Met Gly Pro Vai Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Cys 1 5 10

His He Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe 20 2530

Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 4045

Ser Cys Ser Ser Pro Vai Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 . 5560

Gin Vai Arg Glu Arg Pro Vai Ala Leu Glu Ala Glu Leu Ala LeuThr

70 7580

Leu Lys Vai Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp VaiLeu . 9095

Asp Gin Pro Leu His Thr Leu. His His He Leu Ser Gin Leu Gin Ala 100 105HO

Cys lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120125

His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Ala 130 . 135 140‘.

Gly Cys Leu Glu Ala Ser Vai Thr Phe Asn Leu Phe Arg Leu.LeuThr

145 150 . 155160

Arg Asp Leu Lys Tyr Vai Ala Asp Gly Asp Leu Xaa Leu Arg ThrSer

165 . : . . 170 . '; .175

Thr His Pro Glu Ser Thr -,<

. . '180V/ <210> 98 <211> 546 .

<212> DNA '.

<213> Artificial Sequence /־. <220>

<223> IL29 mutant T10P, C15X, Aspl69 <221> CDS <222* (1)..(546) <221> variation ־ <222> 30, 44, 45 <223> n = A, T, G, or C <400> 98:

ggc cct.gtc ccc act tcc aag ccc acc ccn act ggg aag ggc dnn cac48

Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly XaaHis

5 1015 att ggc agg ttc aaa tct etg tea cca cag gag eta geg age ttc aag96 lie Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser PheLys

2530

cac cct gag tee acc tga .׳ ‘

<td> <213:</td><td> > Artificial. Sequence</td>

<td> <220:</td><td rowspan="2"> > IL29 mutant T10P,.C15X, Aspl69</td>

<221> VARIANT <222> (15)..(15) <223> Xaa = Ser; Ala, Thr, Val, or Asn <400> 99

Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Xaa His 1 5 . 1015

He Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe Lys 20 2530

Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser 35 40 .45

Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gin 50 5560

Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu ThrLeu

70 7580

Lys Val Leu Glu ,Ala Ala Ala Gly Pro Ala Leu Glu Asp Val LeuAsp

9095

Gin Pro Leu His Thr Leu His His He Leu Ser Gin Leu Gin Ala Cys 100 105HO

He Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His 115 120125

His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Ala Gly 130 135140

Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu LeuThrArg !45 150 155160

Aso Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg Thr SerThr

165 170175

His Pro Glu Ser Thr 180

I. . ' <210> 100 <211> 549 <212> DNA <213> Artificial Sequence <220>

<223> Met IL29 mutant T11P, C16X, Aspl70 <221> CDS <222> (1)..(549).

<221> variation , <222> 33, 47, 48 <223> n = A, T, G, or C <400> 100 atg ggc cct gtc ccc act.tcc aag ccc acc ccn act ggg aag ggc dnn48

Met Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Xaa

5 1015 cac att ggc agg ttc aaa tet etg tea cca cag gag eta geg age ttc96 • His lie Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala SerPhe

2530 aag aag gcc agg gac gcc ttg gaa gag tea etc aag etg aaa aac tgg144 ' Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys AsnTrp.

4045 agt tgc age tet cct gtc ttc ccc ggg aat tgg gac etg agg ett etc.192

Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp.Asp Leu Arg LeuLeu . - 55 60 cag gtg agg gag ege cct gtg gcc ttg gag get gag etg gcc etg acg .240

Gin Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr .., .,

189 174797/1 ׳ ׳־ /u 80 כ׳ .

ctg aag gtc ctg gag gcc get get ggc cca gcc ctg gag gac gtc eta288

Leu Lys Vai Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Vai Leu

90gg gac cag ccc ett cac acc ctg cac cac ate etc tee cag etc cag gee336

Asp Gin Pro Leu His Thr Leu His His lie Leu Ser Gin Leu GinAla

100 105no tgt ate cag cct cag ccc aca gca ggg ccc agg ccc egg ggc ege etc384

Cys lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly ArgLeu'

115 120125 cac cac tgg ctg cac egg etc cag gag gcc ccc aaa aag gag tee get432

His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu SerAla

130 135140 ggc tgc ctg gag gca tet gtc acc ttc aac etc ttc ege etc etc acg480

Gly Cys Leu Glu Ala Ser Vai Thr Phe Asn Leu Phe Arg Leu LeuThr i45 150 155.160 ega gac. etc aaa tat gtg gee gat ggg gay ctg tgt ctg aga acg tea '528

Arg Asp Leu Lys Tyr Vai Ala Asp Gly Asp Leu Cys Leu Arg ThrSer

165 170275 acc cac cct gag tee acc tga540

Thr His Pro Glu Ser Thr * .

<210> 101 <211> 182 <212> PRT <213> Artificial Sequence <220>

<223> Met IL29 mutant TUP, C16X, Aspl70 <221> VARIANT <222> (16)..(16) <223> Xaa = Ser,: Ala, Thr, Vai, or Asn <400> 101

Met Gly Pro Vai Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Xaa 1 5 1015

His lie Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe 20 . 2530

Lys Lys Ala Arg Asp Ala .Leu Glu Glu Ser Leu Lys Leu'Lys Asn Trp 35 4045

Ser Cys Ser Ser Pro Vai Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 5560

Gin Vai Arg Glu Arg Pro Vai Ala Leu Glu Ala Glu Leu Ala LeuThr 65 70 7580

Leu Lys Vai Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Vai Leu:

9095

Asp Gin Pro Leu His Thr Leu His His He Leu Ser Gin Leu Gin Ala 100 105., no -, .

Cys lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu

125 י 120 - 115 .״

His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu SerAla 130 .135 .. . 140 ,, . ..

Gly Cys Leu Glu Ala Ser Vai Thr Phe Asn Leu Phe Arg Leu Leu Thr’ 145 . 150 ...' 155 .160

Arg Asp Leu Lys, Tyr Vai Ala Asp Gly Asp Leu Cys Leu Arg Thr Ser.', : 165, .170 , , 175 ;.' '

Thr His Pro Glu Ser Thr <210> 102 <211> 546 <212> DNA <213> Artificial Sequence <220>

<223> IL29 mutant G18D, Asnl69, C171X <221> CDS <222> (1) . . . (5.46) <221> variation.

<222> (512)..(513,) <223> n = A, T, G, or C

<td> <210> <211> <212> <213></td><td> 103 . 181 PRT Artificial Sequence</td>

<td> <223></td><td> IL29 mutant G18D, Asnl69, C171X</td>

<td> <221></td><td> VARIANT</td>

<400> 103

<td> <213></td><td> Artificial Sequence</td>

<td> <223></td><td> Met IL29 mutant G19D, Asnl70, C172X</td>

<td> <221></td><td> variation</td>

30 י י י : 25 ; 20

<td> Gin</td><td> Vai</td><td> Arg</td><td colspan="2"> GluArg</td><td> Pro</td><td> Val</td><td> Ala</td><td> Leu</td><td> Glu</td><td> Ala</td><td> Glu</td><td> Leu</td><td> Ala</td><td> Leu</td><td> Thr</td><td></td>

<td> <212> <213></td><td> PRT .' Artificial Sequence</td>

<td> <223></td><td> Met IL29 mutant G19D, Asnl70, C172X</td>

<td> <221></td><td> VARIANT '</td>

<td> Leu Lys Val</td><td> Leu Glu Ala . 85 ,. ' . .</td><td> AlaAla</td><td> Gly Pro '. 90 '</td><td> Ala Leu Glu</td><td> Asp</td><td> Val 95</td><td> Leu</td>

Asp Gin Pro Leu His Thr Leu His His He Leu Ser Gin Leu Gin Ala

׳1־1־* 105100

Cys He Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu

125 . ׳ ׳ 120115

His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Ala 130 135140

Gly Cys Leu Glu Ala Ser Vai Thr Phe Asn Leu Phe Arg Leu Leu Thr - λ 155 150 שj-ou

Arg Asp Leu Lys Tyr Vai Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser

כ/± 170165

Thr His Pro Glu Ser Thr .

<210> 106 <211> 546 <212> DNA <213> Artificial Sequence <220> ^n <223> IL29 mutant C15X, G18D, Asnl69 <221> CDS <222> (1).-.(546) <221> variation <222> (44) . . . (45) <223> n = A, T, G, or C

<210> 107 <211> 181 <212> PRT <213> Artificial Sequence <220>

<223> IL29 mutant C15X, G18D, Asnl69 <221> VARIANT <222> (15)..(15) <223> Xaa = Ser, Ala, Thr, Val, or Asn

Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa His 1 5 1015

He Asp Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe Lys 20 2530

Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser 35 40 45

Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gin 50 5560

Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu ThrLeu

7° 75

Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val LeuAsp

90 .

