CA2534907A1 - Homogeneous preparations of il-28 and il-29 - Google Patents

Abstract

Homogeneous preparations of IL-28A, IL-28B, and IL-29 have been produced by mutating one or more of the cysteine residues in the polynucleotide sequences encoding the mature proteins. The cysteine mutant proteins can be shown to either bind to their cognate receptor or exhibit biological activity. One type of biological activity that is shown is an antiviral activity.

Description

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-(3 (IFN-(3), l0 interferon-y (IFN-y), IL-10, IL-19 (U.S. Patent 5,985,614), MDA-7 (Jiang et al., Onco~ene 11, 2477-2486, (1995)), IL-20 (Jiang et al., Onco~ene 11, 2477-2486, (1995)), IL-22 (Xie et al., J. Biol. Chem. 275, 31335-31339, (2000)), and AK-(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-ocRl (IFN-otR1), interferon-y-R2 (IFN-~y-R2), interferon-Y R1 (IEN-y R1), 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-20R(3 (Blumberg et al., Cell 104, 9-19, (2001)) (also known as zcytor7 (U.S.
Patent 5,945,511) and CRF2-8 (Kotenko et al., Onco ene 19, 2557-2565, (2000)), IL-20R/3 (Blumberg et al., ibid, (2001)) (also known as DIRS1 (PCT WO 99/46379)), IL-22RA1 (IL-22 receptor-al, submitted to HUGO for approval) (also known as IL-(Xie et al., J. Biol. Chem. 275, 31335-31339, (2000)), zcytorl l (U.S. Patent 5,965,704) and CRF2-9 (Kotenko et al., Onco-gene 19, 2557-2565, (2000)), and tissue factor.
Class II cytokine receptors are typically heterodimers composed of two distinct receptor chains, the a and (3 receptor subunits (Stahl et al., Cell 74, 587-590, (1993)). In general, the a subunits are the primary cytokine binding proteins, and the (3 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 cytokine-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 (Fn111) domains of approximately 100 amino acids each (Bazan J.F. Proc. Natl. Acad.
Sci.
USA 87, 6934-6938, (1990); Thoreau et al., FEBS Lett. 282, 16-31, (1991)).
Each FnI>I
domain contains conserved Cys, Pro, and Trp residues that determine a characteristic folding pattern of seven (3-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 II 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 to produce many cytokines and adaptive immunity to antigens. Cytokines produced by the T cell have been classified as type 1 and type 2 (I~elso, A. Trmmun. Cell Biol. 76:300-317, 1998). Type 1 cytokines include IL-2, interferon-gamma (IFN-y), LT-a, and are involved in inflammatory responses, viral immunity, intracellular parasite immunity and allograft rejection. Type 2 cytokines include 1L-4, IL-5, IL-6, IL-10 and IL-13, and 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 (reviews on interferons are provided by De Maeyer and De Maeyer-Guignard, "Interferons," in The Cytokine HafZdbook, 3rd Edition, Thompson (ed.), pages (Academic Press Ltd. 1998), and by Walsh, Biopharmaceuticals: Biochemistry afzd 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, 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 I" IFNs include IFN-a, IFN-(3,1FN-cn, IFN-~, and interferon-i.
Currently, IFN-y and one subclass of IFN-a are the only type II IFNs.
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 cc-chain of the human IFN-ocJ(3 receptor comprises an extracellular N-terminal domain, which has the characteristics of a class 1I cytokine receptor. IFN-'y does not share significant homology with the type I IFN or with the type II IFN-cc 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 1o myelogenous leukemia, non-Hodgkin's lymphoma, laryngeal papillomatosis, mycosis fungoides, condyloma acuminata, 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-(3 to treat multiple sclerosis, a chronic disease of the. nervous system. IFN-~ 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-y 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 2o IL-10. This new family is fully described in co-owned PCT application WO

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 021086087 and Sheppard et al., supra.) 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 3o molecules having a single intrarnolecular 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-28 and IL-29.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
In the description that follows, a number of terms are used extensively.
The following definitions are provided to facilitate understanding of the invention.
Unless otherwise specified, "a," "an," "the," and "at least one" are used 1o 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 En~~mol. 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
~2:7952-4, 1985), substance P, FIagTM peptide (Hope et al., Biotechnolo~y 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 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.
3o The terms "amino-terminal" and "carboxyl-terminal" are used herein to denote positions within polypeptides. Where the context allows, these terms are used S
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-covalently associated, stable pair under appropriate conditions.
For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair. ~ther exemplary complement/anti-complement l0 pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of <109 M-1.
The term "degenerate nucleotide sequence" denotes a sequence of 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, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked 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 replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
The term "isolated", when applied 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 genetically engineered protein production systems. Such isolated molecules are those 3o 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 in the level of viral infection and includes, but is not limited to, a change in the level of CTLs or NIA cells (as described above), a decrease in viral load, an increase antiviral antibody titer, decrease in serological levels of alanine aminotransferase, or 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 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 the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.

"Paralogs" are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, cc-globin, (3-globin, and myoglobin are paralogs of each other.
A "polynucleotide" is a single- or double-stranded polymer of 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 ifa vitro, or prepared from a combination of natural and synthetic molecules.
Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt"), or l~ilobases ("kb"). Where the context allows, the latter two terms may describe to 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 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 2o 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 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.
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 domain and other molecules) 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 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, 1L-3 receptor, GM-CSF receptor, G-CSF
receptor, erythropoietin receptor and IL-6 receptor).
The term "secretory signal sequence" denotes a DNA sequence that 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 in 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 2o 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 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%.
"zcyto20", "zcyto2l", "zcyto22," are the previous designations for human IL-28A, human IL-29, and human IL-28B, respectively. The nucleotide and amino acid sequence for 1L-28A are shown in SEQ DJ NO:1 and SEQ ~ N0:2, respectively. The nucleotide and amino acid sequences for IL-29 are shown in SEQ ~
N0:3 and SEQ m N0:4, respectively. The nucleotide and amino acid sequence for IL-28B are shown in SEQ m N0:5 and SEQ m N0:6, respectively. These sequences are fully described in PCT application WO 02/086087 commonly assigned to ZymoGenetics, Inc., incorporated herein by reference.
"zcyto24" and "zcyto25" are the previous designations for mouse IL-28, and are shown in SEQ m NOs: 7, 8, 9, 10, respectively. The polynucleotide and polypeptides are fully described in PCT application WO 02/086087 commonly assigned l0 to ZymoGenetics, Inc., incorporated herein by reference.
"zcytorl9" is the previous designation for IL-28 receptor a-subunit, and is shown in SEQ m 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-28 and IL-29 that result in expression of a recombinant IL-28 or IL-29 preparation that is a homogeneous preparation. For the purposes of this invention, a homogeneous preparation of and 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-28 or IL-29, as described herein. For example, the molecules of the present invention can bind to the IL-28 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-28 or 1L-29 Cysteine mutants is present when the molecules are capable of some level of antiviral activity associated with wildtype IL-28 or 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 1L-28A was designated zcyto20 (SEQ m NOs:l and 2), IL-29 was 5 designated zcyto2l (SEQ m NOs:3 and 4), and IL-28B was designated zcyto22 (SEQ
ID NOs:5 and 6). (See, PCT application WO 021086087 and Sheppard et al., supra.) The mouse orthologs for IL-28 were previously designated as zcyto24 (SEQ ~
NOs:7 and 8), zcyto25 (SEQ ID NOs:9 and 10).
Wildtype IL-28A gene encodes a polypeptide of 200 amino acids, as 10 shown in SEQ 117 N0:2. 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 N0:2. The mature peptide for 1L-28A begins at amino acid residue 1 (Val) 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 (Thr) to 65 (Gln);
helix C by amino acid residues 69 (Arg) to 86 (Ala); helix D by amino acid residues 95 (Val) to 114 (Ala); helix E by amino acid residues 126 (Thr) to 142 (Lys); and helix F
by amino acid residues 148 (Cys) to 169 (Ala); as shown in SEQ ID NO: 2.
Wildtype IL-29 gene encodes a polypeptide of 200 amino acids, as shown in SEQ ll~ N0:4. The signal sequence for IL-29 can be predicted as comprising amino acid residue -19 (Met) through amino acid residue -1 (Ala) of SEQ m N0:4, SEQ ~ N0:119, or SEQ ID N0:121. The mature peptide for IL-29 begins at amino acid residue 1 (Gly) of SEQ m N0:4. 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 (Val);
helix C by amino acid residues 70 (Val) to 85 (Ala); helix D by amino acid residues 92 (Glu) to 111 (Gln); helix E by amino acid residues 118 (Thr) to 139 (Lys); and helix F
by amino acid residues 144 (Gly) to 170 (Leu); as shown in SEQ ID N0:4.
Wildtype IL-28B gene encodes a polypeptide of 200 amino acids, as shown in SEQ )D N0:6. The signal sequence for IL-28B can be predicted as 3o comprising amino acid residue -21 (Met) through amino acid residue -1 (Ala) of SEQ
m N0:6. The mature peptide for IL-28B begins at amino acid residue 1 (Val) of SEQ

DJ N0:6. 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 (Thr) to 65 (Gln);
helix C by amino acid residues 69 (Arg) to 86 (Ala); helix D by amino acid residues 95 (Gly) to 114 (Ala); helix E by amino acid residues 126 (Thr) to 142 (Lys); and helix F
by amino acid residues 148 (Cys) to 169 (Ala); as shown in SEQ m N0:6.
The present invention provides mutations in the IL-28 and IL-29 wildtype sequences as shown in SEQ >D NOs: 1, 2, 3, 4, 5, and 6, that result in expression of single forms of the IL-28 or IL-29 molecule. Because the heterogeneity of forms is believed to be a result of multiple intramolecular disulfide bonding patterns, to specific embodiments of the present invention includes mutations to the cysteine residues within the wildtype IL-28 and IL-29 sequences. When IL-28 and IL-29 are expressed in E. coli, an N-terminal or amino-terminal Methionine is present.

NOs:12-17, for example, show the nucleotide and amino acid residue numbering for 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-28B, and IL-29.

Table 1 ~.-2gA 016- 048- 050- C1G7- 016- 016- 048- 050- C115-SEQ ~ 0115 0148 0148 0174 ~48 050 c115 0115 0148 N0:2 Met ~- 017- 049- 051- 0168- 017- 017- 049- 051- ~-116-28A 0116 0149 01498 0175 049 051 0116 ~11G 0149 SEQ m NO:13 SEQ ~ 0112 X145 171 N0:4 Met Il.-C1G- 050- X113-SEQ m NO:15 11..-28BC1G- C4g- C5p- 0167- 016- 016- C4g- 050- C115-SEQ ~ 0115 0148 0148 0174 048 050 X115 0115 0148 NO:6 Met ~..-017- 049- 051- 168- X17' 017- X49- X51- ~11G-28B 0116 0149 0149 X175 049 051 0116 ~11G 0149 SEQ m NO:17 The polynucleotide and polypeptide molecules of the present invention have a mutation at one or more of the Cysteines present in the wildtype IL-28A, IL-29 or IL-28B molecules, yet retain some biological activity as described herein.
Table 2 illustrates exemplary Cysteine mutants, in particular point mutations of cysteine (C) to serine (S).

Tahl P ?.
1L-28A C48S SEQ JD NO:19 Met II,-28A C49S SEQ m N0:21 IL-28A C50S SEQ m N0:23 Met IL-28A C51S SEQ m NO:25 IL-29 C 171 S SEQ m N0:27 Met IL-29 C172S SEQ m N0:29 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 02144209, incorporated by reference herein; Sheppard et al., supra; I~otenko et al., Nature Immunol. 4:69-77, 2003; and PCT
WO/031040345. 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 II cytol~ine receptors include zcytorll (commonly owned US Patent No. 5,965,704), CRF2-4 (Genbank Accession No. 217227), IL-10R
(Genbank Accession No.s U00672 and NM_001558), DIRS1, zcytor7 (commonly owned US Patent No. 5,945,511), and tissue factor. IL-28 receptor, like all known class II receptors except interferon-alphalbeta 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-residue helices, and include IL-6, ciliary neutrotrophic factor (CNTF), leukemia 2o inhibitory factor (LIF) and human growth hormone (hGIT). "Short-helix" form cytokines generally consist of between 18-21 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 lL-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 1o INF-a,1L-10, human growth hormone.
The present invention provides polynucleotide molecules, including DNA and RNA molecules, that encode, for example, Cysteine mutant IL-28 or IL-polypeptides. For example, the present invention provides degenerate nucleotide sequences encoding IL-28A C48S, Met IL-28A C49S, IL-28A C50S, Met IL-28A
C51S, IL-29 C171S and Met IL-29 C172S polypeptides disclosed herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic 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 1L-28A C48S, Met IL-28A C49S, IL-28A C50S, Met IL-28A C51S, IL-29 C171S and Met 1L-29 C172S, respectively. Those skilled in the art will recognize that the degenerate sequence of SEQ m NOs: 30, 31, 32, 33, 34, and 35 also provides all RNA sequences encoding SEQ ID NOs: 30, 31, 32, 33, 34, and 35 by substituting U for T and are thus comtemplated by the present invention.
IL-28A polypeptides of the present invention 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 N0:2 is the cysteine at amino acid position 48, or position 49 (additional N-terminal Met) if expressed in E
coli (see, for example, SEQ )D N0:13). 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, 3o valine, or asparagine. IL-28A C2 mutant molecules of the present invention include, for example, polynucleotide molecules as shown in- SEQ >D NOs:20 and 22, including DNA and RNA molecules, that encode IL-28A C2 mutant polypeptides as shown in SEQ m NOs:21 and 23, respectively. SEQ m NOs:36 and 37 are additional 1L-28A
C2 polypeptides of the present invention.
In addition to the IL-28A C2 mutants, the present invention also includes 5 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 N0:2, is the cysteine at position 50, or position 51 (additional N-terminal Met) if expressed in E. coLi (see, for example, SEQ II7 NO:13). IL-mutant molecules of the present invention include, for example, polynucleotide 10 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 m NOs:25 and 27, respectively. SEQ ID NOs:38 and 39 are additional IL-28A C3 polypeptides of the present invention.
The IL-28A polypeptides of the present invention include, for example, 15 SEQ >D NOs: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 IL-28A polypeptides of the present invention include, for example, SEQ ID NOs:36, 37, 38, and 39.
IL-28B polypeptides of the present invention also include a mutation at 2o the second cysteine, C2, of the mature polypeptide. For example, C2 from the N-terminus or amino-terminus of the polypeptide of SEQ >D N0:6 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 N0:17). This second cysteine (of which there are seven, like IL-28A) or C2 of 1L-28B can be mutated, for example, to a serine, alanine, threonine, valine, or asparagine. 1L-28B C2 mutant molecules of the present invention include, for example, polynucleotide molecules as shown in SEQ )D NOs:122 and 124, including DNA and RNA molecules, that encode IL-28B C2 mutant polypeptides as shown in SEQ ID NOs:123 and 125, respectively. Additional IL-28B C2 mutant molecules of the present invention include polynucleotide molecules as shown in SEQ ll~
NOs:130 3o and 132 including DNA and RNA molecules, that encode IL-28B C2 mutant polypeptides as shown in SEQ ~ NOs:l31 and 133, respectively (PCT publication WO
03/066002 (I~otenko et al.)).
In addition to the IL-28B C2 mutants, the present invention also includes 1L-28B 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 m N0:6, is the cysteine at position 50, or position 51 (additional N-terminal Met) if expressed in E. coli (see, for example, SEQ m N0:17). 1L-mutant molecules of the present invention include, for example, polynucleotide molecules as shown in SEQ ~ NOs:126 and 128, including DNA and RNA molecules, to that encode IL-28B C3 mutant polypeptides as shown in SEQ m NOs:l27 and 129, respectively. Additional IL-28B C3 mutant molecules of the present invention include polynucleotide molecules as shown in SEQ m NOs:134 and 136 including DNA and RNA molecules, that encode IL-28B C3 mutant polypeptides as shown in SEQ m NOs:135 and 137, respectively (PCT publication WO 03/066002 (Kotenko et al.)).
i5 The IL-28B polypeptides of the present invention include, for example, SEQ m NOs:6, 17, 123, 125, 127, 129, 131, 133, 135, and 137, which are encoded by IL-28B polynucleotide molecules as shown in SEQ ~ NOs:S, 16, 122, 124, 126, 128, 130, 132, 134, and 136, respectively.
IL-29 polypeptides of the present invention also include, for example, a 20 mutation at the fifth cysteine, C5, of the mature polypeptide. For example, C5 from the N-terminus of the polypeptide of SEQ m N0:4, is the cysteine at position 171, or position 172 (additional N-terminal Met) if expressed in E. coli. (see, for example, SEQ
m N0:15). 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 25 disulfide bond pattern of C1(Cysl5 of SEQ m N0:4)/C3(Cys112 of SEQ m N0:4) and C2(Cys49 of SEQ m N0:4)/C4(Cys 145 of SEQ m N0:4). Additional IL-29 C5 mutant molecules of the present invention include polynucleotide molecules as shown in SEQ m NOs:26, 28, 82, 84, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, and 160, including DNA and RNA molecules, that encode IL-29 C5 mutant 30 polypeptides as shown in SEQ m NOs:27, 29, 83, 85, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161, respectively. Additional IL-29 C5 mutant molecules of the present invention include polynucleotide molecules as shown in SEQ ~
NOs:86, 88, 94, and 96, including DNA and RNA molecules, that encode IL-29 C5 mutant polypeptides as shown in SEQ m NOs:87, 89, 95, and 97, respectively (PCT
publication WO 03/066002 (Kotenko et al.)). Additional, IL-29 C5 mutant molecules of the present invention include polynucleotide molecules as shown in SEQ m NOs:102, 104, 110, and 112, including DNA and RNA molecules, that encode IL-29 C5 mutant polypeptides as shown in SEQ m NOs:103, 105, 111, and 113, respectively (PCT publication WO 02/092762 (Baum et al.)).
In addition to the 1L-29 C5 mutants, the present invention also includes to IL-29 polypeptides comprising a mutation at the first cysteine position, C1, of the mature polypeptide. For example, C1 from the N-terminus of the polypeptide of SEQ
)D N0:4, is the cysteine at position 15, or position 16 (additional N-terminal Met) if expressed in E. coli (see, for example, SEQ m N0:15). These IL-29 C1 mutant polypeptides will thus have a predicted disulfide bond pattern of C2(Cys49 of SEQ m N0:4)/C4(Cys145 of SEQ m NO:4) and C3(Cys112 of SEQ m N0:4)/C5(Cys171 of SEQ m N0:4). Additional IL-29 C1 mutant molecules of the present invention include polynucleotide molecules as shown in SEQ ~ NOs:74, 76, 78, and 80, including DNA
and RNA molecules, that encode lL-29 C1 mutant polypeptides as shown in SEQ m NOs:75, 77, 79 and 81, respectively. Additional IL-29 C1 mutant molecules of the 2o present invention include polynucleotide molecules as shown in SEQ m NOs:90, 92, 98, and 100, including DNA and RNA molecules, that encode IL-29 C1 mutant polypeptides as shown in SEQ m NOs:9l, 93, 99, and 101, respectively (PCT
publication WO 03/066002 (Kotenko et al.)). Additional, IL-29 C1 mutant molecules of the present invention include polynucleotide molecules as shown in SEQ ~
NOs:106, 108, 114, and 116, including DNA and RNA molecules, that encode IL-29 C 1 mutant polypeptides as shown in SEQ ID NOs:107, 109, 115, and 117, respectively (PCT publication WO 02/092762 (Baum et al.)).
The IL-29 polypeptides of the present invention, for example, SEQ m NOs:4, 15, 27, 29, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161, which are encoded by IL-29 polynucleotide molecules as shown in SEQ ID
NOs:3, 14, 26, 28, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, and 160, may further include a signal sequence as shown in SEQ ID N0:119 or a signal sequence as shown in SEQ m N0:121. In addition, the present invention also includes the polypeptides as shown in SEQ m NOs:40 and 41. A polynucleotide molecule encoding the signal sequence polypeptide of SEQ ID N0:119 is shown as SEQ ID N0:118. A
polynucleotide molecule encoding the signal sequence polypeptide of SEQ ~
N0:120 is shown as SEQ )D N0:121.
Within one aspect the present invention provides an isolated polypeptide to comprising a sequence having at least 90 percent or 95 percent sequence identity to a sequence of amino acid residues selected from the group consisting of SEQ ID
NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161. The polypeptide may optionally comprise at least 15, at least 30, at least 45, or at least 60 sequential amino acids of an amino acid sequence as shown in SEQ ID
NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161. In another embodiment, the isolated polypeptide is an amino acid residues are selected from the group consisting of SEQ ll~ NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161. The polypeptide may have a conservative amino acid change, compared with the amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161.
Within a another aspect the present invention provides a fusion protein comprising a polypeptide that comprises a sequence of amino acid residues selected from the group consisting of SEQ If7 NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161; and a polyalkyl oxide moiety.
The polyalkyl 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:
Within a another aspect the present invention provides a fusion protein 1o comprising a first polypeptide and a second polypeptide joined by a peptide bond, wherein the first polypeptide comprises a sequence of amino acid residues selected from the group consisting of SEQ m NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161; and a second polypeptide. The second polypeptide may optionally be an antibody fragment. The antibody fragment may optionally be F(ab'), F(ab), Fab', Fab, Fv, scFv, and/or minimal recognition unit.
The second polypeptide may optionally be human albumin. The second polypeptide may optionally be a polypeptide selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores.
Table 3 sets forth the one-letter codes used within SEQ m 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.