Gin Pro Leu His Thr Leu His His lie Leu Ser Gin Leu Gin Ala Cys 100 105 ־. HO

He Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His H5 120125

His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Ala Gly 130 1151^0

Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg 145 150 . 1551°°

Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr

כ׳1 170165

His Pro Glu Ser Thr 180 ' <210> 108 <211> 549 <212> DNA <213> Artificial Sequence <220>

<223> Met IL29 mutant C16X, G19D, Asnl70 <221> CDS ..:..יי <222> (1) . . ' (549).

<221> variation <222> (47) - . - (48) .

<223> n = A, T, G, or C <40 0> 108 atg ggc cct gtc ccc act tcc aag ccc

Met Gly Pro Val Pro Thr Ser Lys Pro 5 cac att gay agg ttc aaa tet etg tea

His He Asp Arg Phe Lys Ser Leu Ser

2025 aag aag gcc agg gac gcc ttg gaa gag

Lvs Lys Ala. Arg Asp Ala Leu Glu Glu

3540 agt tgc age tet cct gtc ttc cccggg

Ser Cys Ser Ser Pro Val Phe ProGly cag gtg agg gag ege cct gtg gccttg nin Val Arg Glu Arg Pro Val AlaLeu

6570 etg aag gtc etg gag gee get getggc

Leu Lys Val Leu Glu Ala Ala AlaGly gac cag ccc ett cac acc etg caccac

Asp Gin Pro Leu His Thr Leu HisHis !00 tgt ate cag cct cag ccc aca gca ggg

Cys lie Gin Pro Gin Pro Thr Ala Gly

115 '120 cac cac tgg etg cac egg etc cag gag

His His Trp Leu His Arg Leu Gin Glu

130I ggc tgc etg gag gca tet gtc acc ttc

Gly Cys Leu Glu Ala Ser Val Thr Phe

145150 ega gac etc aaa tat gtg gcc gat ggg

Arg Asp Leu Lys Tyr Val Ala Asp Gly acc cac cct gag tcc acc tga

Thr His Pro Glu Ser Thr * acc aca act ggg aag ggc dim 48 Thr Thr Thr Gly. Lys Gly Xaa

15 cca cag gag eta geg age ttc 96 Pro Gin Glu Leu Ala Ser Phe tea etc aag etg aaa aac tgg 144

Ser Leu- Lys Leu Lys Asn Trp 45 aat tgg gac etg agg ett etc 192 Asn Trp Asp Leu Arg Leu Leu gag get gag etg gcc etg acg 240 Glu Ala Glu Leu Ala Leu Thr

7580 cca gcc etg gag gac gtc eta 288 Pro Ala Leu Glu Asp Val Leu

9095 ate etc tcc cag etc cag gcc 336 lie Leu Ser Gin Leu Gin Ala

110.

ccc agg ccc egg ggc ege etc 384 Pro Arg Pro Arg Gly Arg Leu gcc ccc aaa aag gag tcc get 432 Ala Pro Lys Lys Glu Ser Ala aac etc ttc ege etc etc acg 480 Asn Leu Phe Arg Leu Leu Thr

155 160 aay etg tgt etg aga acg tea 528 Asn Leu Cys Leu Arg Thr Ser 170 . 175 <210> 109 <211> 182 <212> PRT ' <213> Artificial Sequence <220>

<223> Met IL29 mutant C16X, G19D, Asnl70 <221> VARIANT ' ' .י - (16) . . . (16) <222>

<223>. Xaa = Ser,.Ala, Thr, Val, or . Asn .

Met^Gl^Pro Val Pro Thr Ser Lys Pro Thr Thr Thr. Gly.Lys Gly Xaa

י; .1.5 י' 10 '5‘ . ’ 1

His He Asp Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe 20 2530

Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 4045

Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 5560

Gin Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 ' 70 . 75 '80

Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu 85 9095

Asp Gin Pro Leu His Thr Leu His His lie Leu Ser Gin Leu Gin Ala 100 ‘ 105HO

Cys He Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120125

His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Ala 130 135140

Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu LeuThr

145 150 155160

Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg ThrSer

165. 170 .175

Thr His Pro Glu Ser Thr 180

<td> <212> <213></td><td> DNA qArtificial Sequence</td>

<td> <223></td><td> IL29 mutant G18D, Aspl69, C171X</td>

<td> <221></td><td> variation</td>

Gly Pro Val Pro.Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His

5 . 10 15

<210> 111 <211> 181 <212> PRT <213> Artificial Sequence <220>

<223> IL29 mutantG18D, Aspl69, C171X <221> VARIANT , ח (171) . (171) <222>

<223> Xaa = Ser, Ala, Thr, Val, or Asn

Gly Pro val Pro Thr Ser Ly־ Pro Thr Thr Thr Gly Lys Gly Cys His 10 5 ך

He Asp Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe Lys 20 2550

Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser 35 4045

Cvs Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu LeuGin ' 50 . 55 60 η.τ

Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu ThrLeu cc 7075

Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp 85 505Gin Pro Leu His Thr Leu His His lie Leu Ser Gin Leu Gin Ala Cys

He Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His 115 -20125

His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Ala Gly

140 135 ־30 ך

Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg 11c 150 . ׳ I55

Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Xaa Leu Arg Thr Ser Thr . . . 165 .. 170 1/3 . :

His Pro Glu Ser Thr .180 ' ' י'..' '.' :'. . 112 <210>

<211> 549 <212> DNA.

<213> Artificial Sequence <223> Met JL29 mutant G19D, Aspl70, C172X c221> CDS <222> (1)..(549) <221> variation <222> (515) . . . (516) <223> n = A, T, G, or C <400> 112 atg ggc cct gtc ccc act tcc aagccc

Met Gly Pro Vai Pro Thr Ser LysPro cac att gay agg ttc aaa tet etgtea

His He Asp Arg Phe Lys Ser LeuSer

2025 aag aag gcc agg gac gcc ttg gaagag

Lys Lys Ala Arg Asp Ala Leu GluGlu

3540 agt tgc age tet cct gtc ttc cccggg

Ser Cys Ser Ser Pro Vai Phe ProGly

5055 cag gtg agg gag ege cct gtg gccttg

Gin Vai Arg Glu Arg Pro Vai AlaLeu etg aag gtc etg gag gcc get getggc

Leu Lys Vai Leu Glu Ala Ala AlaGly .85 gac cag ccc ett cac. acc etg caccac

Asp Gin Pro Leu His Thr Leu HisHis tgt ate cag cct׳cag ccc aca gcaggg

Cys lie Gin Pro Gin Pro Thr AlaGly

115120 cac cac tgg etg cac egg etc caggag

His His Trp Leu His Arg Leu GinGlu

130135 ggc tgc etg gag gca tet gtc accttc

Gly Cys Leu Glu Ala Ser Vai ThrPhe

145I ega gac etc aaa tat gtg gcc gat ggg

Arg Asp Leu Lys Tyr Vai Ala Asp Gly acc cac cct gag tee acc tga

Thr His Pro Glu Ser Thr * acc aca act ggg aag ggc tgc 48 Thr Thr Thr Gly Lys Gly Cys

15 cca cag gag eta geg age ttc. 96 Pro Gin Glu Leu Ala Ser Phe tea etc aag etg aaa aac tgg 144

Ser Leu Lys Leu Lys Asn Trp aat tgg gac etg agg ett etc 192 Asn Trp Asp Leu Arg Leu Leu gag get gag etg gcc etg acg 240' Glu Ala Glu Leu Ala Leu Thr 80 cca gcc etg gag gac gtc eta 288. Pro Ala Leu Glu Asp Vai Leu'

95 ate etc tcc cag etc cag gcc 336. lie Leu Ser Gin Leu Gin Ala

11° ccc agg ccc egg ggc ege etc 384 Pro Arg Pro Arg Gly Arg Leu

125 gcc ccc aaa aag gag tcc get 432 Ala Pro Lys Lys Glu Ser Ala aac etc ttc ege etc etc acg480

Asn Leu Phe Arg Leu LeuThr

155 :.160 gay etg dnn etg aga acg tea528

Asp Leu Xaa Leu Arg ThrSer

170175

549 <210> 113 <211> 182 ' <212> PRT <213> Artificial Sequence <223> Met IL29 mutant G19D, Aspl70, C172X <221> VARIANT <222> (172)..(172) <223> Xaa = Ser, Ala, Thr, Val, or Asn ' ״ 113 <400>

Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys ! 5 TO15

His lie Asp Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe 20 2530

Lys Lys Ala Arg Asp. Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 4045

Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg LeuLeu.

55 .

Gin Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala LeuThr

80 75 ־ 7065

Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu 85 9095

Aso Gin Pro Leu His Thr Leu His His He Leu Ser Gin Leu Gin Ala 100 1°5110

Cys He Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120125

His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Ala 130 135140

Glv Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu LeuThr !45 150 155160

Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Xaa Leu Arg ThrSer

165 170175

Thr His Pro Glu Ser Thr . 180 <210> 114 .

<211> 546 <212> DNA <213> Artificial Sequence <220>

<223> IL29 mutant C15X, G18D, Aspl69 <221> CDS ' . . ' ' י <222> (1) - -.(546) , .

<221> variation י , (45)..(44) <222>

<223> n = A, T, G, or C <400> 114.

ggc cct gtc ccc act tcc aag ccc acc aca act ggg aag ggc aimcac

Glv Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly XaaHis

- 5 : 10 ,. 15 att gay agg ttc aaa tet etg tea cca cag gag eta geg age ttcaag lie Asp Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser PheLys ' 20 '. .: .:..25 .. -י . .ί 3°, aag gcc agg gac gcc ttg gaa gag tea etc aag etg aaa aac tggagt

Lys Ala Arg Asp Ala, Leu Glu Glu Ser Leu Lys Leu Lys Asn TrpSer

35., 40 . 45 tgc age tet cct gtc ttc ccc ggg aat tgg gac etg agg ett etccag

Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp.Leu Arg Leu LeuGin ׳ - .j /.,. 55 ’ :60 ;.

<210> 115 <211> 181 <212> PRT <213> Artificial Sequence <220>

<223> IL29 mutant C15X, G18D,'Aspl69 <221> VARIANT <222> (15) .(15) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 115

Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa His 1 5 1015

He Asp Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe Lys 20 2530

Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu. Lys Asn Trp Ser. 35 4045

Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu LeuGin • 50 . . 5560

Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu ThrLeu

70 7580

Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp 85 9095

Gin Pro Leu His Thr Leu His His He Leu Ser Gin Leu Gin Ala Cys 100 . , 105 .110 .

He Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His 115 120125

His Trp .Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Ala Gly ' .

. י 140 ' . 135 . /. 130

Cys Leu Glu Ala.Ser Val Thr' Phe Asn Leu Phe Arg Leu Leu Thr Arg 145 ?:.,. . . 150 .י . ή; 160. / . 155. ' . .,<.;.;־

Asp Leu Lys Tyr Vai Ala Asp 165.

His Pro Glu Ser Thr 180

Gly Asp Leu Cys Leu Arg Thr Ser Thr

170 175 <210> 116 <211> 549 <212> DNA <213> Artificial Sequence <220>

<223> Met IL29 mutant C16X, G19D, Aspl70 .<221> CDS <222> (1). - (549) <221> variation <222> (47) . . . (48) <223> n = A, T, G, or C <400> 116 atg ggc cct gtc ccc act tcc aagccc

Met Gly Pro Vai Pro Thr Ser LysPro :

cac att gay agg ttc aaa tct etgtea

His lie Asp Arg Phe Lys Ser LeuSer

2025 aag aag gcc agg gac gcc ttg gaagag

Lys Lys Ala Arg Asp Ala Leu GluGlu

3540 agt tgc age tct cct gtc ttc cccggg

Ser Cys Ser Ser Pro Vai Phe ProGly

55cag gtg agg gag ege cct gtg gccttg

Gin Vai Arg Glu Arg .Pro Vai AlaLeu

6570 etg aag gtc etg gag gcc get getggc

Leu Lys Vai Leu Glu Ala Ala AlaGly gac cag ccc ett cac acc etg caccac

Asp Gin Pro Leu His !Thr Leu HisHis

100105 tgt ate cag.cct cag ccc aca gca ggg

Cys lie Gin Pro Gin Pro Thr Ala Gly 115 ' 120 cac cac tgg etg cac egg etc cag gag • His His Trp Leu His Arg Leu Gin Glu . . 130 . J .,. . 135 ggc tgc etg gag gca .tct gtc acc ttc

Gly Cys Leu Glu Ala Ser Vai Thr Phe

145 '150 cga gac etc aaa tat gtg gcc gat ggg

Arg Asp Leu Lys Tyr Vai Ala Asp Gly ' .. 165 ; ' .?''Π acc aca.act ggg aag ggcdim 48 Thr Thr Thr Gly Lys Gly Xaa

15 .

cca cag gag eta geg age ttc 96 Pro Gin Glu Leu Ala Ser Phe tea etc aag etg aaa aac tgg 144 Ser Leu .Lys Leu Lys Asn Trp aat tgg gac etg agg ett etc 192 Asn Trp Asp Leu Arg Leu Leu gag get gag etg gcc etg acg 240 Glu Ala Glu Leu Ala Leu Thr

80 cca gcc etg gag gac gtc eta 288 Pro Ala Leu Glu Asp Vai Leu

95 ate etc tcc cag etc cag gcc 336 lie Leu Ser Gin Leu Gin Ala

HO ccc agg ccc egg ggc ege etc 384

Pro Arg Pro Arg Gly Arg Leu

125 . . .

gcc ccc aaa aag gag tcc get432

Ala Pro Lys Lys Glu Ser Ala.

140:״ aac etc ttc ege etc etc acg 480 ׳

Asn Leu Phe Arg Leu Leu Thr

155 ,.</ 160 / ' gay etg tgt etg aga acg tea . 528 Asp Leu.Cys Leu Arg Thr Ser. י י 170 .. :- 175 ' .

acc cac cct gag tcc acc tga 549

Thr His Pro Glu Ser Thr * 180

<td> <213></td><td> Artificial Sequence</td>

<td> <220></td><td rowspan="2"> Met IL29 mutant C16X, G19D, Aspl70</td>

<td> <221></td><td> VARIANT .</td>

Thr His Pro Glu Ser Thr <210> 118.

<211> 57 <212> DNA <213> Artificial Sequence <220>

<223> Signal sequence \ <221> CDS .'י, <222> (1). . (57) .'..־.

<400> 118 atg' get gca get tgg acc gtg gtg ctg gtg act ttg gtg eta ggc ttg 48 Met Ala Ala Ala Trp Thr Vai Vai Leu Vai Thr Leu Vai'Leu Gly Leu .

.' .'. 5 .., 10 15 ' gcc gtg gca

Ala Vai Ala <210> 119 <211> 19 <212> PRT <213> Artificial Sequence c220>

<223> Signal sequence

Me^Al^Ala Ala Trp Thr Val Val Leu Val Thr Leu Val Leu Gly Leu 1 5 10 15

Ala Val Ala <210> 120 <211> 66 <212> DNA <213> Artificial Sequence <220>

<223>

Signal sequence <221>

<222>

CDS (1) ..(66) <400> atg Met 1

120 gtg Val ccc

Pro acc

Thr aca

Thr •5.

ttg get

Leu Ala tgg acc

Trp Thr gtg

Val gtg

Val etg gtg

Leu Val act

Thr ttg

Leu gtg

Val eta

Leu ggc

Gly ttg

Leu gec

Ala gtg

Val gca

Ala <210> 121 <211> 22 <212> PRT <213> Artificial Sequence <220>

<223> Signal sequence <400> 121

Met Val Pro Thr Thr 1 5

Leu Gly Leu Ala Val . 20

Leu Ala Trp Thr Val Val ,

Ala

Leu Val Thr Leu Val <210> 122 <211> 528 ' <212> DNA <213> Artificial Sequence <220>

<223> IL-28B C48S <221> CDS <222> (1). -(528) <221> variation <222> (143)..(144) <223>, n = A, T,. G, or C־ <400> •122 ' .'.. /־/

174797/ϊ

<210 123 <211> 175 <212> PRT <213> Artificial Sequence <220 'י <221> VARIANT . . '.'.־ <222> (48) . . .(48) <223> Xaa = Ser, Ala, Thr, Val, or Asn . .'. . ' <223> IL-28B C48S <400> 123

Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly Cys 1 . ;5 10 ' 15

His lie Ala Gin Phe Lys Ser Leu Ser Pro Gin Glu Leu Gin Ala Phe : 25 . .30 ' Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Xaa 35 40 45 . ,.

Lys Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp'Asp Leu Arg Gin Leu־':

Gin Vai Arg Glu Arg 65

Leu Lys Vai Leu Glu

Vai Leu Asp Gin Pro

Arg Ala Cys He Gin .Arg Leu His His Trp

130 ’

Ser Pro Gly Cys Leu 145 .

Leu Thr Arg Asp Leu

Pro Vai Ala Leu Glu 70

Ala Thr Ala Asp Thr

Leu His Thr Leu His

Pro Gin Pro Thr Ala

Leu His Arg Leu Gin

Glu Ala Ser Vai Thr 150

Asn Cys Vai Ala Ser

Ala Glu Leu Ala Leu 75

Asp Pro Ala Leu Gly

His lie Leu.Ser Gin

־ 110

Gly Pro Arg Thr Arg 125

Glu Ala Pro Lys Lys

Phe Asn Leu Phe Arg 155

Gly Asp Leu Cys Vai

Thr 80

Asp

Leu

Gly

Glu

Leu <210> 124 .