Table 3 Nucleoti Resolutio Compleme Resolutio de n nt n A A T T

C C G G

G G C C

T T A A

R AIG Y CIT

Y C~T R AIG

M A~C I~ GIT

I~ G~T M AIC

S CIG S CIG

W AIT W A~T

H AICIT D AIGIT

B CIG~T V AIC~G

V A~CIG B C~GIT

D AIGIT H AICIT

N A~CIG~T N AIC~G~T

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

Table 4 One Amino Letter Codons Degenerate Acid Code Codon Cys C TGC TGT TGY

Ser S AGC AGT TCA TCC TCG TCT WSN

Thr T ACA ACC ACG ACT ACN

Pro P CCA CCC CCG CCT CCN

Ala A GCA GCC GCG GCT GCN

Gly G GGA GGC GGG GGT GGN

Asn N AAC AAT ~Y

Asp D GAC GAT GAY

Glu E GAA GAG GAR

Gln Q CAA CAG CAR

His H CAC CAT CAY

Arg R AGA AGG CGA CGC CGG CGT MGN

Lys K AAA AAG AAR

Met M ATG ATG

Ile I ATA ATC ATT ATH

Leu L CTA CTC CTG CTT TTA TTG YTN

Val V GTA GTC GTG GTT GTN

Phe F TTC TTT TTY

Tyr Y TAC TAT TAY

Trp W TGG TGG

Ter . TAA TAG TGA TRR

Asn I Asp B ~Y

GluIGln Z SAR

~Y K

One of ordinary skill will appreciate that some in the art ambiguity is introduced , representative of all in determining possible codons a degenerate codon encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, and the degenerate codon encode for arginine arginine (AGR), (MGN) can, in some circumstances, ne (AGY). A similar relationship encode seri exists between leucine. Thus, some polynucleotides codons encoding phenylalanine and 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 the amino acid sequence, for example, of SEQ ~ NOs:l9, 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 colon 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 1o Res. 14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As used herein, the term "preferential colon usage" or "preferential colons" is a term of art referring to protein translation colons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible colons encoding each amino acid (See Table 4). For example, the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used colon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr colons may be preferential. Preferential colons 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 colon 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 colon 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 colons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
Within another aspect, the present invention provides an isolated polynucleotide selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, and 160.

Within another aspect, the present invention provides an isolated polynucleotide capable of hybridizing to a sequence selected from the group consisting of SEQ m NOs:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, and 160, or a complement thereof, under hybridization conditions of 50% formamide, SxSSC (lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution (100x Denhardt's solution: 2% (w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin, 10%
dextran to sulfate, and 20 mg/ml denatured, sheared salmon sperm DNA at about 42°C to about 70°C, wherein the isolated polynucleotide encodes a polypeptide having antiviral activity. Optionally, the encoded polypeptide has antiviral activity to hepatitis B and/or hepatitis C. Optionally, the isolated polynucleotide may encode at least a portion ;of a sequence selected from the group of SEQ m NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21; 23~
25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85~ 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161. The isolated polynucleotide may encode a polypeptide represented by SEQ m NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, or 161.
In another aspect, the present invention provides an isolated polynucleotide encoding a polypeptide wherein the encoded polypeptide is selected from the group consisting of SEQ m NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161.
In another aspect, the present invention provides an isolated polynucleotide encoding a polypeptide wherein the encoded polypeptide has at least 90 percent or 95 percent sequence identity to a sequence selected from the group consisting of SEQ m NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161, wherein the encoded polypeptide has antiviral activity.
Optionally, the encoded polypeptide has antiviral activity to hepatitis B
andlor hepatitis C.
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 to by Northern blotting (Thomas, Proc. Natl. Acad. Sci. 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. Sci. 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 1L-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 3o example, SEQ m 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 residue 169 (Asn) as shown in SEQ m 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 5 individuals according to standard procedures. Allelic variants of the DNA
sequence shown in SEQ m NO:1, 3 and 5, including those containing silent mutations and those 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 m NOs:2, 4, and 6. cDNAs generated from alternatively 10 spliced mRNAs, which retain the properties of Cysteine mutant IL-28 or IL-polypeptides, are included within the scope of the present invention, as are polypeptides 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 15 to the polynucleotides encoding cysteines or cysteine residues can be introduced as described herein.
Within embodiments of the invention, 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 m 2o NOs:l8, 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 to nucleic acid molecules having a nucleotide sequence complementary to SEQ m NOs:l8, 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, 25 116, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, and 160. In general, stringent conditions are 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.
3o A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA, can hybridize if the nucleotide sequences have some degree of complementarity. 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 that will 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 T~, for a specific target to sequence is the temperature (under defined conditions) at which 50% of the target sequence will 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 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 Cloning: A Laboratory_Manual, Second Edition (Cold Spring Harbor Press 1989);
Ausubel et al., (eds.), Current Protocols in Molecular Biolo~y (John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to Molecular Cloning Technigues, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. X6:227 (1990)). Sequence analysis software such as ~LIGO 6.0 (LSR; Long Lake, MN) and Prifrzer 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 defined conditions and 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 carried 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.
3o Following hybridization, the nucleic acid molecules can be washed to remove non-hybridized nucleic acid molecules under stringent conditions, or under 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-polypeptides hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NOs:l8, 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 l0 0.1% 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 sequence of SEQ m NOs:l8, 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 2o complement) under highly stringent washing conditions, in which the wash stringency is equivalent to 0.1x - 0.2x SSC with 0.1% SDS at 50 - 65°C, including O.lx SSC with 0.1% SDS at 50°C, or 0.2x SSC with 0.1% SDS at 65°C:
The present invention also provides isolated IL-28 or IL-29 polypeptides that have a substantially similar sequence identity to the polypeptides of the present invention, for example SEQ m NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 or 161. The term "substantially similar sequence identity" is used herein to denote polypeptides comprising at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to the sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 or 161, or their orthologs. The present invention also includes polypeptides that comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to a polypeptide or fragment thereof of the present invention. The present invention further includes polynucleotides that encode such polypeptides. The IL-28 and polypeptides of the present invention are preferably recombinant polypeptides.
In another aspect, the IL-28 and IL-29 polypeptides of the present invention have at least 15, at least 30, at least 45, or at least 60 sequential amino acids. For example, an IL-28 or IL-29 polypeptide of the present invention relates to a polypeptide having a~ least 15, at least 30, at least 45, or at least 60 sequential amino acids from SEQ ~
NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 or 161.
Methods for determining percent identity are described below.
The present invention also contemplates variant nucleic acid molecules that can be identified using two criteria: a determination of the similarity between the encoded polypeptide with the amino acid sequence of SEQ ~ NOs:l9, 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 or 161 respectively, and/or a hybridization assay, as described above. Such variants include nucleic acid molecules: (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ
>D
NOs:lB, 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; or (2) that encode a polypeptide having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99%
sequence identity to the amino acid sequence of SEQ )D NOs:l9, 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 or 161. Alternatively, variants can be characterized as nucleic acid molecules: (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ m NOs:l8, 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, l0 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, or 160, respectively (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; and (2) that encode a polypeptide having at least 80%, at least 90%, at least 91%;: at. least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to the amino acid sequence of SEQ
ID NOs:l9, 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 or 161, respectively.
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. TJSA 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 "BLOSLJM62" scoring matrix of Henikoff and Henilcoff (ibid.) as shown in Table 4 (amino acids are indicated by the standard one-letter codes).
Total number of identical matches x 100 [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences]

I

''S- c--1N M

H I

Lt7N N O
I I

[f~ cH c-IM N N
I I I

pa LW-I r-I~ M N
I I I I I

l0 ~ N N c-IM c-1 I I I I'' Ll7O N c-Ir-Irl i-I,-1 I I I I I

L(1rl M rlO rlM N N
I I I I I I L.

di N N O M N r-IN ~-Iri I I I I I

~

~ H d~N M rl O M N rlM rlM

I~

OO M M rIN ri N ~-IN N N M
I I I I I I I I I I

Z~ ~0N 'd~'diN M M N O N N M M
I I I I I I I I I I I

W ~ N O M M r-IN M r-IO t--IM N N
I I I I I I I I I I

Q! 11lN N O M N r-IO M r-IO rlN c-IN

(~ 01 M ~ M M r-Ic-1M c-iN M rl rlN N rl I I I I I I I I I I I I I I I

lflM O N c-Irl M cH c-IM M rlO r-Idi M M

l0 c-IM O O O rl M M O N M N c-IO d~ N M
I I I I I I I I I

p tSIO N M c--IO N O M N N v-IM N rI v-IM N M
I I I I I I I I I I I I I

~, cN v-IN N O rl~--IO N c-Ic-Irlc-IN r-Irl O M N O

FG fli'-~a~1U OIW U''~'H ~-l"~~i C~ W U1 E-~',~'JiJ

N

Those skilled in the art appreciate that there are many established algorithms available to align two amino acid sequences. The "PASTA" 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 the amino acid sequence of a putative variant IL-28 or IL-29. The PASTA
algorithm is described by Pearson and Lipman, Proc. Nat'1 Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990).
Briefly, PASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID N0:2) and a test sequence that have l0 either the highest density of identities (if the letup variable is 1) or pairs of identities (if letup=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 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 letup 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 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 PASTA
analysis are: letup=1, gap opening penalty=10, gap extension penalty=1, and substitution matrix=BLOSUM62. These parameters can be introduced into a PASTA
program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).
PASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the letup value can range between one to six, preferably from three to six, most 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 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 of from about 146 to 207 amino acid residues that comprise a to sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to the corresponding region of SEQ m NOs:l9, 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 or 161. 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.