<211> 531 <212> DNA <213> Artificial Sequence <220>

<223> Met IL-28B C49S <221> CDS <222> (1).-.(531) <221> variation <222> (146). -(147) <223> n = A,'t, G, or C <400> 124, atg gtt cct gtc gcc agg etc ege ggg get etc ccg gat gca agg ggc48

Met Vai Pro Vai Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala ArgGly

5 10 '15 tgc cac ata gcc cag ttc aag tcc ctg tet cca cag gag ctg cag gcc96

Cvs His He Ala Gin Phe Lys Ser Leu Ser Pro Gin Glu Leu GinAla

25 .30 ttt aag agg gcc aaa gat gcc tta gaa gag teg ett ctg ctg aag gac144

Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu LysAsp ' 40 . . 45 dnn aag tgc ege tcc ege etc ttc ccc agg acc tgg gac ctg agg cag192

Xaa Lys Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu ArgGin

60 .־. 55 - ׳50 ctg cag gtg agg gag ege ccc gtg get ttg gag get. gag ctg gcc ctg240

Leu Gin Vai Arg Glu Arg Pro Vai Ala Leu Glu Ala Glu Leu AlaLeu ״:-;. 70 . ./ 7 י ' 65 .׳y.80 -/ . 75-/, .׳ ./.

acg ctg aag gtt ctg gag gcc acc get gac act gac cca gcc ctg ggg288

Thr Leu Lys Vai Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Gly . . 90 . .' . 95 . , , gat gtc ttg gac cag ccc ett cac acc ctg cac cat ate etc tcc cag 336

Asp Vai Leu Asp Gin Pro Leu His Thr Leu His His lie Leu Ser Gin ־ . V 100 . 105 .:/./?. 110 י .-־./.

etc egg gcc tgt ate cag' cct cag ccc acg gca ggg ccc agg acc egg384 ' Leu Arg Ala Cys He Gin Pro Gin Pro' Thr Ala Gly Pro Arg ThrArg :

. . .120 . . . ' 125 ', ggc cg'c etc cac'cat tgg ctg cac egg etc cag gag gcc cca aaa aag. 432

tga *

531 .

<210> 125 <211> 176 <212> PRT <213> Artificial Sequence <220>

<221> VARIANT <222> (49) . . . (49) <223> Xaa = Ser, Ala, Thr, Val, or Asn <223> Met IL-28B C49S <400> 125

Met Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly 1 5 1015

Cys His He Ala Gin Phe Lys Ser Leu Ser Pro Gin Glu Leu Gin Ala 20 2530

Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp 35 4045

Xaa Lys Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gin 50 5560

Leu Gin Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu AlaLeu

70 7580

Thr Leu Lys Val'Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala LeuGly

9095

Asp Val Leu Asp Gin Pro Leu His Thr Leu His His He Leu Ser Gin 100 105HO

Leu Arg Ala Cys lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Thr Arg 115 120125

Gly Arg Leu His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys 130 - '135140 .

Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu PheArg

145 ' 150 155 .160

Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu CysVal

165 ' 170175 <210> 126 <211> 528 <212> DNA י .

<213> Artificial Sequence <220> :. . ;

<223> IL-28B C50S <221> CDS 7 ' .‘.י <222> (1)..(528).

<221> variation <222>. (149) . . ' (150)' .

<223> η = A, T, G, or C

<210> 127 <211> 175<212> PRT <213> Artificial Sequence <220> ' <221> VARIANT <222> (50) . . . (50) <223> Xaa = Ser, Ala, Thr, Val, or Asn <223> IL-28B C50S . ־ <400> 127

Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly Cys.

.. 5 ' . ' 10 15 :

His lie Ala Giri Phe Lys Ser Leu Ser .Pro Gin Glu.'Leu Gin Ala Phe'

. . 30' :. ... ׳:,{. . .25 .י'.-'.. 20

<td colspan="3"> <213> Artificial Sequence</td><td></td><td></td>

<td colspan="3"> <221> variation</td><td></td><td></td>

<210> 129 <211> 176 <212> PRT <213> Artificial Sequence <220>

<221> VARIANT <222> (51)- . (51) <223> Xaa = Ser, Ala, Thr, Val, or Asn <223> Met IL-28B C51S <400> 129

Met Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly 1 5 1015

Cys His He Ala'Gin Phe Lys Ser Leu Ser Pro Gin Glu Leu Gin Ala 20 2530

Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp 35 4045

Cys Lys Xaa Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gin. 50 5560

Leu Gin Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu AlaLeu

70 7580

Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala LeuGly ,85 9095

Asp Val Leu Asp Gin Pro Leu His Thr' Leu His His lie Leu Ser Gin 100 105HO

Leu Arg Ala Cys He Gin Pro Gin Pro Thr Ala Gly Pro Arg Thr Arg 115 120125

Gly Arg Leu His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys 130 135140

Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu PheArg

145 150 155 .160

Leu LeuThr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu CysVal

165 170175 <210> 130 -.’ <211> 528 <212> DNA <213> Artificial Sequence .

. ' . . ,. / 1:. . .? ' <220>

<223> IL-28B C,48S T87S H135Y <221> CDS 'י',:י <222> (1) . . . (528)־.

<221> variation <222> 143, 144, 261 <223> n = A, T, G, or C

<210> 131 <211> 175 .

<212> PRT <213> Artificial. Sequence <220> .

<221> VARIANT ־ <222> (48) . . . (48) <223> Xaa = Ser, Ala, Thr., Val, or Asn <221> VARIANT . ;

<222> (87) . (87) .;

<223> Xaa =' Ser <223> IL-28B C48S T87S H135Y.

<400> 131

Val Pro Val Ala Arg Leu Arg Gly 15

His He Ala Gin Phe Lys Ser Leu 20 .

Lys Arg Ala Lys Asp Ala Leu Glu

3540

Lys Cys Arg Ser Arg Leu Phe Pro

5055

Gin Val Arg Glu Arg Pro Val Ala

5570

Leu Lys Val Leu Glu Ala Xaa Ala 85

Val Leu Asp Gin Pro Leu His Thr 100

Arg Ala Cys lie Gin Pro Gin Pro

115120

Arg Leu His His Trp Leu Tyr Arg

130135

Ser Pro Gly Cys Leu Glu Ala Ser

145150

Leu Thr. Arg Asp Leu Asn Cys Val

Ala Leu Pro Asp Ala Arg Gly Cys

1015

Ser Pro Gin Glu Leu Gin Ala Phe

2530

Glu Ser Leu Leu Leu Lys Asp Xaa 45

Arg Thr Trp Asp Leu Arg Gin Leu 60 .'

Leu Glu Ala Glu ׳Leu Ala Leu Thr

7580

Asp Thr Asp Pro Ala Leu Gly Asp 9095

Leu His His He Leu Ser Gin Leu

105HO

Thr Ala Gly Pro Arg Thr Arg Gly

Leu Gin Glu Ala Pro Lys Lys Glu 140

Val Thr Phe Asn Leu Phe Arg Leu

155 160

Ala Ser Gly Asp Leu Cys Val

170 175 .

<td> <213></td><td> Artificial</td><td> Sequence</td>

<221> CDS <222> (1)..(531) <221> variation <222> 146, 147', 264 <223> n = A, T, G, or C <400> 132

gat gtc ttg gac cag Asp Val Leu Asp Gin 100 etc egg gcc tgt ate Leu Arg Ala Cys He 115 ggc ege etc cac cat Gly Arg Leu His His 130 ' gag tee cct ggc tgc Glu Ser Pro Gly Cys 145 etc etc acg ega gac Leu Leu Thr Arg Asp 155 tga * .

ccc ett cac acc etg cac pro Leu His. Thr Leu His 105 cag cct cag ccc acg gca

Gin Pro Gin Pro Thr Ala 120 tgg etg tay egg etc cag

Trp Leu Tyr Arg Leu Gin 135 etc gag gcc tet gtc acc Leu Glu Ala Ser Val Thr 150 etg aat tgt gtt gcc age Leu Asn Cys Val Ala Ser 170 cat ate etc tee cag336

His He Leu SerGin ggg ccc agg acc egg384

Gly Pro Arg ThrArg gag gcc cca aaa aag432

Glu Ala Pro LysLys ttc aac etc ttc ege480

Phe Asn Leu PheArg ggg gac etg tgt gtc528

Gly Asp Leu CysVal <210> 133 <211> 176 <212> PRT <213> Artificial Sequence <220>

<221> VARIANT <222> (49) . . . (49) <223> Xaa = Ser, Ala, Thr, Val, or

Asn <221> VARIANT <222> (88). . - (88) <223> Xaa = Ser <223> Met IL-28B C49S T88S H136Y <400> 133

Met Val Pro Val Ala Arg Leu Arg 15

Cys His lie Ala Gin Phe Lys Ser 20 : Phe Lys Arg Ala Lys Asp Ala Leu :40

Xaa Lys Cys Arg Ser Arg LeuPhe

5055

Leu Gin Val Arg Glu Arg ProVal :70

Thr Leu Lys Val Leu Glu Ala Xaa

Asp Val Leu Asp Gin Pro Leu His 100

Leu Arg Ala Cys lie Gin Pro Gin

115 ;.

Gly Arg Leu His His Trp Leu Tyr

Glu Ser Pro Gly Cys Leu Glu Ala 145-50 . Leu Leu Thr Arg Asp Leu Asn Cys

165 -.

Gly Ala Leu Pro Asp Ala Arg Gly

1015

Leu Ser Pro Gin Glu Leu Gin Ala

Glu Glu Ser Leu Leu Leu Lys Asp ' 45.

Pro Arg Thr Trp Asp Leu Arg Gin 60

Ala Leu Glu Ala Glu Leu Ala Leu

7580

Ala Asp Thr Asp Pro Ala Leu Gly

9095

Thr Leu His His He Leu Ser Gin .

105' 110

Pro Thr Ala Gly Pro Arg Thr Arg .125

Arg Leu Gin Glu Ala Pro Lys Lys 140'

Ser Val Thr Phe Asn Leu Phe Arg

16°155 י

Val Ala Ser Gly Asp Leu Cys Val

170 ' .175 ' <210> 134 <211> 528 <212> DNA <213> Artificial Sequence <220>

<223> IL-28B C50S T87S H135Y <221> CDS <222> (1).-(528) <221> variation <222> 149, 150, 261 <223> n = A, T, G, or C <400> 134 gtt cct gtc gcc agg etc ege ggg get val Pro Val Ala Arg Leu Arg Gly Ala 5 cac ata gcc cag. ttc aag tcc ctg tct

His lie Ala Gin Phe Lys Ser Leu Ser 20 aag agg gcc aaa gat gcc tta gaa gag

Lys Arg Ala Lys Asp Ala Leu Glu Glu 35 aag dim ege tcc ege etc ttc ccc agg

Lys Xaa Arg Ser Arg Leu Phe Pro Arg 50 cag gtg agg gag ege ccc gtg get ttg

Gin Val Arg Glu Arg Pro Val Ala Leu .

ctg aag gtt ctg gag gee wsn get gac

Leu Lys Val Leu Glu Ala Xaa Ala Asp 85 gtc ttg gac cag ccc ett cac acc ctg

Val Leu Asp Gin Pro Leu His Thr Leu 100 105 egg gee tgt ate cag cct cag ccc acg • Arg Ala Cys lie Gin Pro' Gin Pro Thr 115 120 ege etc cac cat tgg ctg tay egg etc

Arg Leu His His Trp Leu Tyr Arg Leu 130 ' 135 tee cct ggc tgc. etc gag gcc tct gtc

Ser Pro Gly Cys. Leu Glu Ala Ser Val ' 145 ' I50 etc acg ega gac ctg aat tgt gtt gee Leu Thr Arg Asp ־׳Leu Asn Cys Val Ala 165 etc ccg gat gca agg ggc tgc 48

Leu Pro Asp Ala Arg Gly Cys

15 cca cag gag ctg cag gcc ttt 96

Pro Gin Glu Leu Gin Ala Phe teg ett ctg ctg aag gac tgc 144

Ser Leu Leu Leu Lys Asp Cys acc tgg gac ctg agg cag ctg 192

Thr Trp Asp Leu Arg Gin Leu gag get gag ctg gcc ctg acg 240.

Glu Ala Glu Leu Ala Leu Thr 75 80 act gac cca gcc ctg ggg gat 288

Thr Asp Pro Ala Leu Gly Asp

95 cac cat ate etc tcc cag etc 336

His His He Leu Ser Gin Leu gca ggg ccc agg acc egg ggc 384

Ala Gly Pro Arg Thr Arg Gly '

12.5 cag gag gcc cca. aaa aag gag 432

Gin Glu Ala Pro Lys Lys Glu acc ttc aac etc ttc ege etc480

Thr.Phe Asn Leu Phe ArgLeu

155 ...'.160 age ggg gac ctg tgt gtc tga.528

Ser Gly Asp Leu Cys Val *.

170 . .I <210> 135 <211> 175 <212> PRT' עס/ lut <213 > Artificial Sequence <220>

<221> VARIANT <222> (50). - -(50) <223> Xaa = Ser, Ala, Thr, Val, or Asn <221> VARIANT <222> (87)..(87) <223> Xaa = Ser <223> IL-28B C50S T87S H135Y viJ°Pro3V־l Ala tog Leu tog Gly Al. Leu Pro Asp Ala tog Gly Cy־

His He Ala Gin Phe Lys Ser Leu Ser Pro Gin Glu Leu Gin Ala Phe 20 25

Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys

י־.4 40 35

Lys Xaa Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gin Leu

Gin Val Arg Glu Arg Pro vL Ala Leu Glu Ala Glu Leu. Ala Leu Thr lL Lys Val Leu Glu Ila Xaa Ala Asp Thr Asp Pro Ala Leu Gly Asp θ θ

Val Leu Asp Gin Pro Leu His Thr Leu His His He Leu Ser Gin Leu 1 105 סח רJ־U

Arg Ala Cy־ lie Gin Pro Gin Pro Thr Ala Gly Pro tog Thr tog Gly 120 ף ך ך J-Z□

Arg Leu His His Trp Leu Tyr tog Leu Gin Glu Ala Pro Lys Lys Glu S־r pro Gly Cy־ Leu Glu 21 Ser Val Thr Phe Asn Leu Phe tog Leu 1ar 150 1ככ

Leu Thr tog Asp Leu Asn Cy־ Val Ala Ser Gly Asp Leu Cye Val

165 170 J-3׳ <210> 136 <211> 531 ..

<212> DNA <213> Artificial Sequence <220>

<223> Met IL-28B C51S T88S H136Y <221> CDS <222> (1)---(531) <221> variation <222> 152, 153, 264 <223> n = A; T, G, or C

tgc aag dim cgc tcc cgc etc ttc Cys Lys Xaa Arg Ser Arg Leu Phe 5055 etg cag gtg agg gag cgc ccc gtg

Leu Gin Val Arg Glu Arg Pro Val 6570 acg etg aag gtt etg gag geewsn

Thr Leu Lys Val Leu Glu Ala Xaa 85.

gat gtc ttg gac cag ccc ettcac

Asp Val Leu Asp Gin Pro LeuHis etc egg gee tgt ate cag cctcag

Leu Arg Ala Cys He Gin ProGin ggc cgc etc cac cat tgg etg tay gly Arg Leu His His Trp Leu Tyr 130 gag tcc cct ggc tgc etc gag gcc Glu Ser Pro Gly Cys Leu Glu Ala 145 ccc agg acc tgg gac etg agg cag

Pro Arg Thr Trp Asp Leu Arg Gin 60 get ttg gag get gag etg gcc etg

Ala Leu Glu Ala Glu Leu Ala Leu 7580 get gac act gac cca gcc etgggg

Ala Asp Thr Asp Pro Ala LeuGly

9095 acc etg cac cat ate etc tcccag

Thr Leu His His He Leu SerGin

105110 ccc acg gca ggg ccc agg acc egg

Pro Thr Ala Gly Pro Arg Thr Arg egg etc cag gag gee cca aaa aag

Arg Leu Gin Glu Ala Pro LysLys tct gtc acc ttc aac etc ttccgc

Ser Val Thr Phe Asn Leu PheArg

155160 etc etc acg ega gac etg aat tgt gtt Leu Leu Thr Arg Asp Leu Asn Cys Val 165 gcc age ggg gac etg tgt gtc Ala Ser Gly Asp Leu Cys Val 170 175 tga <210> 137 ’ <211> 176 .

<212> PRT <213> Artificial Sequence <220> .

<221> VARIANT ' <222> (51) . . . (51) <223> Xaa = Ser, Ala, Thr, Val, or Asn <221> VARIANT <222> (88) . . . (88) <223> Xaa = Ser <223> Met IL-28B C51S T88S H136Y . .י....,.;.. . 137 <400>

Met Val' Pro Val Ala Arg Leu Arg Gly Ala

51°

Cys His He Ala Gin Phe Lys Ser Leu Ser

Phe Lys Arg Ala Lys Asp Ala Leu.Glu Glu

40/ ־. ' 35 . . ..

.Cys Lys Xaa Arg Ser. Arg Leu Phe Pro'Arg

50. 55

Leu׳Gin Val Arg Glu Arg Pro Val Ala Leu . . . ..70 . :. .

Thr Leu Lys Val Leu Glu Ala Xaa Ala Asp . . . ... 85 , . ., , 90 .