Amino acid sequence changes are made in Cysteine mutant IL-28 or IL-29 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 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., Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995).
Other 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 associations (Bean et al., Anal. Biochem. 201:216-226, 1992; Gray, Protein Sci.
2:1732-1748, 1993; and Patterson et al., Anal. Chem. 66:3727-3732, 1994). It is generally believed that if 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 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 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:l9, 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 or 161 can be generated (Hopp et al., Proc. Natl.
Acad.
Sci.78:3824-3828, 1981; Hopp, J. Immun. Meth. X8:1-18, 1986 and Triquier et al., Frotein En~ineerin~ 11:153-169, 1998). The profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored.
Those skilled in the art will recognize that hydrophilicity or hydrophobicity will be taleen into account when designing modifications in the amino acid sequence of a 5 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 Val, Leu and Ile or the group consisting of Met, Gly, Ser, Ala, Tyr and Trp.
The identities of essential amino acids can also be inferred from analysis 10 of sequence similarity between IFN-ec 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 15 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 20 244:1081 (1989), Bass et al., Proc. Natl Acad. Sci. USA 88:4498 (1991), Coombs and Corey, "Site-Directed Mutagenesis and Protein Engineering," in Proteins:
Analysis and De~ sign, 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 25 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).
The present invention also includes functional fragments of Cysteine mutant 1L-28 or IL-29 polypeptides and nucleic acid molecules encoding such functional fragments. A "functional" Cysteine mutant IL-28 or IL-29 or fragment 3o 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 1L-29 polypeptides 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. Thus, the present invention further provides fusion proteins encompassing: (a) polypeptide molecules comprising one or more of the helices described above; and (b) functional fragments comprising one or more of these helices. The other polypeptide portion of the fusion protein may be contributed by another helical-bundle cytokine or interferon, such as IFN-oc, or by a non-native and/or an unrelated secretory signal peptide that 1o facilitates secretion of the fusion protein.
The Cysteine mutant IL-28 or 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 2o manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning A
Laborator~Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., eds., Current Protocols in Molecular Biolo~y, 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.
The present invention also provides an expression vector comprising an isolated and purified DNA molecule including the following operably linked elements:
a transcription promoter; a DNA segment encoding a polypeptide having at least percent or 95 percent sequence identity with a polypeptide selected from the group consisting of SEQ ID NOs:l9, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161; and a transcription terminator. The DNA molecule may further comprise a secretory signal sequence operably linked to the DNA segment. The encoding polypeptide may further comprise an affinity tag as described herein. The present invention also provides a cultured cell containing the above-described expression vector. The encoded polypeptide may optionally comprise at least 15, at least 30, at least 45, or at least 60 sequential amino acids of an amino acid sequence as shown in SEQ ID NOs:l9, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and. 161.
The encoded polypeptide may optionally have antiviral activity, e.g., hepatitis B and hepatitis C.
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-28 or 1L-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 slulled 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 slcill in the art. Many such elements are described in the literature and are available through commercial suppliers.
To direct a Cysteine mutant IL-28 or 1L-29 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of Cysteine mutant IL-28 or IL-29, e.g., SEQ ID
N0:119 or SEQ >D N0:121, or may be derived from another secreted protein (e.g., t-PA;
see, U.S.
Patent No. 5,641,655) or synthesized de novo. The secretory signal sequence is operably linked to the Cysteine mutant IL-28 or IL-29 DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the~DNA
sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743;
Holland, et al., .
U.S. Patent No. 5,143,830).
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, Kl2-derived strains MM294, TG-1, JM-107, BL21, and UT5600. Other suitable strains include: BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, .DHl, 2o DH4I, DHS, DHSI, DHSIF', DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, ER1647, E. coli K12, E. coli K12 RV308, E. coli K12 C600, E. coliHB 101, E. coli K12 R_{k-M lc-}, E. coli K12 RRl (see, for example, Brown (ed.), Molecular Biolo~y Labfax (Academic Press 1991)). Other gram-negative prokaryotic hosts can include Serratia, PseudomofZas, Caulobacter. Prokaryotic hosts can include gram-positive organisms such as Bacillus, for example, B. subtilis and B. tlaurifzgieuesis, and B.
thuringienesis var. israelensis, as well as Streptomyces, for example, S.
lividafzs, S.
ambofaciens, S. fradiae, and S. griseofuscus. Suitable strains of Bacillus subtilus include BR151, YB886, MI119, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning Methods," in DNA Cloni~: A Practical Approach, Glover (ed.) (IRL 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 Biolo~y, 3r~ Editiofa (John Wiley & Sons 1995); Wu et al., Methods in Gene Biotechnolo~y (CRC Press, Inc. 1997)). In one embodiment, the methods of the present invention use Cysteine mutant IL-28 or 1L-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-28 or 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-28 or IL,-29 in a suitable medium to in shake flask culture to allow for growth to an optical density (OD) of between 5 and 20 at 600 nm. A suitable medium would contain nitrogen from a sources) 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-28 or IL-29 protein. The Cysteine mutant IL-28 or 1L-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, re-suspended 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 fermentor 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) containing sodium lauryl sulfate (0.1-2°70). In the process for recovering purified Cysteine mutant IL-28 or 1L-29 from transformed E. coli host strains in which the Cysteine mutant IL-28 or 1L-29 is accumulates as refractile inclusion bodies, the cells are disrupted and the inclusion bodies are recovered by centrifugation. The inclusion bodies are then solubilized and denatured in 6 M guanidine hydrochloride containing a 5 reducing agent. The reduced Cysteine mutant IL-28 or IL-29 is then oxidized in a controlled renaturation step. Refolded Cysteine mutant IL-28 or 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-28 or 1L-29 protein is refolded and concentrated, the refolded 10 Cysteine mutant IL-28 or 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 15 Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virolo~y 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-5, 1982), DEAF-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, 2o 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
25 (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-Kl; 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, 30 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 resistance 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, CDB, Class I MHC, 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.
(Ban alore 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 Autographs califorraica nuclear polylaedrosis vzrus (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
3o Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biolo~y, Totowa, NJ, Humana Press, 1995. The second method of mal~ing 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, pFastBaclTM (Life Technologies) containing a Tn7 transposon to move the DNA encoding the Cysteine mutant IL-28 or IL-29 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid." The pFastBaclT"" transfer vector utilizes the AcNPV
polyhedrin promoter to drive the expression of the gene of interest, in this case Cysteine mutant IL-28 or IL-29. However, pFastBaclTM can be modified to a considerable degree.
The l0 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 (Invitrogen, Carlsbad, CA), or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native IL-28 or 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 1L-28 or 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-28 or IL-29 is transformed into E. Coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus.
The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells.
3o Recombinant virus that expresses Cysteine mutant 1L-28 or IL-29 is subsequently produced. Recombinant viral stoclcs 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 Biotechnolo~y: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994. Another suitable cell line is the High FiveOT"" cell line (Invitrogen) 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 Piclzia methanolica. Methods for transforming S.
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 Saccharo~rzyces cerevisiae is the P~Tl 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 polymorplza, Sc7zizosaccharonzyces pombe, Kluyveromyces lactis, I~luyveronzyces fragilis, Z7stilago maydis, Pichia pastoris, Pichia rnetlzanolica, Pichia guillernzondii and Cazzdida 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 Acremoniurn chrysogenunz 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 nzetlzanolica as host for the 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 >_80% 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 1o those of animal origin.
Expressed recombinant Cysteine mutant IL-28 or 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 Chromato r~aphy: Principles & Methods, Pharmacia LKE 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 lL-28 or 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, IRL Press, Oxford, 1989.
In vitro synthesis is particularly advantageous for the preparation of smaller polypeptides.

Using methods known in the art, Cysteine mutant IL-28 or II,-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-28 or IL-29 conjugates used for 5 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)).
10 Suitable water-soluble polymers include polyethylene glycol (PEG), monomethoxy-PEG, mono-(C1-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 15 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 2o daltons, 20,000 daltons, 30,000 daltons, and 40,000 daltons, which can be linear or branched. A Cysteine mutant IL-28 or IL-29 conjugate can also comprise a mixture of such water-soluble polymers.
One example of a Cysteine mutant IL-28 or IL-29 conjugate comprises a Cysteine mutant IL-28 or IL-29 moiety and a polyalkyl oxide moiety attached to the N
25 terminus of the Cysteine mutant IL-28 or 1L-29 moiety. PEG is one suitable polyalkyl oxide. As an illustration, Cysteine mutant IL-28 or IL-29 can be modified with PEG, a process known as "PEGylation." PEGylation of Cysteine mutant IL-28 or 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 Dru~'Carrier S std 9:249 30 (1992), Duncan and Spreafico, Clin. Pharmacokinet. 27:290 (1994), and Francis et al., Int J Hematol 68:1 (1998)). For example, PEGylation can be performed by an acylation reaction or by an alkylation reaction with a reactive polyethylene glycol molecule. In an alternative approach, Cysteine mutant IL-28 or IL-29 conjugates are 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 1L-28 or 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 IL-28 or IL-29 and a water-soluble polymer: amide, carbamate, urethane, and the like.
Methods for preparing PEGylated Cysteine mutant IL-28 or 1L-29 by acylation will typically comprise the steps of (a) reacting an Cysteine mutant IL-28 or 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-28 or IL-29, and (b) obtaining the reaction product(s). Generally, the optimal reaction conditions for acylation reactions will be determined based upon known parameters and desired results. For example, the larger the ratio of PEG: Cysteine mutant IL-28 or IL-29, the greater the percentage of polyPEGylated Cysteine mutant IL-28 or IL-29 product.
PEGylation by alkylation generally involves reacting a terminal 2o aldehyde, e.g., propionaldehyde, butyraldehyde, acetaldehyde, and the like, derivative of PEG with Cysteine mutant IL-28 or 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 ~-amino groups of the lysine residues and the a-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 N terminus of the protein without significant modification of other reactive groups such as the lysine side chain amino groups.

Reductive allcylation to produce a substantially homogenous population of monopolymer Cysteine mutant IL-28 or IL-29 conjugate molecule can comprise the steps of: (a) reacting a Cysteine mutant IL-28 or IL-29 polypeptide with a reactive PEG
under reductive alkylation conditions at a pH suitable to permit selective modification of the oc-amino group at the amino terminus of the Cysteine mutant IL-28 or 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 borohydride, sodium cyanoborohydride, dimethylamine borane, l0 trimethylamine borane, and pyridine borane.
For a substantially homogenous population of monopolymer Cysteine mutant IL-28 or 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.-28 or IL-29. Such reaction conditions generally provide for pKa differences between the lysine amino groups and the a-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 a-group, the more polymer is needed to achieve optimal conditions. If the pH is higher, the polymer: Cysteine mutant IL-28 or 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-will generally be in the range of 1:1 to 100:1. Typically, the molar ratio of water-soluble polymer to Cysteine mutant IL-28 or IL-29 will be 1:1 to 20:1 for polyPEGylation, and l:l to 5:1 for monoPEGylation.
30 General methods for producing conjugates comprising interferon and water-soluble polymer moieties are known in the art. See, for example, Karasiewicz et 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 or 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-28 or 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-28 or ll-29 polypeptides to the IL-28 receptor can be assayed using established approaches. Cysteine mutant IL-28 or IL-29 can be iodinated using an iodobead (Pierce, Rockford, IL) according to manufacturer"s directions, and the lasl-IL-28 or l2sl-1L-29 can then be used as described below.
In a first approach fifty nanograms of l2sl-IL-28 or lasl-IL-29, can be combind with 1000ng 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 2o them, and the reactions are incubated another hour at 4°C. The protein-G sepharose is then collected, washed three times with PBS and lasl-IL-28 or laSl-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 izsl-~-28 or lzsl-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 ~,l of 1 glmL 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 lzSI-IL-28 or lasl-IL-29 with or without increasing concentrations of potential binding competitors including, Cystein mutant IL-28, cysteine mutant IL-29,1L-28 and IL-29, and 100 ,ul of the solution added to appropriate wells of the plate. Following a one hour incubation at 4°C the plate is washed and the amount l2sl-IL-28 or l2sl-IL-29 bound determined by counting (Topcount, Packard Instruments, Downers grove, IL). The specificity of binding of l2sl-IL-28 or lasl-IL-29 can be defined by receptor molecules used in these binding assays as well as by the molecules used as inhibitors.
In addition to pegylation, human albumin can be genetically coupled to a polypeptide of the present invention to prolong its half-life. Human albumin is the most prevalent naturally occurring blood protein in the human circulatory system, persisting in circulation in the body for over twenty days. Research has shown that therapeutic proteins genetically fused to human albumin have longer half lives. An 1L28 or IL29 albumin fusion protein, like pegylation, may provide patients with.long-acting treatment options that offer a more convenient administration schedule, with similar or improved efficacy and safety compared to currently available treatments (U.S. Patent No. 6,165,470; Syed et al., Blood, 89(9):3243-3253 (1997); Yeh et al., Proc: Natl. Acad. Sci. USA, X9:1904-1908 (1992); and Zeisel et al., Horm.
Res., 37:5-13 (1992)).
Like the aforementioned peglyation and human albumin, an Fc portion of the human IgG molecule can be fused to a polypeptide of the present invention. The resultant fusion protein may have an increased circulating half-life due to the Fc moiety (U.S. Patent No. 5,750,375, U.S. Patent No. 5843,725, U.S. Patent No.

6,291,646;
Barouch et al., Journal of Immunolo~y, 61:1875-1882 (1998); Barouch et al., Proc.
Natl. Acad. Sci. USA, 97(8):4192-4197 (April 11, 2000); and I~im et al., Transplant Proc., 30(8):4031-4036 (Dec. 1998)).
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 i~z vivo.
If the infection 3o is in vivo, 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.

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 levels, immunofluorescence and enzyme-linked immunosorbant assays, 5 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 IL-29 may be given parentally to prevent viral infection or to reduce ongoing to 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.
Of particular interest is the use of Cysteine mutant IL-28 or IL-29 as an 15 antiviral therapeutic for viral leukemias (HTLV), AIDS (HIV), or gastrointestinal viral infections caused by, for example, rotavirus, calicivirus (e.g., Norwalk Agent) and certain strains of pathogenic adenovirus, Hepatitis B and C.
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 20 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 25 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;
3o 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., Hepatolog_y 27:1700-1702, 1998). Qualitative and quantitative assays generally use polymerase chain reaction techniques, and are preferred for assessing viremia and treatment response to (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 MonitorTM, Roche Molecular Systems, Branchburg, NJ) and a branched DNA (deoxyribonucleic acid) signal amplification assay (QuantiplexTM
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. 1):2S-lOS, 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 ire 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(5498):1972-1974 (Dec. 8, 2000); and Lohmann et al., Science, 285(5424):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 3o infected with HBV can accessed using a woodchuck model. Briefly, woodchucks chronically infected with woodchuck hepatitis virus (WHV) develop hepatitis and 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.
Dis. 147:531-534, 1983.) The chimpanzee model has been used in evaluating vaccines (Prince et al., In: Vaccines 97 Cold Spring Harbor Laboratory Press, 1997.) Therapies 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):5113-5120, 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 Virolo~y 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 (Money et al., Antiviral Therapy 3:59-68, 1998).
Moreover, Cysteine mutant IL-28 or 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-28 or IL-29 polypeptides and proteins is assayed using in vitro or ira vivo assays designed to detect 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 cells are most convenient for screening, such as for determining the effects of amino acid substitutions, deletions, or insertions.
Activity of Cysteine mutant TL-28 or IL-29 proteins can be measured ifa 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 x:347-354, 1990), incorporation of radiolabelled nucleotides (as disclosed by, to e.g., Raines and Ross, Methods Enz, m~ol. 109:749-773, 1985; 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. T_m_m__unol. Methods 65:55-63, 1983; Alley et al., Cancer Res.
48:589-601, 1988; Marshall et al., Growth Rep. 5:69-84, 1995; and Scudiero et al., Cancer Res.
48:4827-4833, 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 2o morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporated herein by reference).
Cysteine mutant 1L-28 or 1L-29 polypeptide activity may also be detected using assays designed to measure IL-28- and IL-29-induced production of one or more additional growth factors or other macromolecules. Certain members of the protein family comprising IL-28 and IL-29 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 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-28 or IL-29 will be useful as an anti-infectious agent, enhancing innate, cell-mediated and humoral immune responses. Increases in ICAM staining in CD14+ monocytes was seen suggesting that IL-28 and 1L-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-28 or IL-29 proteins to further stimulate monocyte activation, and (2) to examine the ability of Cysteine mutant IL-28 or IL-29 proteins to modulate attachment-induced or endotoxin-induced monocyte activation (Fuhlbrigge et al., J.
Immunol. 138: 3799-3802, 1987). IL-1a and TNFoc 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 2o immnunocompetency, for example, after chemotherapy. Thus, administration of Cysteine mutant IL-28 or IL-29 to patients receiving chemotherapy could promote their 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 vivo. 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 3o known to clinicians, physicians, and other persons spilled the art.
Monocyte cells are included in the myeloid lineage of hematopoietic cells, so affects on other cells in that 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 5 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 10 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.
15 (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; Corning Costar Corp.). The test sample, diluted in medium .
20 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. Non-migrating 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 25 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, 3o 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 BSA, and cells (such as smooth muscle cells, leukocytes, or endothelial cells) are plated at a density of approximately 104 - 105 cells/well. The wells are incubated at 37°C (typically for about 60 minutes), then non-adherent 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.
l0 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 ifa 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., Meth.
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 1L-29 gene, and mice that exhibit a complete absence of Cysteine mutant IL-28 or 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 IL-29 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 IL-29 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 sclerosis, 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 may be useful in modulating the immune system, whereby Cysteine mutant IL-28~
or IL-29 antagonists may be used for reducing graft rejection, preventing graft-vs-host , disease, boosting immunity to infectious diseases, treating immunocompromised patients (e.g., HIV+ 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, chronic 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 1L-28 and IL,-29 activity to treat such conditions, as well as to treat retinopathy. The present invention also contemplates the 3o use of Cysteine mutant IL-28 or IL-29 proteins, polypeptides, and peptides having IL-28 and 1L-29 activity to treat lymphoproliferative disorders, including B-cell lymphomas, 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., Hepatolo~y 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 vitf-o. 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 l0 using anti-tumor antigen antibodies (Guadagni et al., Cancer Immunol.
Irnrnunother.
26:222 (1988); Guadagni 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 if2 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 B 16 melanoma (ATCC No. CRL-6323), 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 10~ cells are implanted under the dorsal skin. Alternatively, the cells themselves may be infected with recombinant 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 1o for histopathological examination, immunohistochemistry, and ifZ situ hybridization, using methods known in the art and described herein. The influence of the expressed polypeptide in question, e.g., Cysteine mutant 1L-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 2o 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 14:349-361, 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 autoirnmune myocarditis. Interferon dosages can be extrapolated using a autoimmune 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 5 beta cells early during viral infection (Flodstroem et al., Nature Imrnunolo~y 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 1L-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 1L-28 and IL-29 may play a role in to 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 viral-induced 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.
15 Cysteine mutant IL-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.
2o 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 i 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 25 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.
3o Within another aspect, the present invention provides a pharmaceutical formulation comprising an isolated polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161, and a pharmaceutically acceptable vehicle.
For pharmaceutical use, Cysteine mutant IL-28 or 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-28 or IL-29 polypeptide in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5°Io dextrose in l0 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 Remimgtom: Th.e Science amd Practice of Pharmacy, Gennaro, ed:, Mack Publishing Co., Easton, PA, 19'x' ed., 1995. Cysteine mutant IL-28 or IL-29 will preferably be used in a concentration of about 10 to 100 ~,g/ml of total volume, although concentrations in the range of 1 ng/ml to 1000 p,g/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 p,g/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 IL-28 or IL-29 Cysteine mutant is an amount sufficient to produce a clinically significant change in the treated 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 IL-28 or 1L29 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 IL-28 or IL29 polypeptide formulation is contraindicated in patients with known hypersensitivity to IL-28 or IL29 polypeptide.
Within another aspect the present invention provides a method of producing an antibody to a polypeptide comprising: inoculating an animal with a polypeptide selected from the group consisting of SEQ ID NOs:l9, 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, wherein the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal. Within another aspect the present invention provides an antibody-(e'.g., neutralizing antibody) produced by the method as disclosed above, wherein the antibody binds to a polypeptide selected from the group consisting of SEQ ID
NOs:l9, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161. In one embodiment, the antibody disclosed above specifically binds to a polypeptide polypeptide selected from the group consisting of SEQ ID NOs:l9, 21, 23, 25, 27, 29, a 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. Within another aspect, the present invention provides an antibody or antibody fragment that specifically binds to a polypeptide as described herein. In one embodiment, the antibody is selected from the group consisting of a polyclonal antibody, a murine monoclonal antibody a humanized antibody derived from a murine monoclonal antibody, an antibody fragment, and human monoclonal antibody. In one embodiment, the antibody fragment is as described herein, wherein said antibody fragment is selected from the group consisting of F(ab'), F(ab), Fab', Fab, Fv, scFv, and minimal recognition unit.