Leu Pro Asp

Pro Gin Glu

Ser Leu Leu 45

Thr Trp Asp 60 .

Glu Ala Glu 75 ,

Thr Asp Pro

Ala Arg Gly

. י 15

Leu Gin Ala 30

Leu Lys Asp

Leu Arg Gin

Leu Ala Leu

־80

Ala Leu Gly .' 95 ,

Asp Val Leu Asp Gin Pro Leu His ' 100

Leu Arg Ala Cys He Gin Pro Gin

115120

Gly Arg Leu His His Trp Leu Tyr

ו, 5יל

Glu Ser Pro Gly Cys Leu Glu Ala

145 '

Leu Leu Thr Arg Asp Leu Asn Cys

JSO/llW

Thr Leu His His He Leu

105 110

Pro Thr Ala Gly Pro Arg

Arg Leu Gin Glu Ala Pro 140

Ser Val Thr Phe Asn Leu

Val Ala Ser Gly Asp Leu

Ser

Thr

Lys

Phe

Cys

Gin

Arg

Lys

Arg 160 Val <210> 138 <211> 543 <212> DNA <213> Artificial Sequence <223> IL-29 C17OX, truncated after N-tenninal Methionine and Glycine <221> variation <222> (509) . . -(510) <223> n = A, T, G, or C <221> CDS <222> (1)-.(543) <400>

cct

Pro

138 gtc Val ccc act

Pro

Thr tcc

Ser aag

Lys ccc

Pro acc

Thr aca

Thr act ggg

Thr Gly ggc

Gly agg

Arg ttc

Phe aaa

Lys tet

Ser ctg

Leu tea

Ser cca

Pro cag

Gin gag

Glu eta

Leu gcc

Ala age

Ser

ctg

Leu ctg

Leu agg

Arg aag

Lys aaa

Lys geg

Ala

217 / 174797/1

<210> 139 <211> 180 <212> PRT <213> Artificial Sequence <220>

<221> VARIANT <222> (170) . . . (170) » <223> Xaa = Ser, Ala, Thr, Val, or Asn <223> IL-29 C170X, truncated after N-terminal Methionine and Glycine <400> 139

<210> 140 <211> 540 .

<212> DNA ';..י <213> Artificial Sequence

..־.'. י'-. . ־ ל־ <220>

<223> IL-29 C169X,'truncated after N-terminal Methionine, Glycine, arid Proline <221> variation

־. ;.'(507) . . .'(506) <222>

<223> n = A, T, G, or C <221> CDS .

//IV*♦ <222> (1) . . .(540) <400> 140 gtc ccc act tcc aag

Val Pro Thr Ser Lys 5 ccc acc aca act ggg aag Pro Thr Thr Thr Gly Lys 10 ggc tgc. cac att ggc Gly Cys His He Gly agg ttc aaa tct etg Arg Phe Lys Ser Leu 20 tea cca cag gag eta geg Ser Pro Gin Glu Leu Ala 25 age ttc aag aag gcc Ser Phe Lys Lys Ala

384' gag tee acc tga Glu Ser Thr * <210> 141 .

<211> 179 ' <212> PRT <213> Artificial Sequence

׳ ׳ ' :. .? . .' ׳: ' ׳ ’ ׳'׳ '$ <220>

<221> VARIANT <222> (169).--(169) <223> Xaa = Ser, .Ala, Thr, Val, or Asn <223> L-29 C169X, truncated after N-terminal Methionine,' Glycine,, and Proline .

<400> 141 י'''.- .י :'.'..Λ.'’.י' '.'.'.,

Val Pro Ite ־« W־ Pro Thr Thr Thr Gly Lys Gly Cys His 11־ Gly «g Ph־ Lys Ser llu Ser Pro Gin Glu L־u Ma Ser Phe Lye Lys Ma Arg Asp Ala Leu Glu Glu Ser Leu Ly־ Leu Lys Asn Trp Ser CyB S־r Ser pro vll Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gin Val Arg Glu W Pro val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val ‘L Glu Ala Ala ^Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gin Ph־ Leu His Thr Leu His His !1־ Leu Ser Gin Leu Gin Ala Cys He Gin l.00״י״ Λτ-rr Pro Ara Gly Arg Leu His His Trp Pro Gin Leu His 130 Glu Ala 145 Lys Tyr Glu Ser

Pro 115 Arg

Leu Gin

Glu

Ser Val Thr Phe 150 Asp Gly 165

Val Ala

Thr

Ala 135

Asn Leu

Pro Lys

Asn Leu

Phe

Xaa

Lys

Glu

Arg

Leu 170

Leu 155 Arg

Ser 140 Leu

Thr

125 Ala

Gly

Cys

Leu

Thr

Arg

Asp

Ser

Thr

His 175

Leu 160 Pro <210> 142 <211> 537 <212> DNA <213> Artificial Sequence <220>

<223>

IL-29 C168X,

Methionine, truncated after N terminal Glycine, Proline, and Valine.

<221> variation <222> (503) . -(504) <223> n = A, T, G, or C <221> CDS <222> (1)-.-(537) <400> 142 ' , ccc act tcc aag ccc acc Pro Thr Ser Lys Pro Thr 5 - .

ttc aaa tet etg tea cca Phe Lys Ser Leu Ser Pro gac gcc ttg gaa gag tea Asp Ala Leu Glu Glu Ser , . . . 35 cct gtc'ttc cc.c ggg aat ׳ Pro Val Phe Pro Gly Asn 50 ege cct gtg gcc ttg gag

Ara Pro Val Ala Leu Glu

70 ׳:;. ;:;.;65 .׳ gag gcc get get ggc cca 'Glu Ala Ala Ala GlyPro 85 aca act ggg aag ggc tgc Thr Thr Gly. Lys Gly Cys 10 cag gag eta geg age ttc

Gin Glu Leu Ala Ser Phe . . . 25 etc aag etg aaa aac tgg

Leu Lys Leu Lys Asn Trp .40 tgg gac etg agg ett etc

Trp Asp Leu Arg Leu Leu 60 get gag etg gcc etg acg

Ala Glu Leu Ala Leu Thr ,... 75 gcc etg gag gac gtc eta

Ala Leu Glu Asp Val Leu 90 cac att ggc agg 48

His Tie Gly Arg aag aag gcc agg 96

Lys Lys Ala Arg agt tgc age.tet 144

Ser Cys Ser Ser 45 cag gtg agg gag 192

Gin Val Arg Glu etg aag gtc etg 240

Leu Lys .Val Leu 80 .

gac. cagccc ett . 288 Asp Gin Pro Leu . 95 .I /220

tcc acc tga

Ser Thr * <210> 143 <211> 178 <212> PRT <213> Artificial' Sequence ו . . > <220>

<223> IL-29 C168X, truncated after N-terminal Methionine‘, Glycine, Proline, and Valine <221> VARIANT <222> (168)..(168) <223> Xaa = Ser, Ala, Thr, Val, <400> 143

Pro Thr Ser Lys Pro Thr Thr Thr 15

Phe Lys Ser Leu Ser Pro Gin Glu

Asp Ala Leu Glu Glu Ser Leu Lys ’40

Pro Val Phe Pro Gly Asn Trp Asp

Arg Pro Val Ala Leu Glu Ala Glu

6570

Glu Ala Ala Ala Gly Pro Ala Leu

His Thr Leu His His lie Leu Ser . .100 ,

Gin Pro Thr Ala Gly Pro Arg Pro

His Arg Leu Gin Glu Ala Pro Lys ' 130135

Ala Ser Val Thr Phe Asn Leu Phe

145 .150

Tyr Val Ala Asp Gly Asn Leu Xaa

Ser Thr or Asn

Gly Lys Gly Cys His He Gly Arg 15

Leu Ala Ser Phe Lys Lys Ala Arg

׳ 30 25

Leu Lys Asn Trp Ser Cys Ser Ser 45

Leu Arg Leu Leu Gin Val Arg Glu 60

Leu Ala Leu Thr Leu Lys Val. Leu

7580

Glu Asp Val Leu Asp Gin Pro Leu 9095

Gin Leu Gin Ala Cys He Gin Pro

105!1°

Arg Gly Arg Leu His His Trp Leu 125 .

Lys Glu Ser Ala Gly Cys Leu Glu . . 140 .