Within another aspect, the present invention provides an anti-idiotype antibody that specifically binds to the antibody as described herein.
As used herein, the term "antibodies" includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and Fab fragments, single chain antibodies, and the like, including genetically engineered antibodies. Non-human antibodies 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. ~ne 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 lL-28 or IL-29 polypeptide or protein with an affinity at least 10-fold greater than the binding affinity to control (non-Cysteine mutant ' 1L-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. Sci. 51: 660-672, 1949).
Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see for example, Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Technidues 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 axe 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 immunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations, enzyme-s linlced 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 ih vivo diagnostic or therapeutic applications(e.g., inhibition of cell proliferation). See, in general, Ramakrishnan et al., Cancer Res. 56:1324-1330, 1996.
The present invention is further illustrated by the following non-limiting examples.
2o EXAMPLES
Example 1 Mammalian Expression plasmids An expression plasmid containing zcyto20 and zcyto2l was constructed via homologous recombination. Fragments of zcyto20 and zcyto2l cDNA were generated using PCR amplification. The primers for PCR were as follows:
zcyto20/pZMP2l: zc40923, and zc43152 SEQ ID NOs:42 and 43, respectively; and zcyto2l/pZMP2l: 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 QIAquickT""
Gel Extraction Kit (Qiagen, Valencia, CA).
The plasmid pZMP2l, which was cut with BgIII, was used for 5 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;
1o 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

15 2209, under Accession No. 77145), an internal ribosome entry site (IRES) element from poliovirus, and the extracellular domain of CD8 truncated at the C-terminal end of the transmembrane domain.
One hundred microliters of competent yeast (S. cerevisiae) cells were independently combined with 10 ~,1 of the insert DNA and 100ng of the cut pZMP21 2o 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 ,uF. Six hundred ~,1 of 1.2 M sorbitol was added to the cuvette, and the yeast was plated in a 100-~,l and 300,1 aliquot onto two URA-D plates arid incubated at 30°C. After about 72 hours, the Ura+
25 yeast transformants from a single plate were resuspended in 1 ml H20 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 ~,1 acid-washed glass beads and 300 ~,1 phenol-chloroform, was vortexed for 3 minutes, 3o 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 ~,1 ethanol (EtOH) and 30,1 3M sodium acetate, followed by centrifugation for 30 minutes at maximum speed. The DNA pellet was resuspended in 30 ,ul TE.
Transformation of electrocompetent E. coli host cells (MC1061) was done using 5 ,ul of the yeast DNA prep and 50 ~,l of cells. The cells were electropulsed at 2.0 kV, 25 ,uF, and 400 ohms. Following electroporation, 1 ml SOC (2%
BactoTM
Tryptone (Difco, Detroit, MI), 0.5% yeast extract (Difco), 10 mM NaCI, 2.5 mM
KCI, mM MgCl2, 10 mM MgS04, 20 mM glucose) was added and then the cells were ' plated in a 50 ,ul and 200 ~,1 aliquot on two LB AMP plates (LB broth (Lennox), 1.8%
to BactoTM 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 zcyto2l/pZMP2l.
Example 2 Expression of Mammalian Constructs in CHO cells 200p,g of a zcyto20/pZMP21 and zcyto2l/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 ~,1 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 ~,F, high capacitance, and 300 V.
3o 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% C02, and shaking at 120 RPM.
Example 3 Purification and Anal~is of zc~rto20-CHO Protein A. Purification of Zcyto20-CHO Protein Recombinant zcyto20 (IL-2~A) protein was produced from a pool of DXB 11-CHO cell lines. Cultures were harvested, and the media were sterile filtered using a 0.2 [um 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 6505 column,; (TosoH, Montgomeryville, PA), and a Superdex 75 column (Amersham Biosciences, Piscataway, NJ). Culture media from DXB 111-CHO were adjusted to pH 6.0 before loading onto a Poros 50 HS column. The column was washed with 50 mM MES (2-Morpholinoethanesulfonic acid), 100 mM NaCI, pH 6 and the bound protein was eluted with a 10 column volumes (CV) linear gradient to 60% of 50 mM MES, 2 M NaCI, 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 NaCI, pH 6, and 7% of 50 mM MES, 2 M NaCI, pH 6. The bound protein was eluted with a 25-CV linear gradient from 7% to 50%
of 50 mM MES, 2 M NaCI, 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 6505 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 through a 0.2 ~,m 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 Wester~a blotting analysis of zcyto20-CHO protein Recombinant zcyto20 protein was analyzed by SDS-PAGE (Nupage 4-12% Bis-Tris, Invitrogen, Carlsbad, CA) and Western blot using rabbit anti-zcyto2l-CEE-BV IgG as the primary antibody that cross-reacts to zcyto20-CHO protein.
The gel was electrophoresed using Invitrogen's Xcell II mini-cell (Carlsbad, CA) and transferred to a 0.2 ~,m nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA) using Invitrogen's Xcell II 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 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, 1L) and the signal was captured using a Lumi-Imager (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 4 Purification and Analysis of zcyto2l-CHO Protein A. Purification of Zcyto2l -OHO Protein Recombinant zcyto2l was produced from stable DXB 11-CHO cell lines.
Cultures were harvested, and the media were sterile filtered using a 0.2 ~,m 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. DXB 111-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 zcyto2l 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 zcyto2l protein were then adjusted to about 127 mS
with ammonium sulfate and loaded onto a Toyopearl Phenyl 6505 column (TosoH, Montgomeryville, PA). Zcyto2l protein was eluted with a decreasing ammonium sulfate gradient and the fractions containing the pure zcyto2l were pooled and concentrated for injection into a Superdex 75 column (Amersham Biosciences, 2o 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.
B. SDS-PAGE and Westeryc blotting analysis of .zcyto2l -OHO proteifZ
Recombinant zcyto2l protein was analyzed by SDS-PAGE (Nupage 4-12% Bis-Tris, Invitrogen, Carlsbad, CA) and Western blot using rabbit anti-zcyto2l-CEE-BV IgG as the primary antibody. The gel was electrophoresed using Invitrogen's Xcell II mini-cell (Carlsbad, CA) and transferred to a 0.2 ~,m nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA) using Invitrogen's Xcell II 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 transferred blot was blocked with 10% non-fat dry milk in 1x 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 anti-rabbit IgG-HRP (Pierce Chemical Co., Rockford, 5 1L) for one hour. The blot was washed three times with 1x 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).
l0 C. Summary of proteifa puri,~catiou ahd analysis The purified zcyto2l 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 15 O-linked glycosylation sites.
Example 5 Identification of 1L-29 Forms 20 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 in-line to a quadrupole-time of flight hybrid mass spectrometer (Micromass, Milford MA).
25 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 -30 C145 with 0171 observed as a S-glutathionyl cysteine (all referring to SEQ
m N0:4).

The material from fraction #51 showed the disulfide pattern C49 - C 145 and C

C171 with C15 observed as an S-glutathionyl cysteine (referring to SEQ ~
N0:4).
Example 6 E. coli Expression Plasmids Construction of expression vector, pTAP237 Plasmid pTAP237 was generated by inserting a PCR-generated linker into the SmaI site of pTAP186 by homologous recombination. Plasmid pTAP186 was l0 derived from the plasmids pRS316 (a Sacclaaromyces cerevisiae shuttle vector) and pMAL-c2, an E. coli expression plasmid derived from pKI~223-3 and comprising the tac promoter and the ~r~cB terminator.. Plasmid pTAP186 contains a kanamycin resistance gene in which the Sma I site has been destroyed and has NotI and SfiI sites flanking the yeast ARS-CEN6 and URA3 sequences, facilitating their removal from the plasmid by digestion with NotI. The PCR-generated linker replaced the expression coupler sequence in pTAP186 with the synthetic RBS II 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 -products were concentrated by precipitation with two times the volume of 100%
ethanol. Pellet was resuspended in 10 ~,L water to be used for recombining into the recipient vector pTAP186 digested with SmaI to produce the construct containing the synthetic RBS II sequence. Approximately 1 ,ug of the PCR-generated linker and ng of pTAP186 digested with SmaI were mixed together and transformed into competent yeast cells (S. ceYevisiae). 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 H20 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 MC1061. Clones were screened by colony PCR as disclosed above using 20 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 7 Codon ORtimization of IL-29 Cysteine mutant A. Codon Optimi.zatiofa Generation of the IL-29 wildtype expressioyz cofzstruct 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.
3 :1281-95, 1987). Genes coding for highly expressed proteins tend to have high 15 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.
x:494-500, 1995).
It has been shown that the expression level of proteins whose genes v 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.
66:3166-3173, 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.

The codon optimized IL-29 coding sequence was constructed from sixteen overlaying oligonucleotides: zc44,566 (SEQ I~ N0:48), zc44,565 (SEQ >D
N0:49), zc44,564 (SEQ ~ N0:50), zc44,563 (SEQ m NO:51), zc44,562 (SEQ m N0:52), zc44,561 (SEQ m N0:53), zc44,560 (SEQ m N0:54), zc244,559 (SEQ m N0:55), zc44,558 (SEQ m N0:56), zc44,557 (SEQ m N0:57). 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 1o 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. Gefaeratiofa of the codon optiy~aized zcyto2l C172S CysteifZe rrauta~ct expressiof2 cofzstruct The strategy used to generate the zcyto2l C172S Cysteine mutant is based on the QuikChange Site-Directed Mutagenesis ~ Kit (Stratagene). Primers were 2o designed to introduce the C172S mutation based on manufacturer's suggestions. These primers were designated ZG44,340 (SEQ m NO:58) and ZG44,341 (SEQ m N0:59).
PCR was performed to generate the zcyto2l C172S Cysteine mutant according to QuikChange Mutagenesis instructions. Five identical 50 pl reactions were set-up. 2.5 ~,1 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 ~,1 10x PCR buffer, 125 ng (27.42 ~,1) ZG44,340, 125 ng (9.18 [ul) ZG44,341, 6 ~,l dNTP, 6 ~,1 Pfu Turbo polymerase (Stratagene, La Jolla, CA), and 206.4 ~.1 water. 47.5 ~,l 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 3o 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 manufacturer's instructions, 5 ~,l DpnI 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 carried-out 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 ~,l water. DNA resulting from PCR was transformed into E.coli strain DHlOB. 5~,1 DNA was mixed with 40 ~,l ElectroMAX DH10B cells (Invitrogen). Cells and DNA mixture were then electroporated in a O.lcm cuvette (Bio-Rad) using a Bio-Rad Gene Pulser IITM
set to 1.75 kV, 100 SZ, and 25 ~,F. Electroporated cells were then outgrown at 37°C for 1 to hour. Mixture was plated on an LB + 25 ~.g/ml kanamycin plate and incubated at 37°C
overnight. Ten clones were screened for presence of zcyto2l C172S insert. DNA
was isolated from all ten clones using the QIAprepTM Spin Miniprep Kit (Qiagen, Valencia, CA) and analyzed for presence of insert by cutting with XbaI and PstI
restriction enzymes. Nine clones contained insert and were sequenced to insure the zcyto2l C172S mutation had been introduced. A clone was sequence verified and was subsequently labeled pSDHl88.
Example 8 2o 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 by corresponding to the amino terminus of IL-29, and primer zc41,041 (SEQ m NO:61) contained 38 by corresponding to the 3' end of the vector which contained the zcyto2l 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 ~L) of the PCR
sample was run on a 1% agarose gel with 1X TBE buffer for analysis, and the expected band of approximately 500 by fragment was seen. The remaining volume of the 100 ~uL
3o reaction was precipitated with 200 ~L, absolute ethanol. The pellet was resuspended in 10 ~.L water to be used for recombining into recipient vector pTAP238 cut with SmaI to produce the construct encoding the zcyto2l as disclosed above. The clone with correct sequence was designated as pTAP377. Clone pTAP377 was digested with Not1/Ncol (10,1 DNA, 5~u1 buffer 3 New England BioLabs, 2 p,L Not l, 2 p.L Ncol, 31 p.L
water for 1 hour at 37°C) and relegated with T4 DNA ligase buffer (7 p.L of the previous 5 digest, 2 ~L of 5X buffer, 1 ~,L 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 Pst1 to confirm the absence of the yeast sequence.
PltaP377 DNA was transformed into E. coli strain W3110lpRARE, host strain carrying extra copies of rare E. coli tRNA genes.
Example 9 E. eoli IL-28A expression construct A DNA fragment containing the wildtype sequence of zcyto20 (as shown in SEQ ID NO: 1) was isolated using PCR. Primers zc43,431 (SEQ ID N0:62) containing 41 by of vector flanking sequence and 24 by corresponding to the amino terminus of zcyto20, and primer zc43,437 (SEQ ID NO:63) contained 38 by 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 72°C for 1 minute; followed by a 4°C soak. A small sample (2-4 p,L) of the PCR
sample was run on a 1% agarose gel with 1X TBE buffer for analysis, and the expected ' band of approximately 500 by fragment was seen. The remaining volume of the 100 p,L, reaction was precipitated with 200 p,L absolute ethanol. The pellet was resuspended in 10 ~tI. water to be used for recombining into recipient vector pTAP238 cut with SmaI to produce the construct encoding the zcyto20 as disclosed above. The clone with correct sequence was designated as pYEL7. It was digested with Notl/Nco1 (l0pl DNA, 5~,1 buffer 3 New England BioLabs, 2 ~L Notl, 2 p.L Ncol, 31 ~,L water for 1 hour at 37°C) and relegated with T4 DNA ligase buffer (7 p.I. of the previous digest, 2 ~,L
of 5X
buffer, 1 p,L of T4 DNA ligase). This step removed the yeast sequence, CEN-ARS, to 3o streamline the vector. The relegated pYEL7 DNA was diagnostically digested with Pvu2 and Pst1 to confirm the absence of the yeast sequence. PYEL7 DNA was transformed into E. ccli strain W3110/pRARE.
Example 10 zcyto2l C172S Cysteine mutant expression construct The strategy used to generate the zcyto2l 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 to 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 zcyto2l C172S Cysteine mutant according to QuikChange Mutagenesis instructions. Five identical 50 ~,l reactions were set-up. 2.5 ~.1 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 ~.1 10x PCR buffer, 125 ng (27.42 ~,l) ZG44,327 (SEQ ID NO: 64), 125 ng (9.18 ~.l) ZG44,328 (SEQ ID NO: 65), 6 ~.1 dNTP, 6 ~,l Pfu Turbo polymerase (Strategene), and 206.4 ~.1 water. 47.5 ~.l 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 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 manufacturer's instructions, 5 ~,1 DpnI restriction enzyme was added to the PCR reaction and incubated at 37°C for 2 hours. DNA was precipitated my adding 10°l0 3 Molar Sodium Acetate and two volumes of 100°Io ethanol (Aaper Alcohol, Shelbyville, KY).
Precipitation was carried-out at -20°C for 20 minutes. DNA was spun at 14,000 rprn for 5 minutes and pellet was speed-vac dried. DNA pellet was resuspended in 20 ~.1 water. DNA
resulting from PCR was transformed into E.coli strain DH10B. 5 ~,l DNA was mixed with 40 ~.l ElectroMAX DH10B cells (Invitrogen, Carlsbad, CA). Cells and DNA mixture were then electroporated in a O.lcm cuvette (Bio-Rad, Hercules, CA) using a Bio-Rad Gene 3o Pulser IITM set to 1.75kV, 100SZ, and 25 ~.~F. Electroporated cells were then outgrown at 37°C for 1 hour. Mixture was plated on an LB + 25 ~,g/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 QIAprepTM Spin Miniprep Kit (Qiagen) and analyzed for presence of insert by cutting with XbaI (Roche) and PstI (New England Biolabs) restriction enzymes. Nine clones contained insert and were sequenced to insure the zcyto2l 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 zcyto2l C15S mutant.
to Example 11 zcyto20 C49S Cysteine mutant expression construct The zcyto20 C49S Cysteine mutant coding sequence was generated by overlap PCR (SEQ ID N0:20). The first 187 bases of the wildtype IL-28A
sequence (SEQ ID NO:l) was generated by PCR amplification using pYEL7 (SEQ 117 N0:67) 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 N0:67) as template and oligonucleotide primers zc45,398 (SEQ ID NO: 68) and zc43,437 (SEQ ID N0:63). Primers zc45,399 (SEQ ID
N0:66) and zc45,398 (SEQ ID NO:68) contained the specific modified sequence which 2o changed the cysteine 49 to a serine. These two PCR products were combined and PCR
overlap amplified using oligonucleotide primers zc43,431 (SEQ ID N0:62) and ' zc43,437 (SEQ ID N0:63). The final PCR product was inserted into expression vector pTAP238 by yeast homologous recombination (Raymond et al. Biotechniques. Jan.
26 1 :134-8, 140-l, 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 W3110/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 N0:24). The first 193 bases of the wildtype IL-28A
sequence was generated by PCR amplification using pYEL7 (SEQ ID N0:67) as template and oligonucleotide primers zc43,431 (SEQ ID N0:62) and zc45,397 (SEQ ID N0:63).
The second DNA fragment from base 111 to 531 was generated by PCR
amplification using pYEL7 (SEQ ID N0:67) as template and oligonucleotide primers zc45,396 (SEQ
ID NO:70) and zc43,437 (SEQ )D NO:63). Primers zc45,397 (SEQ ID N0:69) and zc45,396 (SEQ )D NO:70) contained the specific modified sequence which changed the to cysteine5l 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 N0:63). The final PCR product was inserted into our in-house expression vector pTAP238 by yeast homologous recombination (Raymond et al. su ra . 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 pCHANIO.
Example 13 Expression of Il-28A, IL-29 and Cys to Ser C~teine mutants in E. coli In separate experiments, E. coli transformed with each of the expression vectors described in Examples 6-9 were inoculated into 100 mL Superbroth II
medium (Becton Dickinson, San Diego, CA) with 0.01% Antifoam 289 (Sigma Aldrich, St.
Louis, MO), 30 ~,g/ml kanamycin , 35 ~.glml 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 14 Refolding and Purification of IL-28 A. Inclusion body preparatio~z 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 APV-Gaulin homogenizes (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 IL-29 was diluted slowly into 50 mM Tris, pH 8, 0.75 M
Arginine, 0.05% PEG3350, 2 mM MgCl2, 2 mM CaCl2, 0.4 mM KCI, 10 mM NaCI, 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 ~,m 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

NaCI, pH 5. Solid (NHq.)2504 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 6505 HIC column (Tosoh Biosep, Montgomery, PA). The column was then washed with 3 column volumes of 50 mM NaOAc, 1 M (NH4)2504, 5 pH 5. A linear gradient of 10 column volumes from 50 mM NaOAc, 1 M
(NH4)ZS04, pH 5 to 50 mM NaOAc, pH 5 was used to elute the bound zcyto2l. 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 15 Refolding and Purification of IL-29 Cysteine mutant As described in Example 3, purification of IL-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 ~% and 9% for the C15-0112 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 C15-0112 form of the isomer is homologous in disulfide bond patterns to type I INF's. The C15-form also demonstrated 30-fold higher bioactivity than the C 112-C 171 form in an ISRE
assay (see below).
Refolding and purification of .zcyto2l Cys172Ser muteifa The inclusion body preparation, refolding and purification of zcyto2l C172S polypeptide (SEQ ID N0:29) is essentially the same as those of IL-29 wild-type (SEQ ID N0:4). 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 "peale shaving". The final yield for the entire process is close to 50%.

The zcyto2l Cys172Ser polypeptide (SEQ ID N0:29) showed equivalent bioactivity to the C 15-C 112 form of wild-type lL-29 in ISRE assay shown in Example 16.
Example 16 Antiviral Activity: C t~opathic 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 l0 human or murine 1L-28A, IL-28B, or IL-29 was cloned into the pzp7Z vector using standard procedures. The human or murine IL-28A, lL-28B, or IL-29 constructs were transfected into 293 HEK cells. Briefly, for each construct 700,000 cellslwell (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 2o 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-288, 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 Cvtonathic Effect Design-Observation of Cytopathic Effect (CPE) ation - No CPE

+/- Possible CPE (about 1 % of monola er surface) + CPE limited to one la ue (about 5% of the surface) +1 CPE is limited to three la ues, affectin less than 25% of the monola er 1 25 % CPE

1-2 37% CPE

2 50% CPE

2-3 62% CPE

3 75% CPE

3-4 87% CPE

4 100% CPE

Table 8 shows that conditioned medium containing human or murine IL-28A, IL-288, or IL-29 inhibited viral infection (%CPE) in HeLa cells in a dose-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 1L-28A, IL-28B, or IL-29 did not inhibit viral infection in L929 cells. In both experiments purified interferon showed positive antiviral activity.

Table 8: Percentage Cytopathic Effect of human or murine IL-28A, IL-28B, or IL-29 in HeLa Cells using Conditioned Medium (CM) Relative CM Control zcyto20 zcyto2l zcyto22 zcyto24 zcyto25 hIFN- hIFN-a-2a Concentration GFP IL-28A IL-29 IL-28B mouse mouse a-2a Concentration (CM) (CM) (CM) 1L-28 IL-28 (CM) (CM) No Add 87 87 87 87 87 87 87 0 ng/ml .008X 87 10 56 0 0 10 15 .0001 ng/ml .0156X 87 2.5 31 0 0 5 8.3 .001 ng/ml .0325X 87 5 10 0 0 5 1.7 .O1 ng/ml .0625X 87 2.5 10 0 0 0 0 .1 ng/ml .125X 87 0 5 0 0 0 0 1 ng/ml .25X 87 0 0 0 0 0 0 10 ng/ml .5X 87 0 0 0 0 0 0 100 ng/ml Table 9: Percentage Cytopathic Effect of human or murine IL-28A, IL-28B, or IL-29 in L929 Cells usin Conditioned Medium (CM) Relative Control zcyto20 zcyto2 zcyto2 zcyto24 zcyto25 mIFN- mIFN-alpha CM GFP (CM) 1 2 (CM) (CM) alpha Conc.
Conc. (CM) (CM) No Add 87 87 87 87 87 87 87 0 ng/ml .008X 87 87 87 87 87 87 87 .0001 ng/ml .0156X87 87 87 87 87 87 87 .001 ng/ml .0325X87 87 87 87 87 87 87 .O1 ng/ml .0625X87 87 87 87 87 87 58 .1 ng/ml .125X 87 87 87 87 87 87 6.7 1 ng/ml .25X 87 87 87 87 87 87 0 10 ng/ml .5X 87 87 87 87 87 87 0 100 ng/ml Example 17 Signaling Via Interferon-Response Pathway to 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 (ISG-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 IL-28A. No induction was seen for ICAM-1 or the non-interferon stimulated gene control, G3PDH.
Example 18 2o Signal Transduction Reporter Assax 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 II cytokine receptors (including human DIRS1, IFNaRl, IFNaR2 and IL-28 receptor). Luciferase activity following stimulation of transfected cells with class II ligands (including lL-28A (SEQ ID N0:2), IL-29 (SEQ 117 N0:4), IL-28B (SEQ ID N0:6), zcytol0, 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 HEIR cells were transfected as follows: 700,000 293 cells/well (6 well plates) were plated approximately 18h prior to transfection in 2 milliliters DMEM
5 + 10% fetal bovine serum. Per well, 1 microgram pISRE-Luciferase DNA
(Stratagene), 1 microgram cytokine receptor DNA and 1 microgram plRES2-EGFP DNA (Clontech,) were added to 9 microliters Fugene 6 reagent (Roche Biochemicals) in a total of 100 microliters DMEM. Two micrograms pIRES2-EGFP DNA was used when cytokine receptor DNA was not included. This transfection mix was added 30 minutes later to 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.
15 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 dilutions (in DMEM + 0.5%FBS) of the following class II ligands; IL-28A, IL-29, IL-288, zcytol0, huILlO and huIFNa-2a. Following a 4-hour incubation at 37°C, the 2o 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 10 shows that IL-28A, IL-29, and IL-28B induce ISRE signaling in 293 cells transfected with ISRE-25 luciferase giving a 15 to 17-fold induction in luciferase activity over medium alone.
The addition of IL-28 receptor alpha subunit DNA (SEQ ID N0:11), using the endogenous CRF2-4 (SEQ ID N0:71) to the transfection mix results in a 6 to 8-fold further induction in ISRE signaling by 1L-28A, IL-29, and 1L-28B giving a 104 to 125-fold total induction. None of the other transfected class II cytokine receptor DNAs 30 resulted in increased ISRE signaling. These results indicate that IL-28A,1L-29, and IL-28B 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-signaling.
Table 10 Interferon Stimulated Response Element (ISRE) Signaling of Transfected 293 Cells Following Class II Cytokine Stimulation (Fold Induction) Li and ISRE-Luc. ISRE-Luc./IL-28R

IL-28A (125n ml) 15 125 IL-29 (125n ml) 17 108 IL-28B (125n ml) 17 104 HuIFNa-2a (100n 205 18 ml) Zc o10 (125n ml) 1.3 1 Hu1L10 (100n ml) 1 0.5 Exam lp a 19 Signal Transduction Assays with IL-29 Cysteine mutants Cell Transfectiofas To produce 293 HEIR cells stably overexpressing human IL-28 receptor, 293 cells were transfected as follows: 300,000 293 cellslwell (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/milliliter 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 6418 (Geneticin, Life Technologies). Puromycin and 6418 resistant to 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-ED'TA 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%FSS) of the different forms of 2o E.coli-derived zcyto2l containing different cysteine binding patterns.
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 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. coli-derived IL-29 is better able to induce ISRE signaling than wild-type C3-C5 form 0113-C172) or a mixture of wild-type C1-C3 form and C3-C5 form (C16-C113, C 113-C 172), all referring to SEQ ID N0:15.
Table 12 shows that C1-C3 (C16-C113) of wild-type E. coli-derived IL-29 and C1-C3 (C16-C113; SEQ ID N0:15) of Cysteine mutant (C172S) E. coli-derived IL-29 (SEQ ID N0:29) are equally able to induce ISRE signaling in 293HEI~
cells overexpressing human 1L-28 receptor.
Table 11 ISRE Signaling by different forms of E.coli-derived IL-29 (Fold Induction) Cytokine C1-C3 form C3-C5 form Mixture of Concentration(C16-C113) (C113-C172) C1-(n ml) C3 and C3-C5 0.1 10 2 5 0.01 3 1 1 0.001 1 1 1 Table 12 1o ISl~E Signaling by different forms of E.coli-derived IL-29 (Fold Induction) Cytokine Wild-type Cysteine ConcentrationCl-C3 mutant C172S
(n ml) Cl-C3 1000 9.9 8.9 100 9.3 8.7 10 9.3 8.1 1 7.8 7 0.1 4.6 3.3 0.01 1.9 1.5 0.001 1.3 0.9 Example 20 Induction of IL-28A, IL-29, IL-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 ~.glml) (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 (Invitrogen).
Low to undetectable amounts of human IL-28A, IL-28B, and IL,-29, IFN-oc and IFN-(3 RNA were seen in untreated cells. In contrast, the amount of 1L-28A, to 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,1L-29, IL-28B, like type I interferons, can be induced by double-stranded RNA or viral infection.
Example 21 IL-28, IL-29 signalin_ a~ ctivity compared to IFNa in HepG2 cells A. Cell Transfections HepG2 cells were transfected as follows: 700,000 HepG2 cells/well (6 2o 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 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 3o 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 100 ng/ml IL-28A, IL-29, IL-28B, zcyto24, zcyto25 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 5 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, zcyto24 and zcyto25 induce ISRE signaling in human HepG2 liver cells transfected with ISRE-luciferase.

Table 13: Fold Induction of Cytokine-dependent ISRE Signaling in HepG2 Cells Cytokine Fold Induction IL-28A 5.6 IL-28B S.s Zcyto24 4.7 Zcyto25 HuIFN-a2a 5' g Example 22 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 Enzym. 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 37oC
for 2 hours. A solubiliser solution was added, incubated overnight at 37oC 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-(3 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 nglml; for IFN-2o a2a, the EC50 was 0.57 ng/ml; and for IFN-(3, the EC50 was 0.46ng/ml.
Example 23 IL-28RA mRNA expression in liver and lymphocyte subsets In order to further examine the mRNA distribution for IL-28RA, semi-quantitative RT-PCR was performed using the SDS 7900HT system (Applied Biosystems, CA). One-step RT-PCR was performed using 100ng total RNA for each 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.
to Low levels are seen in dendritic cells and most monocytes.
Table 14 Cell/Tissue IL-28RA IFNAR2 CRF2-4 Dendritic Cells unstim .04 5.9 9.8 Dendritic Cells +IFNg .07 3.6 4.3 Dendritic Cells .16 7.85 3.9 CD14+ stim'd with LPS/IFNg .13 12 27 CD 14+ monocytes resting .12 11 15.4 Hu CD14+ Unact. 4.2 TBD TBD

Hu CD14+ 1 ug/ml LPS act. 2.3 TBD TBD

H. Inflamed tonsil 3 12.4 9.5 H. B-cells+PMA/Iono 4 & 24 3.6 1.3 1.4 hrs Hu CD19+ resting 6.2 TBD TBD

Hu CD19+ 4 hr. PMA/Iono 10.6 TBD TBD

Hu CD19+ 24 hr Act. PMA/Iono3.7 TBD TBD

IgD+ B-cells 6.47 13.15 6.42 IgM+ B-cells 9.06 15.4 2.18 IgD- B-cells 5.66 2.86 6.76 NKCells + PMA/Iono 0 6.7 2.9 Hu CD3+ Unactivated 2.1 TBD TBD

CD4+ resting .9 8.5 29.1 CD4+ Unstim 18 hrs 1.6 8.4 13.2 CD4+ +Poly IlC 2.2 4.5 5.1 CD4+ + PMA/Iono .3 1.8 .9 CD3 neg resting 1.6 7.3 46 CD3 neg unstim 18 hrs 2.4 13.2 16.8 CD3 neg+Poly I/C 18 hrs 5.7 7 30.2 CD3 neg+LPS 18 hrs 3.1 11.9 28.2 CD8+ unstim 18 hrs 1.8 4.9 13.1 CD8+ stim'd with PMA/Ion .3 .6 1.1 18 hrs As shown in Table 14, normal liver tissue and liver derived cell lines display substantial levels of IL-28RA and CRF2-4 mRNA.

Table 15 Cell/Tissue IL-28RA IFNARZ CRF2-4 HepG2 1.6 3.56 2.1 HepG2 UGAR 5/10/02 1.1 1.2 2.7 HepG2, CGAT HI~ES081501C 4.3 2.1 6 HuH7 5/10/02 1.63 16 2 HuH7 hepatoma - CGAT 4.2 7.2 3.1 Liver, normal - CGAT #HXYZ020801I~11.7 3.2 8.4 Liver, NAT - Normal adjacent4.5 4.9 7.7 tissue Liver, NAT - Normal adjacent2.2 6.3 10.4 tissue Hep SMVC hep vein 0 1.4 6.5 Hep SMCA hep. Artery 0 2.1 7.5 Hep. Fibro 0 2.9 6.2 Hep. Ca. 3.8 2.9 5.8 Adenoca liver 8.3 4.2 10.5 SK-Hep-1 adenoca. Liver .1 1.3 2.5 AsPC-1 Hu. Pancreatic adenocarc..7 .8 1.3 IHu. Hep. Stellate cells .025 4.4 9.7 As shown in Table 15, primary airway epithelial cells contain abundant levels of IL-28RA and CRF2-4.
Table 16 Cell/Tissue IL-28RA IFNAR2 CRF2-4 U87MG - glioma 0 .66 .99 NHBE unstim 1.9 1.7 8.8 NHBE + TNF-alpha 2.2 5.7 4.6 NHBE + poly I/C 1.8 nd nd Small Airway Epithelial Cells3.9 3.3 27.8 INHLF - Normal human lung 0 nd nd fibroblasts As shown in Table 16, IL-28RA is present in normal and diseased liver l0 specimens, with increased expression in tissue from Hepatitis C and Hepatitis )3 infected specimens.

Table 17 CelllTissue IL-28RA CRF2-4 IFNAR2 Liver with Coagulation Necrosis8.87 15.12 1.72 Liver with Autoimmune He 6.46 8.90 3.07 atitis Neonatal He atitis 6.29 12.46 6.16 Endsta a Liver disease 4.79 17.05 10.58 ulminant Liver Failure 1.90 14.20 7.69 ulminant Liver failure 2.52 11.25 8.84 Cirrhosis, rimar biliar 4.64 12.03 3.62 Cirrhosis Alcoholic (Laennec's)4.17 8.30 4.14 Cirrhosis, Cr to enic 4.84 7.13 5.06 a atitis C+, with cirrhosis3.64 7.99 6.62 a atitis C+ 6.32 11.29 7.43 ulminant he antis secondar 8.94 21.63 8.48 to He A

a atitis C+ 7.69 15.88 8.05 a atitis B+ 1.61 12.79 6.93 ormal Liver 8.76 5.42 3.78 ormal Liver 1.46 4.13 4.83 fiver NAT 3.61 5.43 6.42 fiver NAT 1.97 10.37 6.31 a Fetal Liver 1.07 4.87 3.98 a atocellular Carcinoma 3.58 3.80 3.22 denocarcinoma Liver 8.30 10.48 4.17 he . SMVC, he . Vein 0.00 6.46 1.45 a SMCA he . Arter 0.00 7.55 2.10 a . Fibroblast 0.00 6.20 2.94 uH7 he atoma 4.20 3.05 7.24 a G2 He atocellular carcinoma3.40 5.98 2.11 SIB-He -1 adenocar. Liver 0.03 2.53 1.30 a G2 Unstim 2.06 2.98 2.28 a G2+zc to21 2.28 3.01 2.53 a G2+IFNa 2.61 3.05 3.00 ormal Female Liver - de 1.38 6.45 4.57 aded ormal Liver - de aded 1.93 4.99 6.25 ormal Liver - de aded 2.41 2.32 2.75 isease Liver - de aded 2.33 3.00 6.04 rimar He atoc tes from Clonetics9.13 7.97 13.30 As shown in Tables l~-22, IL-2~RA is detectable in normal B cells, B
lymphoma cell lines, T cells, T lymphoma cell lines (Jurkat), normal and transformed lymphocytes (B cells and T cells) and normal human monocytes.