Arg Leu Leu Thr Arg Asp Leu Lys

155 160

Leu' Arg Thr Ser Thr His Pro Glu 170 175 <210 144 <211> 534 <212> DNA <213> Artificial Sequence

[-terminal

Valine, and Proline <223> IL-29 C167X, truncated after Methionine, Glycine, Proline, <221> variation <222> (500)..(501) <223> n = A, T, G, or C <221> CDS <222> (1)..(534) <400> 144 act tcc aag ccc acc aca act ggg aag Thr Ser Lys Pro Thr Thr Thr Gly Lys 1 5 aaa tct ctg tea cca cag gag eta geg

Lys Ser Leu Ser Pro Gin Glu Leu Ala gcc ttg gaa gag tea etc aag ctg aaa Ala Leu Glu Glu Ser Leu Lys Leu Lys gtc ttc ccc ggg aat tgg gac ctg agg Val Phe Pro Gly Asn Trp Asp Leu Arg

5055 cct gtg gCc ttg gag get gag ctg gcc Pro Val Ala Leu Glu Ala Glu Leu Ala 70..

gcc get get ggc cca gcc ctg gag gac

Ala Ala Ala Gly Pro Ala Leu Glu Asp acc ctg cac cac ate etc tcc cag etc

Thr Leu His His lie Leu Ser Gin Leu

100 ‘ 1°5 ccc aca gca ggg ccc agg ccc egg ggc

Pro Thr Ala Gly Pro Arg Pro Arg Gly

115 120 .

egg etc cag gag gcc ccc aaa aag gag

Arg Leu Gin Glu Ala Pro Lys Lys Glu

130 . 135 Λ/ tct gtc acc ttc aac etc ttc ege etc

Ser Val Thr Phe־’Asn Leu Phe Arg Leu

145 150 gtg gcc gat ggg aac ctg dnn ctg aga

Val Ala Asp Gly , Asn Leu Xaa Leu Arg

I. :165;

acc tga . ’.

Thr. . * ׳' י יי . ׳ י׳ ' ggc tgc cac att ggc agg ttc 48 Gly Cys His He Gly Arg Phe.

10. . 15 .' .

age ttc aag aag gcc agg gac 96

Ser Phe Lys Lys Ala Arg Asp aac tgg agt tgc age tct cct 144

Asn Trp Ser Cys Ser Ser Pro ett etc cag gtg agg gag ege 1.92 .

Leu Leu Gin Val Arg Glu Arg ctg acg ctg aag gtc ctg gag 240.

Leu Thr Leu Lys Val Leu Glu . 80 gtc eta gac cag ccc ett cac 288'

Val Leu Asp Gin Pro Leu His 95 qag gcc tgt ate cag cct cag 336

Gin Ala Cys He Gin Pro Gin

110 ege etc cac cac tgg ctg cac 384

Arg Leu His His Trp Leu His • 125 tcc get ggc tgcctg gag gca 432

Ser Ala Gly Cys Leu Glu Ala

14° .'.

etc acg ega gac etc aaa tat 480 Leu Thr Arg Asp Leu Lys Tyr. .

155. I acg tea acc cac cct gag tee 528

Thr Ser Thr His Pro Glu Ser !70 ’ ;I75 .

534 ־ :.

145 <כ210>

<211> 177 .

<212> PRT <213> Artificial Sequence <223> IL-29 C167X, truncated after N-terminal

Methionine, Glycine, Proline, Valine, and

Proline <221> VARIANT <222> (167)..(167) <223> Xaa = Ser, Ala, Thr, Vai, or Asn

Pro Thr Thr Thr Gly Lys Gly Cys His He Gly «3

Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp

25Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser SerPro

Vai Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gin Vai Arg GluArg

RO 550 ם

Pro Vai Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Vai Leu Glu ,- 7075 .

Ala Ala Ala Gly Pro Ala Leu Glu Asp Vai Leu Asp Gin Pro Leu His 85 90.”

Thr Leu His His lie Leu Ser Gin Leu Gin Ala Cys He Gin Pro Gin

100 _ iJ-u

Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His

□12 120 5 ו ו

Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu A a

ט14 ' 135 חוו

Ser Vai Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys yr 150155

Vai Ala Asp Gly Asn Leu Xaa Leu Arg.Thr Ser Thr His Pro Glu Ser 165170

Thr ',... ....־.

<210> 146 <211> 531 <212> DNA!

<213> Artificial Sequence <223> IL-29 C166X, truncated after N-terminal

Methionine, Glycine, Proline, Valine, Proline, and

Threonine .' ., <221> variation <222> (497) . . . (498). .

<223> n = A, T, G, or C <221> CDS . י' ..'.. 'י ־ <222> (1) - . (531) ;'י <400> 146 ..'.י ' ' tcc aag ccc acc aca act ggg aag ggc tgc Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys tct ctg tea cca;cag gag eta gcg.agc ttc

Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe

25 . . 20 ־ cac att ggc agg ttc aaa.

His He Gly Arg Phe Lys aag aag gcc agg gac gcc

Lys Lys Ala Arg Asp Ala .

<210> 147 <211> 176 <212> PRT <213> Artificial Sequence <220>.

<223> IL-29 C166X, truncated after N-terminal

Methionine,‘ Glycine, Proline, Valine, Proline, and . Threonine <221> VARIANT <222> (166),. . . (166) <223> Xaa = Ser, Ala, Thr, Vai, or Asn.

<400> 147 ..

Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His lie Gly Arg Phe Lys.

15 '. 10 . 5 ׳. 1

Ser Leu Ser Pro Gin Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala 20 ' 25 . 30.

Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Vai 35 4045

Phe Pro Gly Asn Trp.Asp Leu Arg Leu Leu, Gin Vai Arg Glu Arg Pro

60 י; 55 ' 50 . .

Vai Ala Leu Glu,Ala Glu Leu Ala Leu Thr Leu Lys Vai Leu Glu Ala / 65 י. .. .,Λ -../<. .' 70 75 .;: ...80

Ala Ala Gly Pro־ Ala Leu 85

Leu His His lie;Leu Ser

10.0

Thr Ala Gly Pro Arg Pro 115

Leu Gin Glu Ala Pro Lys 130

Val Thr Phe Asn Leu Phe

145 .150

Ala Asp Gly Asn Leu Xaa 165

Glu Asp Val Leu Asp Gin 90

Gin Leu Gin Ala Cys lie

Arg Gly Arg Leu His His

Lys Glu Ser Ala Gly Cys

135 140

Arg Leu Leu Thr Arg Asp

Leu Arg Thr Ser Thr His

170 , pro Leu His Thr 95

Gin Pro Gin Pro

Trp Leu His Arg

Leu Glu Ala Ser

Leu Lys Tyr Val

Pro Glu Ser Thr <210> 148 <211> 528 <212> DNA <213> Artificial Sequence <220>

<223> IL-29 C165X, truncated after N-terminal Methionine, Glycine, Proline, Valine, Proline, Threonine, and Serine <221> variation.

<222> (494)..(495) <223> n = A, T, G, or C <221> CDS <222> (1) . . - (528)

acc ttc aac etc Thr Phe Asn Leu 145 gat ggg aac etg Asp Gly Asn Leu ttc ege etc etc

Phe Arg Leu Leu dnn etg aga acg Xaa Leu Arg Thr 165

1v*t acg ega gac etc

Thr Arg Asp Leu 155 tea acc cac cct Ser Thr His Pro 170.

aaa tat gtg gcc

Lys Tyr Val Ala 160 gag tee acc tga Glu Ser Thr * 175 <210> 149 <211> 175 <212> PRT <213> Artificial Sequence <220>

<223>

TL-29 C165X, truncated after N-terminal Methionine, Glycine, Proline, Valine, Proline, Threonine, and Serine <221> VARIANT <222> (165) . . .(165) <223> Xaa = Ser, Ala, Thr, Val, or Asn

Lys°Pro4Thr Thr Thr Gly Lys Gly Cys His He Gly

Leu Ser Pro Giri Glu Leu Ala Ser Phe Lys Lys Ala 20

Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser

3540

Pro Gly Asn Trp Asp Leu Arg Leu Leu Gin Val Arg

Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val 7075

Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gin Pro 8590

His His He Leu Ser Gin Leu Gin Ala Cys lie Gin .100,

Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp

Gin Glu Ala Pro Lys.Lys Glu Ser Ala Gly Cys Leu

130 135140

Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu

145 150

Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro

165 . .. 170

Arg

Ser 45 Glu

Leu

Pro

Leu 125 Glu

Lys

Glu

Phe Lys Ser

Asp Ala Leu

Pro Val Phe

Arg Pro Val

Glu Ala Ala

His Thr Leu

Gin Pro Thr

His Arg Leu

Ala Ser Val

Tyr Val Ala

Ser Thr <210> 150 <211> 552 <212> DNA <213> Artificial Sequence <223> il-29 Leu insert'after N-terminal Met, C173X <221> variation .,.:i <222> (518)..(519) . .

<223> n.= A, T, G, or C :־; .י <221> CDS ;..''י..’. '.. ’ ..׳ .<:.;;'.

<222> (1)..(552) \ .1L.... : .:, ; .

>3/1υ.+ <400> 150

<210> 151 <211>.183 <212> PRT <213> Artificial Sequence <220>

<223> IL-29 Leu insert.after N-terminal Met, C173Xי <221> VARIANT <222> (173) . . .(173) .