Table 18 MeanMean norm IFNAR2norm CRF2-4Norm CD14+ 24hr unstim 13.168.9 5.2 92.3 7.0 199.8 15.2 #A38 CD14+ 24 hr stun 6.9 7.6 1.1 219.5 31.8 276.6 40.1 #A38 CD14+ 24 hr unstim 17.540.6 2.3 163.8 9.4 239.7 13.7 #A112 CD14+ 24 hr stim 11.86.4 0.5 264.6 22.4 266.9 22.6 #A112 CD 14+ rest #X 32.0164.2 5.1 1279.739.9 699.9 21.8 CD14++LPS #X 21.440.8 1.9 338.2 15.8 518.0 24.2 CD14+ 24 hr unstim 26.386.8 3.3 297.4 11.3 480.6 18.3 #A39 CD14+ 24 hr stim 16.612.5 0.8 210.0 12.7 406.4 24.5 #A39 HL60 Restin 161.20.2 0.0 214.2 1.3 264.0 1.6 HL60+PMA 23.62.8 0.1 372.5 15.8 397.5 16.8 U937 Restin 246.70.0 0.0 449.4 1.8 362.5 1.5 U937+PMA 222.70.0 0.0 379.2 1.7 475.9 2.1 Jurkat Restin 241.7103.0 0.4 327.7 1.4 36.1 0.1 Jurkat Activated 130.7143.2 1.1 Co1o205 88.843.5 0.5 HT-29 ~ ~ 30.51.2 26.5~

Table 19 SD

Mono 24hr unstim0.6 2.4 #A38 Mono 24 hr stun0.7 0.2 #A38 Mono 24 hr unstim2.0 0.7 #A112 Mono 24 hr stim0.3 0.1 #A112 Mono rest #X 5.7 2.2 Mono+LPS #X 0.5 1.0 Mono 24 hr unstim0.7 0.8 #A39 Mono 24 hr stun0.1 0.7 #A39 HL60 Restin 19.7 0.1 HL60+PMA 0.7 0.4 U937 Restin 7.4 0.0 U937+PMA 7.1 0.0 Jurkat Restin 3.7 1.1 Jurkat Activated2.4 1.8 Co1o205 1.9 0.7 HT-29 2.3 1.7 Table 20 Mean Mean IL-Mean IFNAR2 28RA Mean H rt CRF

CD3+/CD4+ 0 10.1 85.9 9.0 ' 294.6 CD4/CD3+ Unstim 18 12.9 108.7 20.3 170.4 hrs CD4+/CD3+ +Pol I/C 24.1 108.5 52.1 121.8 18 hrs CD4+/CD3+ + PMA/Iono47.8 83.7 16.5 40.8 18 hrs CD3 ne 0 15.4 111.7 24.8 706.1 CD3 ne unstim 18 15.7 206.6 37.5 263.0 hrs CD3 ne +Pol I/C 18 9.6 67.0 54.7 289.5 hrs CD3 ne +LPS 18 hrs 14.5 173.2 44.6 409.3 CD8+ Unstim. 18 hrs 6.1 29.7 11.1 79.9 CD8+ + PMA/Iono 18 78.4 47.6 26.1 85.5 hrs 12.8.1 - NHBE Unstim47.4 81.1 76.5 415.6 12.8.2 - NHBE+TNF-al42.3 238.8 127.7 193.9 ha SAEC 15.3 49.9 63.6 426.0 Table 21 Norm Norm Norm SD SD SD

CD3+/CD4+ 0 0.9 29.1 8.5 0.1 1.6 0.4 CD4/CD3+ Unstim 1.6 13.2 8.4 0.2 1.6 1.4 18 hrs CD4+/CD3+ +Pol I/C 2.2 5.1 4.5 0.1 0.3 0.5 18 hrs CD4+/CD3+ + PMA/Iono0.3 0.9 1.8 0.0 0.1 0.3 18 hrs CD3 ne 0 1.6 46.0 7.3 0.2 4.7 1.3 CD3 ne unstim 18 2.4 16.8 13.2 0.4 2.7 2.3 hrs CD3 ne +Pol I/C 5.7 30.2 7.0 0.3 1.7 0.8 18 hrs CD3 ne +LPS 18 hrs 3.1 28.2 11.9 0.4 5.4 2.9 CD8+ Unstim. 18 1.8 13.1 4.9 0.1 1.1 0.3 hrs CD8+ + PMA/Iono 0.3 1.1 0.6 0.0 0.1 0.0 18 hrs 12.8.1 - NHBE Unstim1.6 8.8 1.7 0.1 0.4 0.1 12.8.2 - NHBE+TNF-al3.0 4.6 5.7 0.1 0.1 0.1 ha SAEC 4.1 27.8 3.3 0.2 1.1 0.3 Table 22 SD H rt SD IFNAR2SD IL-28RASD CRF

CD3+/CD4+ 0 0.3 3.5 0.6 12.8 CD4/CD3+ Unstim 18 1.4 13.7 1.1 8.5 hrs CD4+/CD3+ +Pol I/C 1.3 9.8 1.6 3.4 18 hrs CD4+/CD3+ + PMAIIono4.0 10.3 0.7 3.7 18 hrs CD3 ne 0 1.4 16.6 1.6 28.6 CD3 ne unstim 18 2.4 16.2 2.7 12.6 hrs CD3 ne +Pol I/C 18 0.5 7.0 1.0 8.3 hrs CD3 ne +LPS 18 hrs 1.0 39.8 5.6 73.6 CD8+ Unstim. 18 hrs 0.2 1.6 0.5 6.1 CD8+ + PMA/Iono 18 1.3 1.7 0.2 , 8.1 hrs 12.8.1 - NHBE Unstim2.4 5.6 2.7 2.8 12.8.2 - NHBE+TNF-al0.5 3.4 3.5 3.4 ha SAEC 0.5 4.8 1.8 9.9 Example 24 Mouse IL-28 Does Not Effect Daudi Cell Proliferation Human Daudi cells were suspended in RPMI + 10%FBS at 50,000 to cells/milliliter and 5000 cells were plated per well in a 96 well plate. IL-29-CEE (IL-29 conjugated with glu tag), IFN-y 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-y was used at a concentration range from 125 ng/ml to 0.06 ng/ml. IEN-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 were read on the FmaxTM plate reader (Molecular Devices, Sunnyvale, CA) using the SoftMaxTM 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 25 Mouse IL-28 Does Not Have Antiproliferative Effect on Mouse B cells to 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 LPS, 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 3H-thymidine incorporation was measured after 72 hrs. culture.
IFNa at 10 ng/ml inhibited 3H-thymidine incorporation by mouse B cells stimulated with either LPS or anti-IgM. However mouse IL-28 did not inhibit 3H-thymidine 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.
2o These data demonstrate that mouse IL-28 unlike lFNa 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 IL-28 differs from IFNa in that mouse IL-28 does not display antiproliferative activity on mouse B cells, even at high concentrations. In addition, mouse lL-28 enhances proliferation in the presence of anti-CD40 monoclonal antibodies.
Example 26 Bone marrow expansion assax Fresh human marrow mononuclear cells (Poietic Technologies, 3o Gaithersburg, Md.) were adhered to plastic for 2 hrs in aMEM, 10% FBS, 50 micromolar (3-mercaptoethanol, 2 ng/ml FLT3L at 37oC. Non adherent cells were then plated at 25,000 to 45,000 cells/well (96 well tissue culture plates) in aMEM, 10%
FES, 50 micromolar (3-mercaptoethanol, 2 ng/ml FLT3L in the presence or absence of 1000 ng/ml 1L-29-CEE, 100 ng/ml IL-29-CEE, 10 ng/ml IL-29-CEE, 100 ng/ml IFN-a2a, 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 IC,-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, Ill.) was added at 20 microliters/well. Plates were further incubated at 37 oC, 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.
IFN-a2a caused a significant inhibition of bone marrow expansion under l all conditions tested. In contrast, IL-29 had no significant effect on expansion of bone ma~~row 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.
2o Example 27 Inhibition of IL-28 and IL-29 si n~ cling with soluble receptor (zcytoRl9/CRF2-4) 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, zcyto2l and zcyto24 signaling. Human embryonal l~idney (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 N0:2), zcyto2l (SEQ
ID

N~:4), zcyto24 (SEQ ID N0:8)) reflects the interaction of the ligand with soluble receptor.
B. Cell Transfections 293 HEIR 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 1o 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, zcyto2l or zcyto24 and 10 micrograms/ml of the following 2o soluble receptors; human zcytorl9-Fc homodimer, human zcytorl9-Fc/human Fc 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 (RLIT) 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, zcyto2l and zcyto24-induced signaling between 16 and 45% of control. The human zcytorl9-Fc homodimeric soluble receptor is also able to inhibit zcyto2l-induced signaling by 45%. No significant effects were seen with huCRF2-4-Fc or muzcytorl9-Ig homodimeric soluble receptors.

Table 23: Percent Inhibition of Ligand-induced Interferon Stimulated Response FIPmPnt (T~RRI ~iunalin~ by Seluhle ReCet7tors Ligand Huzcytorl9- Huzcytorl9-FcHuCRF2-4-Fc Muzcytorl9-Ig Fc/huCRF2-4-Fc Zc o20 16% 92% 80% 91%

Zc o21 16% 45% 79% 103%

Zc o24 47% 90% 82% 89%

Example 28 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 irc vitro, PBMCs from normal donors were infected with the HIV virus in the presence of IL-28, 1o IL-29 and MetIL-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; cellgroTM 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 cells/mL in RPMI 1640 supplemented with 15% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 4 ~,g/mL PHA-P. The cells were allowed to incubate for 48-72 hours at 37°C. After incubation, PBMCs were centrifuged and resuspended in with 15% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 ~,g/mL streptomycin, ~,g/mL gentamycin, and 20 U/mL recombinant human 1L-2. PBMCs were maintained in the medium at a concentration of 1-2 x 10~ 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 10~
cells/mL, and plated in the interior wells of a 96 well round bottom microplate at 50 ~,L/well (5 x 104 cells/well). Test dilutions were prepared at a 2X
concentration in microtiter tubes and 100 ~.L 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 ~,g/ml, usually in 1/2 log dilutions. 50 ~I, of a predetermined dilution of virus 1o 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 IL-28, 1L-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.
ExamRle 29 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 ifs 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 3o 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 lL-28, IL,-29 and MetIL-29C 1725-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,-29C 1725-PEG polypeptide in triplicate. Untreated cultures serve as the cell and virus controls. Both RIBAVIRIN~ (200 pg/ml at the highest test concentration) and IFN-oc (5000 IU/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-PEG, 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°Io 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 30 1L-28 1L-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 Hepadfaaviridae family of viruses. To test the antiviral activities of IL-28 and IL-29 against HBV, IL-28, IL-29 and MetIL-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 MetIL-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 Chemother.
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 2o 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 (ICso). A dye uptake assay is then employed to measure cell viability which is used to calculate toxicity (TCso). The therapeutic index (TI) is calculated as TCSO/ICSO.
IL-28, 1L-29 and MetIL-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 3o value for treating HBV in human patients.

Example 31 IL-28, IL-29 and MetIL-29C172S-PEG inhibit BVDV replication in bovine kidney cells HCV is a member of the Flaviviridae family of RNA viruses. Other viruses belonging to this family are the bovine viral diarrhea virus (BVDV) and yellow fever virus (YFV). HCV does not replicate well in either ex vivo or ifa 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 1L-28, IL-29 and MetIL-29C172S-PEG were to 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 1L-28, IL-29 and MetIL-PEG. The MDBK cells were propagated in Dulbecco's modified essential medium (DMEM) containing phenol red with 10% horse serum, 1 % glutamine v and 1 %
i5 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 pl/well. The next day, the medium was removed and a pre-20 titered 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 7 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 25 each, diluted in assay medium in a half-log series. IFNa, and R1BAVIRIN~
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 ICso calculated relative to the TCso.
3o IL-28, IL-29 and MetlL-29C172S-PEG inhibited cell death induced by BVDV in MDBK bovine kidney cells. IL-28 inhibited cell death with an ICso of 0.02 p.g/ml, IL-29 inhibited cell death with an ICSO of 0.19 ~.g/ml, and MetIL-inhibited cell death with an ICSO of 0.45 ~,g/ml. This demonstrated that IL-28 and IL-29 have antiviral activity against the Flavivif~ida family of viruses..
Example 32 Induction of Interferon Stimulated Genes by 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 primer's 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 1L-29 induces Interferon Stimulated Gene Expression in human peripheral blood mononuclear cells at all time-points tested.

Table 24 Fold inductionPkr Fold OAS Fold MxA Induction Induction G hr _ 2.1 2.5 IL29 3.1 6 hr 17.2 9.6 16.2 IFNa2a 4 hr 19.2 5.0 8.8 4 hr 57.2 9.4 22.3 IFNa2a 8 hr 7.9 3.5 3.3 8hr IFNa2a18.1 5.0 17.3 2 hr 9.4 3.7 9.6 IL,29 2 hr 29.9 6.4 47.3 IFNa2a 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 anti-CD28 (0.5ug/ml), (Pharmingen, San Diego, CA) and Interleukin 2 (lL-2; 100 U/ml) (R&D
Systems, Minneapolis, MN), washed and then expanded for a further 5 days with IL-2.
to Following activation and expansion, cells were stimulated with IL-28A (20 ng/ml), IL-29 (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.

Table 25 MxA Fold InductionPkr Fold OAS Fold Induction Induction onor #1 3 5.2 2.8 4.8 hr IL28 onor #1 3 5.0 3.5 6.0 hr IL29 onor #1 6 5.5 2.2 3.0 hr IL28 onor #1 6 6.4 2.2 3.7 hr IL,29 onor #1 18 4.6 4.8 4.0 hr IL28 onor #1 18 5.0 3.8 4.1 hr IL29 onor #2 3 5.7 2.2 3.5 hr IL28 onor #2 3 6.2 2.8 4.7 hr IL29 onor #2 6 7.3 1.9 4.4 hr IL28 onor #2 6 8.7 2.6 4.9 hr IL29 onor #2 18 4.7 2.3 3.6 hr IL,28 onor #2 18 4.9 2.1 3.8 hr IL,29 C. Primary Human Hepatocytes Freshly isolated human hepatocytes from two separate donors (Cambrex, 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 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 MxA Fold Induction Pkr Fold OAS Fold Induction Induction onor #1 31.4 6.4 30.4 onor #1 31.8 5.2 27.8 onor #1 63.4 8.2 66.7 IFN-a2a onor #2 41.7 4.2 24.3 onor #2 44.8 5.2 25.2 onor #2 53.2 4.8 38.3 IFN-a2a D HepG2 and IiuH7~ Human Liver Hepatoma Cell Lines HepG2 and HuH7 cells (ATCC NOS. 8065, Manassas, VA) were 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 MetlL-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 semi-to 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.

Table 27 MxA Pkr Fold OAS Fold Induction Fold InductionInduction HepG2 24 hr _ 0.7 3.3 IL,28 12.4 a G2 24 hr 36.6 2.2 6.4.

a G2 24 hr 12.2 1.9 3.2 IFNa2a a G2 24 hr 93.6 3.9 19.0 a G2 48hr 2.7 0.9 1.1 a G2 48hr 27.2 2.1 5.3 a G2 48 hr 2.5 0.9 1.2 IFNa2a a G2 48hr 15.9 1.8 3.3 IFN

uH7 24 hr 132.5 5.4 52.6 uH7 24 hr 220.2 7.0 116.6 uH7 24 hr 157.0 5.7 67.0 IFNa2a uH7 24 hr 279.8 5.6 151.8 IFN

uH7 48hr II,2825.6 3.4 10.3 uH7 48hr IL29143.5 7.4 60.3 uH7 48 hr 91.3 5.8 32.3 IFNa2a uH7 48hr IFN 65.0 4.2 35.7 Table 28 MxA Fold InductionOAS Fold InductionPkr Fold Induction MetIL-29-PEG 36.7 6.9 _ 2.2 MetIL-29C 1725-PEG46.1 8.9 2.8 Data shown is for 20 ng/ml metIL-29-PEG and metIL-29C172S-PEG versions of IL-after culture for 24 hours.
Data shown is normalized to HPRT and shown as fold induction over unstimulated cells.
Example 33 l0 Antiviral Activi~ of IL-28 and IL-29 in HCV Replicon System The ability of antiviral drugs to inhibit HCV replication can be tested ifz 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, K.J. et al. Science 290:1972-1974, 2000). Treatment of replicon clones with IFNa at 10 lU/ml reduces the amount of HCV RNA by 85°lo 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 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 ~,g/ml 2. IL29 alone at increasing concentrations (6)* up to 1.0 ~,g/ml 3. PEGIL29 alone at increasing concentrations (6)* up to 1.0 ~.g/ml 4. IFNoc2A alone at 0.3, 1.0, and 3.0 ICT/ml to 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 ~.g/ml, 0.32 ~,g/ml, 0.10 ~,g/ml, 0.032 ~,g/ml, 0.010 ~.g/ml, 0.0032 ~,g/ml.
The replicon clone (BB7) is treated 1X per day for 3 consecutive days with the doses listed above. Total HCV RNA is measured after 72 hours.
Example 34 IL-28 and IL-29 have antiviral activity against pathogenic viruses Two methods are used to assay in vitro antiviral activity of IL-28 and IL
29 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 virus-induced 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 (1og10 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 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 (M~I) 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 to (EC50) determined by plotting compound concentration versus percent inhibition on semilogarithmic graph paper. The EC50/IC50 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.

Table 29: Visual Assav Virus Cell Drug EC50 VisualIC50 VisualSI Visual line -(IC50/

EC50) AdenovirusA549 1L-28A >10 ~. ml >10 ~. 0 ml AdenovirusA549 1L-29 >10 ~ ml >10 ~, 0 ml AdenovirusA549 MetIL-29>10 ~,g/ml >10 ~g/ml 0 ParainfluenzaMA-104 IL,-28A >10 p,g/ml >10 ~g/ml 0 virus ParainfluenzaMA-104 IL-29 >10 ~g/ml >10 ~.g/ml0 virus ParainfluenzaMA-104 MetIL-29>10 ~gJml >10 ~g/ml 0 virus C 172S-PEG

RespiratoryMA-104 IL-28A >10 ~g/ml >10 pg/ml 0 s nc tial virus RespiratoryMA-104 lL-29 >10 ~,g/ml >10 ~g/ml 0 s nc tial virus RespiratoryMA-104 MetIL-29>10 p,g/ml >10 p,g/ml0 s nc tial C172S-PEG
virus Rhino 2 KB IL-28A >10 ~ ml >10 ~, 0 ml Rhino 2 KB IL-29 > 10 p ml > 10 ~ 0 ml Rhino 2 KB MetIL,-29> 10 [~g/ml> 10 ~g/ml0 Rhino 9 HeLa IL-28A >10 ml >10 ~, 0 ml Rhino 9 HeLa lL-29 > 10 ~ ml > 10 ~, 0 /ml Rhino 9 HeLa MetIL-29>10 pg/ml >10 ~g/ml 0 CoxsaclcieKB IL-28A >10 ~g/ml >10 pg/ml 0 virus Coxsaclue KB lL-29 >10 ~.g/ml >10 ~,g/ml0 virus CoxsaclcieKB MetIL-29>10 p,g/ml >10 p,g/ml0 virus C 1725-PEG

Influenza Maden- IL-28A >10 ~g/ml >10 p.g/ml0 (type A [H3N2]) Darby Canine Kidne Influenza Maden- IL-29 >10 ~g/ml >10 p,g/ml0 (type A [H3N2]) Darby Canine Kidne Influenza Maden- MetIL-29 >10 p,g/ml>10 p,g/ml0 (type A [H3N2]) Darby C172S-PEG

Canine Kidne Influenza Vero IL-28A 0.1 ~glml >10 ~.g/ml>100 (type A [H3N2]) Influenza Vero IL-29 >10 p.g/ml>10 ~g/ml 0 (type A [H3N2]) Influenza Vero MetIL-29 0.045 p,g/ml>10 ~g/ml >222 (type A [H3N2]) C172S-PEG

Vaccinia Vero IL-28A >10 ml >10 p, 0 virus ml Vaccinia Vero IL-29 >10 p, >10 ml 0 virus ml ~Vaccinia Vero MetIL-29 >10 pg/ml >10 ~g/ml 0 virus West Nile Vero IL-28A 0.00001 >10 ~,glml>1,000,0 p,g/ml virus 00 West Nile Vero IL.-29 0.000032 >10 p,g/ml>300,000 virus p, ml West Nile Vero MetIL-29 0.001 p,g/ml>10 ~.g/ml>10,000 virus C 172S-PEG

Dengue Vero IL-28A 0.01 p >10 ~ /ml >1000 virus ml Dengue Vero IL-29 0.032 ml >10 p. >312 virus ml Dengue Vero MetlL-29 0.0075 >10 ~g/ml >1330 virus pg/ml VenezuelanVero IL-28A 0.01 ~.g/ml>10 p,g/ml>1000 equine encephalitis virus VenezuelanVero IL-29 0.012 ~,g/ml>10 ~g/ml >833 equine encephalitis virus VenezuelanVero MetIL-29 0.0065 >10 p,g/ml>1538 ~tg/ml equine C 1725-PEG

encephalitis virus Pichinde BSC-1 IL-28A >10 ml >10 ~ ml 0 virus Pichinde BSC-1 IL-29 >10 ~ ml >10 ~. 0 virus ' ml Pichinde BSC-1 MetIL-29 >10 p,g/ml>10 ~g/ml 0 virus C 1725-PEG' Polio virusVero IL-28A >10 p,g/xnl>10 ~g/ml 0 Polio virusVero IL-29 >10 ~g/ml >10 ~,g/ml0 Polio virusVero MetIL-29 >10 p,g/ml>10 p,g/ml0 Table 30: Neutral Red Assay V'xzus~ ~ C~IInIine~Drug ~ECSt2,1'~R' rC50NR, ~ NR.' ~ ''' ~w a ; ~ ~ = J ;, , (~~C50I
~ ". ~'~
~ r~ ~
j _ ~ ~ ' EG50),' < < r ,, ~ ~ ~~

Adenovirus A549 IL-28A >10 p, ml >10 ml 0 Adenovirus A549 IL-29 >10 ~ ml >10 ~ ml 0 Adenovirus A549 MetIL-29>10 ~g/ml >10 ~g/ml 0 ParainfluenzaMA-104 IL-28A >10 pg/ml >10 p.g/ml0 virus ParainfluenzaMA-104 IL-29 >10 ~g/ml >10 ~.g/ml0 virus ParainfluenzaMA-104 MetIL-29>10 p,g/ml >10 ~,g/ml0 virus C172S-PEG

Respiratory MA-104 IL,-28A >10 ~g/ml >10 ~,g/ml0 s nc tial virus Respiratory MA-104 IL-29 >10 p,g/ml >10 ~g/ml 0 s nc tial virus Respiratory MA-104 MetIL-295.47 ~g/ml >10 ~g/ml >2 s nc tial C 1725-PEG
virus Rhino 2 KB IL-28A >10 ml >10 ~ ml 0 Rhino 2 KB IL-29 > 10 p, > 10 /ml 0 ml Rhino 2 I~B MetIL-29>10 ~g/ml >10 ~,g/ml0 Rhino 9 HeLa IL-28A 1.726 ml >10 ~ ml >6 Rhino 9 HeLa IL,-29 0.982 ml >10 ~, >10 ml Rhino 9 HeLa MetIL-292.051 ~g/ml>10 l.tg/ml>5 Coxsackie KB IL-28A >10 ml >10 ml 0 virus Coxsackie KB IL-29 >10 ~g/ml >10 p,g/ml0 virus Coxsackie KB MetIL-29 >10 ~,g/ml>10 ~g/ml 0 virus C 172S-PEG

Influenza Maden- IL-28A >10 ~,g/ml>10 pg/ml 0 (type A

[H3N2]) Darby Canine Kidne Influenza Maden- IL-29 >10 ~,g/ml>10 p,g/ml0 (type A

[H3N2]) Darby Canine Kidne Influenza Maden- MetIL-29 >10 ~.g/ml>10 pg/ml 0 (type A

[H3N2]) Darby C172S-PEG

Canine Kidne Influenza Vero IL-28A 0.25 ~g/ml>10 pg/ml >40 (type A

[H3N2]) Influenza Vero 1L-29 2 pg/ml >10 pg/ml >5 (type A

[H3N2]) Influenza Vero MetIL-29 1.4 ~g/ml >10 ~g/ml >7 (type A

[H3N2]) C172S-PEG

Vaccinia Vero IL-28A >10 ~ ml >10 ~ ml 0 virus Vaccinia Vero IL,-29 >10 ~, >10 p. 0 virus ml /ml Vaccinia Vero MetIL-29 >10 ~,g/ml>10 pg/ml 0 virus West Nile Vero IL-28A 0.0001 >10 ~, >100,000 virus ml ml West Nile Vero IL-29 0.00025 >10 p, >40,000 virus ~. ml ml West Nile Vero MetIL-29 0.00037 >10 pg/ml >27,000 virus ~iglml Dengue virusVero II,-28A 0.1 ~ ml >10 p ml >100 Dengue virusVero IL-29 0.05 ~, >10 ~ ml >200 ml Dengue virusVero MetIL,-290.06 pg/ml>10 pg/ml >166 Venezuelan Vero IL-28A 0.035 ~g/ml>10 ~.g/ml>286 equine ence halitis virus Venezuelan Vero IL-29 0.05 ~g/ml>10 pg/ml >200 equine ence halitis virus Venezuelan Vero MetIL-29 0.02 ~,g/ml>10 ~g/ml >500 equine C 172S-PEG

ence halitis virus Pichinde BSC-1 IL-28A >10 lml >10 ml 0 virus Pichinde BSC-1 II,-29 >10 ml >10 ml 0 virus Pichinde BSC-1 MetIL-29 >10 ~g/ml >10 p,g/ml0 virus Polio virus Vero IL-28A >1.672 >10 pg/ml >6 p,g/ml Polio virus Vero IL-29 >10 ~glml >10 ~,g/ml0 Polio virus Vero MetIL-29 > 10 ~g/ml> 10 ~g/ml0 Example 35 IL-28 1L-29, metlL-29-PEG and metlL-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 IFN~i) 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 IL-29 have effects on mouse cells, ISG induction by human IL-28 and IL-29 was evaluated 1o 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 1L-28 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 CHO-derived human IL-28A (SEQ ID N0:2) or IL-29 (SEQ ID N0:4) . 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 IL-28A and IL-29 stimulated ISG induction in the mouse hepatocyte cell line AML-12 and demonstrated that unlike type I IFNs, the IL-family proteins showed cross-species reactivity.

Tahle 31 OAS PkR Mx1 Stimulation None 0.001 0.001 0.001 Human IL-28 0.04 0.02 0.06 Human IL-29 0.04 0.02 0.07 Mouse IL,-28 0.04 0.02 0.08 Mouse IFNa 0.02 0.02 0.01 All data shown were expressed as fold relative to HPRT gene expression n~ 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 erar »~~
Mxl Fold InductionOAS Fold InductionPkr Fold Induction MetIL-29-PEG 728 614 8.

MetIL-29C 1725-PEG761 657 8 Cells were stimulated with 20 ng/ml metIL-29-PEG or metIL-29C 1725-PEG for 24 to hours.
Data shown is normalized to HPRT and shown as fold induction over unstimulated cells.
Example 36 ISGs are Efficient Induced in Spleens of Transg_enic Mice Expressing Human IL-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 IL-29 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-Elmer 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 Mx1 compared to their non-Tg littermate controls suggesting that human IL-29 is biologically active in vivo in mice.

TahlP ~"~
OAS PkR Mxl Mice Non-Tg 4.5 4.5 3.5 IL,-29 Tg ~ 12 8 ~ 21 All data shown are fold expression relative to tiE'lt'1' gene expression.
The average expression in two mice is shown Example 37 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 ifZ vivo, CHO-derived human IL.-28A and IL-29 protein were injected into mice. In addition, E. coli derived 1L-29 was also tested in in vivo assays as described above using MetIL-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 ~.g - 250 ~,g) of CHO-derived human IL-28A and 1L-29 or MetIL-29C172S-PEG and MetIL-29C16-C113-PEG. Mice were sacrificed at various time points (1hr - 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 Injection OAS- 1hr OAS-6hr OAS-24hr OAS-48hr None - liver 1.6 1.6 1.6 1.6 IL-29 liver 2.5 4 2.5 2.8 None - s Teen1.8 1.8 1.8 1.8 IL-29 - s 4 6 3.2 3.2 leen None - blood 5 5 5 5 IL-29 blood 12 18 11 10 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 ~.g i.v. is shown. The data shown is the average expression from 5 different animals/group., Table 35 In'ection OAS (24hr) None _ 1.8 IL-29 10 ~, 3.7 IL-29 50 ~. 4.2 IL-29 250 ~. 6 Table 36 MetIL-29-PEG MetIL-29C Naive 3hr 6hr l2hr 4hr 3hr 6hr l2hr 4hr 41~r KR 18.2 13.93 4.99 3.77 5.29 5.65 3.79 3.55 3.70 OAS 91.29 65.93 54.0 20.81 13.42 13.02 10.5 8.72 6.60 xl 537.51124.9933.5835.82 27.89 29.3 16.610.0 10.98 ice 100 Data M were ~,g shown injected of is with proteins fold i.v.

expression over HPRT expression from livers of mice. Similar data was obtained from blood and spleens of mice.
Example 38 2o 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 ~,g-250 ~,g) 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.
l0 Table 37 OAS-lhr OAS-6hr OAS-24hr OAS-48hr Injectiota None 80 80 80 80 OAS activity is shown at pmol/dL of plasma for a single concentration (250 ~,g) of human IL-29.
Example 39 IL,-28 and IL-29 inhibit Adenoviral pathology in mice To test the antiviral activities of IL-28 and 1L-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 ~,g mouse IL-28 (zcyto24 as shown in SEQ ID N0:8) 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 10~ plaque-forming units (pfu)). Following this, every other day mice were given an additional dose of PBS, mouse IL-28 or metlL-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 to 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:r. 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 2o 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 IL-28 and IL-29 have antiviral properties against a liver-trophic virus.
Example 40 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 3o 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. x:540-547, 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 1L-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 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 1L-28 or IL-29 treated mice, a 20°Io increase in the percentage of tetramer positive T cells that proliferate, make cytokine, or display a mature phenotype 2o 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 41 Influenza Model of Acute Viral Infection A. Preliminar~x~eriment to test antiviral activity To determine the antiviral activity of IL-28 or II,-29 on acute infection by Irifluen.za virus, an in vivo study using influenza infected c57B 1/6 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 to (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 ~,g, i.p. starting 2 hours after infection) (5) IL-28 or 1L-29 (25 ~,g, i.p. starting 2 hours after infection) (6) IL-28 or lL-29 (125 ~,g, 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.) 2o 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/CDB, CD3/CD8/CDllb, CD8/CD441CD62L, CD3/DXS, GR-1/F480, and CD19.
3o Study No.2 Efficacy study of IL-28 or 1L-29 in C57B1/6 mice infected with mouse-adapted 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/LTSSR (H1N1) (Chemicon International, Temecula, CA); 40 ~.glmouse at 2 h and 4 h post infection (10 ~,1 intranasal) Group 3: IL-28 or IL-29 (5 ~.g, i.p.) Group 4: IL-28 or IL-29 (25 ~.g, i.p.) Group 5: IL-28 or IL-29 (125 ~.g, i.p.) to 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).
2o Tetramer staining: The number of CD8+ T cells binding MHC Class I
tetramers containing influenza A nucleoprotein (NP) epitope are assessed using complexes of MHC class I with viral peptides: FLU-NP3~6-37a~b (ASNENMETM), (LMCV peptide/Db ).
Quantitative immunophenotyping of the following: CDB, tetramer, intracellular IFN~y, NK1.1, CDB, tetramer, CD62L, CD44, CD3(+ or -), NKl.l(+), intracellular IEN~y, CD4, CDB, NK1.1, DXS, CD3 (+ or -), NK1.1, DXS, 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-challen e~ study:
Day 0: Both groups will be infected with A/PR virus (1LD30).
Group 6 will not be treated.
l0 Group 7 will be treated for 9 days with 125 ~,g 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 AIPR 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 42 IL-28 and IL-29 have Antiviral Activity Against Hepatitis B virus (HBV) in vivo A transgenic mouse model (Guidotti et al., J. Virolo~y 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 3o effectiveness of lamivudine and IFN-a in reducing HBV viral titers..

HBV TG mice (male) are given intraperitoneal injections of 2.5, 25 or 250 micrograms IL-28 or 1L-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. ~ne hour following the final dose of 1L-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 lL-28 or IL-29 reflects antiviral activity of these compounds against HBV.
to Example 43 IL-28 and IL-29 inhibit human herpesvirus-8 (HI3V-8) replication in BCBL-1 cells The antiviral activities of IL-28 and IL-29 were tested against HHV-8 in an ifz 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 2o 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 HIiV-8 DNA levels (ICSO). A novel dye uptake assay was then employed to measure cell viability which was used to calculate toxicity (TCso). The therapeutic index (TI) is calculated as TCSO/IC5o~
IL-28 and TL-29 inhibit HIiV-8 viral replication in BCBL-1 cells. 1L-28A had an ICso of 1 ~,g/ml and a TCso of >10 ~.g/ml (TI >10). IL-29 had an ICso of 6.5 ~,g/ml and a TC$o of >10 ~.g/ml (TI >1.85). MetlL-29C172S-PEG had an ICso of 0.14 ~,g/ml and a TCso of >10 ~,g/ml (TI >100).
Example 44 IL-28 and IL-29 antiviral activity against Epstein Bam 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 (ICso). A novel dye uptake assay was then employed to measure cell viability which was used to calculate toxicity (TCSO). The therapeutic index (TI) is calculated as TCso/ICso.
Example 45 IL-28 and II,-29 antiviral activity against Heroes Simplex Virus-2 (HSV-27 The antiviral activities of IL-28 and IL-29 were tested against HSV-2 in an ifi 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 1L-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. ~n 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 ~.1/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 IC5o calculated relative to the TCso.
IL-28A, IL-29 and MetIL-29C172S-PEG did not inhibit cell death (ICSo of >l0ug/ml) in this assay. There was also no antiviral activity of IFND 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 3o 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.
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<222> .(25) (1)..