<223> Xaa = Ser,Ala, Thr, Val, or. Asn י'׳.,'' ׳’ <400> 151 : . ' ‘-:.י.'. יי

Met Leu Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly

.. 5. 10 . 15

Cys His He Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala Ser

174797/ϊ

2530

Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn 35 4045

Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu 50 55 60.

Leu Gin Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu AlaLeu

65: 70 7580

Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu AspVal

9095

Leu Asp Gin Pro Leu His Thr Leu His His He Leu Ser Gin Leu Gin 100 1°5

Ala Cys He Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg 115 120125

Leu His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser 130 4.35440

Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg LeuLeu

145 150 155160

Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu ArgThr

165 170175

Ser Thr His Pro Glu Ser Thr. 180 <210> 152 <211> 549 <212> DNA <213> Artificial Sequence <220>

<223> IL-29 G2L C172X <221> variation <222> (515). . .(516) <223> n = A, T, G, or C <221> CDS <222> (1)..(549) : .

<400> 152 atg ytn cct gtc ccc act tcc aag ccc acc aca act ggg aag ggc tgc48 .

Met Leu Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys GlyCys

5 1° 15. .

cac att ggc agg ttc aaa tet etg tea cca cag gag eta geg age ttc96

His He Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala SerPhe

2530 aag aag gcc agg gac gcc ttg gaa gag tea etc aag etg aaa aac tgg144

Lvs Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys AsnTrp

4045 agt tgc age tet cct gtc ttc ccc ggg aat tgg gac etg agg ett etc192

Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg LeuLeu

55 . . . 60 .

cag gtg agg gag ege cct gtg gcc ttg gag get gag etg gcc etg acg240

Gin Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr. .

. 65 70 75 / . . 80 .-.

ctq aag gtc etg gag gcc׳get get ggc cca gcc etg gag gac gtc eta288

Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp ValLeu

־.- י' . 95 ;י. . 90. .. . ' 85 . .

gac cag ccc ett cac acc etg. cac'cac ate etc tcc cag etc cag gcc336.

Asn Gin Pro Leu His Thr Leu His His He Leu Ser Gin Leu Gin Ala. ״ . . , ./. ., 100 :. / /',< 105 J.//. 110::;.;.,.

tgt ate cag cct cag ccc aca gca ggg ccc agg ccc egg ggc cgc etc384

Cys lie Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly ArgLeu

115 120125 cac cac tgg etg cac egg etc cag gag gcc ccc aaa aag gag tee get432

His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu SerAla

130 135140 ggc tgc etg gag gca tct gtc acc ttc aac etc ttc cgc etc etc acg480

Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu LeuThr

145 150 155160 ega gac etc aaa tat,gtg gcc gat ggg aac etg dnn etg aga acg tea528

Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser

165 1 170175

<td> <213></td><td> Artificial Sequence</td>

<220>

<223> IL-29 G2L C172X <221> VARIANT <222> (172)..(172) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 153

־ 154 <210> .

' <211> 552 .

<212> DNA

- . <213> Artificial) Sequence <223> IL-29 lie insert after N-terminal Met, C173X <221> variation <222> (518)..(519) <223> n = A, T, G,. or C <221> CDS <222>.(1)..(552) <400> 154

<210> 155 <211> 183 ;

<212> PRT <213> Artificial Sequence.

<220>

<223> IL-29 He insert after N-terminal Met, C173X <221> VARIANT <222> (173)..(173) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 155

Ser Thr His Pro Glu:Ser Thr <210> 156 <211> 549 <212> DNA <213> Artificial Sequence <220>

<223> IL-29 G21 C172X <221> variation <222> (515)..(516) <223> n = A, T, G, or C <221> CDS..

. <222> (1) . . - (549) .;.

<400> 156 atg ath cct gtc ccc act tcc aag ccc acc aca act ggg aag ggc tgc48

Met lie Pro Val Pro. Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys.'

5 1015 cac att ggc agg ttc aaa tct ctg tea cca cag gag. eta geg age ttc96

His He Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala SerPhe

25 30' . aag aag gcc agg gac gcc ttg gaa gag tea etc aag ctg aaa aac tgg144

Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp ; . : . 4045 agt tgc age tct cct gtc ttc ccc ggg aat tgg gac ctg agg ett etc192

Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg LeuLeu . 50 7 5560 , cag gtg agg gag cgc. cct gtg gcc ttg gag get gagctg gcc׳ ctg acg . 240

acc cac cct gag tcc acc tga Thr His Pro Glu Ser Thr * 180 <210> 157 <211> 182 <212> PRT <213> Artificial Sequence <220>

<223> IL-29 G2I C172X <221> VARIANT <222> (172)..(172) <223> Xaa = Ser, Ala, Thr,. Val, or Asn.

<400> 157

Met He Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys GlyCys .1 5 10 15 .

His lie Gly Arg Phe Lys Ser Leu Ser Pro Gin Glu Leu Ala SerPhe

2530

Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35. 4045

Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 5560

Gin Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu . Thr

80 . . . 75 ׳: .70 .'־65

Leu Lys Val Leu Glu Ala Ala 'Ala Gly Pro Ala Leu Glu Asp Val Leu 85 9095

Asp Gin Pro Leu His Thr Leu His His He Leu Ser Gin Leu Gin Ala 100 105 .:HO

Cys He Gin Pro Gin Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120 . . 125

His His Trp Leu His Arg Leu Gin Glu Ala Pro Lys Lys Glu Ser Ala 130 135 .140 .

Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu. Thr

160 155 150 ן. . 145

'.Arg Asp Leu .Lys Tyr Val Ala Asp Gly'Asn Leu Xaa Leu. Arg Thr Ser

165 170

Thr- His Pro Glu ;Ser Thr 180 : <210> 158 <211> 531 <212> DNA <213> Artificial Sequence <220>

<223> IL-29 after N-terminal Met amino acid residues 2-7 deleted, C166X <221> variation <222> (497)..(498) <223> n = A, T, G, or C <221> CDS <222> (1)..(531) <400> 158

175 170 .. י. 165 tga * <210> 159 <211> 176 <212> PRT <213> Artificial Sequence <220> . . .

<223> IL-29 after N-terminal Met amino acid residues 2-7 deleted, C166X <221> VARIANT <222> (166)..(166)

<210> 160 <211> 558 <212> DNA <213> Artificial Sequence <220> ' <223> IL-29 Glu, Ala, and Glu inserted after N-terminal

Met, C175X <221> variation <222> (524) . . . (525) <223> n = A, T, G, or C <221> CDS .

<222> (1) . . . (558).

<400> 160 .

.234

IVJ/lVf : 25 30

180 185 <210> 161 <211> 185 <212> PRT <213> Artificial Sequence <220> .

<223> IL-29 Glu, Ala, and Glu inserted after N-terminal

Met, C175X .

<221> VARIANT ' <222> (175)..(175) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 161

Ala

His

Ala

Gin

Thr 155 Asp ct

Ala Leu Thr Leu Lys Vai Leu Glu Ala Ala 85

Asp Vai Leu Asp Gin Pro Leu His Thr Leu

Leu Gin 'Ala ^s lie Gin Pro Gin Pro Thr

ט115

Gly ArgLeu His His Trp Leu His Arg Leu Glu Ser Ala Gly Cys Leu Glu Ala Ser Vai ttu Leu Thr Arg Asp Leu Lys Tyr Vai Ala Arg Thr Ser Thr His Pro Glu Ser Thr

כט1 180

BU

Gly Pro Ala Leu Glu 55

His He Leu Ser Gin

Gly Pro Arg Pro Arg

Glu Ala Pro Lys Lys

Phe Asn Leu Phe Arg

Gly Asn Leu Xaa Leu 175

Contents31

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Priority claims
Document	Office	Kind	Date
2004025864	United States of America	W	2004-08-09
2004025864	United States of America	W	2004-08-09
PCTUS2004025864	–	–	–
WO2004US25864	–	–	–

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US7157559B2	United States of America	B2	2007-01-02
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Homogeneous preparation of il-29 polypeptide and method for producing such

Abstract

Term

BACKGROUND OF THE INVENTION

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

PCI7US2004/025864

EXAMPLES

PCT7US2004/025864

PCI7US2004/025864

PCI7US2004/025864

PCT7US2004/025864

PCT7US2004/025864

PCT7US2004/025864

PCT7US2004/025864

PCI7US2004/025864

PCI7US2004/025864

PCI7US2004/025864

PCI7US2004/025864

PCI7US2004/025864

PCT7US2004/025864

SIGNAL (1) . - - (25)

HO115

DNA

130 135I

165 170I

145 150I

145I

105.-:.HO:

DNA

105HO

IL-29 C168X,

I. :165;

BU

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