<400>

Met LysLeuAsp MetThrGly AspCysThr ProValLeu ValLeu Met Ala AlaValLeu ThrValThr GlyAlaVal ProValAla ArgLeu His Gly AlaLeuPro AspAlaArg GlyCysHis IleAlaGln PheLys Ser Leu SerProGln GluLeuGln AlaPheLys ArgAlaLys AspAla Leu Glu GluSerLeu LeuLeuLys AspCysArg CysHisSer ArgLeu Phe Pro ArgThrTrp AspLeuArg GlnLeuGln ValArgGlu ArgPro Met Ala LeuGluAla GluLeuAla LeuThrLeu LysValLeu GluAla Thr Ala AspThrAsp ProAlaLeu ValAspVal LeuAspGln ProLeu His Thr LeuHisHis IleLeuSer GlnPheArg AlaCysIle GlnPro Gln Pro ThrAlaGly ProArgThr ArgGlyArg LeuHisHis TrpLeu Tyr Arg LeuGlnGlu AlaProLys LysGluSer ProGlyCys LeuGlu Ala Ser ValThrPhe AsnLeuPhe ArgLeuLeu ThrArgAsp LeuAsn Cys Val AlaSerGly AspLeuCys Val <210> 3 <211> 856 <212> DNA
<213> Homo Sapiens <220>
<221> sig~eptide <222> (98)...(154) <221> mat~eptide <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 Met Ala Ala Ala Trp Thr , gtg gtg ctg gtg act ttg gtg cta ggc ttg gcc gtg gca ggc cct gtc 163 Val Val Leu Val Thr Leu Val Leu Gly Leu Ala Val Ala Gly Pro Val ccc act tcc aag ccc acc aca act ggg aag ggc tgc cac att ggc agg 211 Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg ttc aaa tct ctg tca cca cag gag cta gcg agc ttc aag aag gcc agg 259 Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg agt tgc agc tct 307 Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser cct gtc ttc ccc ggg aat tgg gac ctg agg ctt ctc cag gtg agg gag 355 Pro Val Phe Pro G1y Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu cgc cct gtg gcc ttg gag get gag ctg gcc ctg acg ctg aag gtc ctg 403 Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu gag gcc get get ggc cca gcc ctg gag gac gtc cta gac cag ccc ctt 451 Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu CaC aCC Ctg CaC CaC atC CtC tCC Cag CtC Cag gCC tgt atC Cag cct 499 His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro cag ccc aca gca ggg ccc agg CCC Cgg ggC CgC CtC CaC CaC tgg ctg 547 Gln Pro Thr Ala G1y Pro Arg Pro Arg Gly Arg Leu His His Trp Leu cac cgg ctc cag gag gcc ccc aaa aag gag tcc get ggc tgc ctg gag 595 His Arg Leu Gln Glu Ala Pro Lys Lys G1u Ser Ala Gly Cys Leu Glu gca tct gtc acc ttc aac ctc ttC CgC CtC CtC aCg cga gac ctc aaa 643 Ala Ser Va1 Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys tat gtg gcc gat ggg aac ctg tgt ctg aga acg tca acc cac cct gag 691 Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr His Pro G1u tcc acc tga caccccacac cttatttatg cgctgagccc tactccttcc 740 Ser Thr ttaatttatt tcctctcacc ctttatttat gaagctgcag ccctgactga gacatagggc 800 tgagtttatt gttttacttt tatacattat gcacaaataa acaacaagga attgga 856 <210> 4 <211> 200 <212> PRT
<213> Homo Sapiens <220>
<221> SIGNAL
<222> (1)...(19) <400> 4 Met Ala Ala Ala Trp Thr Val Val Leu Val Thr Leu Val Leu Gly Leu Ala Val Ala Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg G1u Arg Pro Val Ala Leu Glu Ala Glu Leu A1a Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly, Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 5 <211> 734 <212> DNA
<213> Homo Sapiens <220>
<221> sig~eptide <222> (53)...(127) <221> mat_peptide <222> (128)...(655) <221> CDS
<222> (53j...(655) <400> 5 tgggtgacag cctcagagtg tttcttctgc tgacaaagac cagagatcag ga atg aaa 58 Met Lys cta gac atg acc ggg gac tgc atg cca gtg ctg gtg ctg atg gcc gca 106 Leu Asp Met Thr Gly Asp Cys Met Pro Val Leu Val Leu Met Ala Ala gtg ctg acc gtg act gga gca gtt cct gtc gcc agg ctc cgc ggg get 154 Val Leu Thr Val Thr Gly Ala Val Pro Va1 Ala Arg Leu Arg Gly Ala ctc ccg gat gca agg ggc tgc cac ata gcc cag ttc aag tcc ctg tct 202 Leu Pro Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser cca cag gag ctg cag gcc ttt aag agg gcc aaa gat gcc tta gaa gag 250 Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp A1a Leu Glu Glu tcg ctt ctg ctg aag gac tgc aag tgc cgc tcc cgc ctc ttc ccc agg 298 Ser Leu Leu Leu Lys Asp Cys Lys Cys Arg Ser Arg Leu Phe Pro Arg acc tgg gac ctg agg cag ctg cag gtg agg gag CgC CCC gtg get ttg 346 Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Val Ala Leu gag get gag ctg gcc ctg acg ctg aag gtt ctg gag gcc acc get gac 3,,94 Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp act gac cca gcc ctg ggg gat gtc ttg gac cag ccc ctt cac acc ctg 442.
Thr Asp Pro Ala Leu Gly Asp Val Leu Asp Gln Pro Leu His Thr Leu cac cat atc ctc tcc cag ctc cgg gcc tgt atc cag cct cag ccc acg 490 His His Ile Leu Ser Gln Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr gca ggg ccc agg acc cgg ggc cgc ctc cac cat tgg ctg cac cgg ctc 538 Ala G1y Pro Arg Thr Arg Gly Arg Leu His His Trp Leu His Arg Leu cag gag gcc cca aaa aag gag tcc cct ggc tgc ctc gag gcc tct gtc 586 G1n Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val acc ttc aac ctc ttC CgC CtC CtC aCg cga gac ctg aat tgt gtt gcc 634 Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Va1 A1a agc ggg gac ctg tgt gtc tga cccttccgcc agtcatgcaa cctgagattt 685 Ser Gly Asp Leu Cys Val tatttataaa ttagccactt ggcttaattt attgccaccc agtcgctat 734 <210> 6 <211> 200 <212> PRT
<213> Homo Sapiens <220>
<221> SIGNAL
<222> (1)...(25) <400> 6 Met Lys Leu Asp Met Thr Gly Asp Cys Met Pro Val Leu Val Leu Met Ala Ala Val Leu Thr Val Thr Gly Ala Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg G1y Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys Lys Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr A1a Asp Thr Asp Pro Ala Leu Gly Asp Val Leu Asp G1n Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val <210> 7 <211> 633 <212> DNA

<213> Mus musculus <220>

<221> sig~eptide <222> (22)...(105) <221> mat~eptide <222> (106)...(630) <221> CDS

<222> (22)...(630) <400> 7 tcacagaccc cggagagcaa atg aagccagaa acagetggg ggccacatg 51 c Met LysProGlu ThrAlaGly GlyHisMet ctc ctc ctg ctg cctctg ctgctggcc gcagtgctg acaagaacc 99 ttg Leu Leu Leu Leu ProLeu LeuLeuAla AlaValLeu ThrArgThr Leu caa get gac cct cccagg gccaccagg ctcccagtg gaagcaaag 147 gtc Gln Ala Asp Pro ProArg AlaThrArg LeuProVal G1uAlaLys Val gat tgc cac att cagttc aagtctctg tccccaaaa gagctgcag 195 get Asp Cys His Ile GlnPhe LysSerLeu SerProLys GluLeuG1n Ala gcc ttc aaa aag aaggat gccatcgag aagaggctg cttgagaag 243 gcc Ala Phe Lys Lys LysAsp AlaIleGlu LysArgLeu LeuGluLys Ala gacctgaggtgc agttcccac ctcttc cccagggcc tgggacctg aag 291 AspLeuArgCys SerSerHis LeuPhe ProArgAla TrpAspLeu Lys cagctgcaggtc caagagcgc cccaag gccttgcag getgaggtg gcc 339 GlnLeuGlnVal GlnGluArg ProLys A1aLeuGln AlaGluVal Ala ctgaccctgaag gtctgggag aacatg actgactca gccctggcc acc 387 LeuThrLeuLys ValTrpGlu AsnMet ThrAspSer AlaLeuAla Thr atcctgggccag cctcttcat acactg agccacatt cactcccag ctg 435 IleLeuGlyGln ProLeuHis ThrLeu SerHisIle HisSerGln Leu cagacctgtaca cagcttcag gccaca gcagagccc aggtccccg agc 483 GlnThrCysThr GlnLeuGln AlaThr AlaGluPro ArgSerPro Ser cgccgcctctcc cgctggctg cacagg ctccaggag gcccagagc aag 531 ArgArgLeuSer ArgTrpLeu HisArg LeuGlnGlu AlaG1nSer Lys gagacccctggc tgcctggag gcctct gtcacctcc aacctgttt cgc 579 GluThrProGly CysLeuGlu AlaSer ValThrSer AsnLeuPhe Arg ctgctcacccgg gacctcaag tgtgtg gccaatgga gaccagtgt gtc 6<27 '~
' LeuLeuThrArg AspLeuLys CysVal AlaAsnGly AspGlnCys Val tgacct 633 <210>

<211>

<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 Leu Leu Leu Ala Ala Val Leu Thr Arg Thr Gln Ala Asp Pro Val Pro Arg Ala Thr Arg Leu Pro Val Glu Ala Lys Asp Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Lys Glu Leu Gln Ala Phe Lys Lys Ala Lys Asp Ala Ile G1u Lys Arg Leu Leu Glu Lys Asp Leu Arg Cys Ser Ser His Leu Phe Pro Arg Ala Trp Asp Leu Lys Gln Leu Gln Val G1n Glu Arg Pro Lys A1a Leu Gln Ala Glu Val Ala Leu Thr Leu Lys Val Trp Glu Asn Met Thr Asp Ser Ala Leu Ala Thr Ile Leu Gly Gln Pro Leu His Thr Leu Ser His Ile His Ser Gln Leu Gln Thr Cys Thr Gln Leu Gln Ala Thr Ala Glu Pro Arg Ser Pro Ser Arg Arg Leu Ser Arg Trp Leu His Arg Leu Gln Glu Ala Gln Ser Lys Glu Thr Pro Gly Cys Leu G1u Ala Ser Val Thr Ser Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Cys Val A1a Asn Gly Asp Gln Cys Val <210> 9 <211> 632 <212> DNA

<213> Mus musculus <~20>

<221> sig~eptide <222> (22)...(105) ' <221> mat,~eptide <222> (106)...(630) <221> CDS

<222> (22)...(630) <400> 9 tcacagaccc atg aagccagaa acagetggg ggccacatg 51.
cggagagcaa c Met LysProGlu ThrAlaGly GlyHisMet ctc ctc ctgttg cctctg ctgctggcc gcagtgctg acaagaacc 99 ctg Leu Leu LeuLeu ProLeu LeuLeuAla AlaValLeu ThrArgThr Leu caa get cctgtc cccagg gccaccagg ctcccagtg gaagcaaag 147 gac Gln Ala ProVal ProArg AlaThrArg LeuProVal GluAlaLys Asp gat tgc attget cagttc aagtctctg tccccaaaa gagctgcag 195 cac Asp Cys IleAla GlnPhe LysSerLeu SerProLys GluLeuGln His gcc ttc aaggcc aagggt gccatcgag aagaggctg cttgagaag 243 aaa Ala Phe LysAla LysGly AlaIleGlu LysArgLeu LeuGluLys Lys gac atg tgcagt tcccac ctcatctcc agggcctgg gacctgaag 291 agg Asp Met CysSer SerHis LeuIleSer ArgAlaTrp AspLeuLys Arg cag ctg gtccaa gagcgc cccaaggcc ttgcagget gaggtggcc 339 cag Gln Leu ValGln GluArg ProLysAla LeuGlnAla GluValAla Gln ctg acc aaggtc tgggag aacataaat gactcagcc ctgaccacc 387 ctg Leu Thr LysVal TrpGlu AsnI1eAsn AspSerAla LeuThrThr Leu atc ctg cagcct cttcat acactgagc cacattcac tcccagctg 435 ggc Ile Leu GlnPro LeuHis ThrLeuSer HisIleHis SerGlnLeu Gly cag acc acacag cttcag gccacagca gagcccaag cccccgagt 483 tgt Gln Thr ThrGln LeuGln A1aThrAla GluProLys ProProSer Cys cgc cgc tcccgc tggctg cacaggctc caggaggcc cagagcaag 531 ctc Arg Arg Leu Ser Arg Trp Leu His Arg Leu Gln Glu A1a Gln Ser Lys gag act cct ggc tgc ctg gag gac tct gtc acc tcc aac ctg ttt caa 579 Glu Thr Pro Gly Cys Leu Glu Asp Ser Val Thr Ser Asn Leu Phe Gln ctg ctc ctc cgg gac ctc aag tgt gtg gcc agt gga gac cag tgt gtc 627 Leu Leu Leu Arg Asp Leu Lys Cys Val Ala Ser Gly Asp Gln Cys Val tga cc 632 <210> 10 <211> 202 <212> PRT
<213> Mus musculus <220>
<221> SIGNAL
<222> (1)...(28) <400> 10 Met Lys Pro Glu Thr Ala Gly Gly His Met Leu Leu Leu Leu Leu Pro Leu Leu Leu Ala Ala Val Leu Thr Arg Thr Gln Ala Asp Pro Val Pro Arg Ala Thr Arg Leu Pro Val Glu Ala Lys Asp Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Lys Glu Leu Gln Ala Phe Lys Lys Ala Lys Gly A1a Ile Glu Lys Arg Leu Leu Glu Lys Asp Met Arg Cys Ser Ser His Leu Ile Ser Arg Ala Trp Asp Leu Lys Gln Leu Gln Val Gln Glu Arg Pro Lys Ala Leu Gln Ala Glu Val Ala Leu Thr Leu Lys Val Trp Glu Asn Ile Asn Asp Ser Ala Leu Thr Thr Ile Leu Gly Gln Pro Leu His Thr Leu Ser His Ile His Ser Gln Leu Gln Thr Cys Thr Gln Leu Gln Ala Thr Ala Glu Pro Lys Pro Pro Ser Arg Arg Leu Ser Arg Trp Leu His Arg Leu Gln Glu Ala Gln Ser Lys Glu Thr Pro Gly Cys Leu Glu Asp Ser Val Thr Ser Asn Leu Phe Gln Leu Leu Leu Arg Asp Leu Lys Cys Va1 Ala Ser Gly Asp Gln Cys Val <210> 11 <211> 520 <212> PRT
<213> Homo sapiens <400> 11 Met A1a G1y Pro Glu Arg Trp Gly Pro Leu Leu Leu Cys Leu Leu Gln Ala Ala Pro Gly Arg Pro Arg Leu Ala Pro Pro Gln Asn Val Thr Leu Leu Ser Gln Asn Phe Ser Val Tyr Leu Thr Trp Leu Pro Gly Leu Gly Asn Pro G1n Asp Val Thr Tyr Phe Val Ala Tyr Gln Ser Ser Pro Thr Arg Arg Arg Trp Arg Glu Val Glu Glu Cys Ala Gly Thr Lys Glu Leu Leu Cys Ser Met Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys Phe Lys G1y Arg Val Arg Thr Val Ser Pro Ser Ser Lys Ser Pro Trp Val Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Val Glu Pro A1a Pro Pro Val Leu Val Leu Thr Gln Thr Glu Glu Ile Leu Ser A1a Asn Ala Thr Tyr Gln Leu Pro Pro Cys Met Pro Pro Leu Asp Leu Lys Tyr Glu Val A1a Phe Trp Lys Glu Gly Ala Gly Asn Lys Thr Leu Phe Pro Val Thr Pro His Gly Gln Pro Val Gln I1e Thr Leu Gln Pro Ala Ala Ser Glu His His Cys Leu Ser Ala Arg Thr Ile Tyr Thr Phe Ser Val Pro Lys Tyr Ser Lys Phe Ser Lys Pro Thr Cys Phe Leu Leu Glu Val Pro Glu Ala Asn Trp Ala Phe Leu Val Leu Pro Ser Leu Leu Ile Leu Leu Leu Val Ile Ala Ala Gly Gly Val Ile Trp Lys Thr Leu Met Gly Asn Pro Trp Phe Gln Arg Ala Lys Met Pro Arg Ala Leu Asp Phe Ser Gly His Thr His Pro Val Ala Thr Phe Gln Pro Ser Arg Pro Glu Ser Val Asn , Asp Leu Phe Leu Cys Pro Gln Lys Glu Leu Thr Arg Gly Val Arg Pro Thr Pro Arg Val Arg Ala Pro Ala Thr Gln Gln Thr Arg Trp Lys Lys Asp Leu Ala Glu Asp Glu Glu Glu Glu Asp Glu Glu Asp Thr Glu Asp Gly Val Ser Phe Gln Pro Tyr Ile Glu Pro Pro Ser Phe Leu Gly Gln Glu His Gln Ala Pro Gly His Ser Glu Ala Gly Gly Val Asp Ser Gly Arg Pro Arg Ala Pro Leu Val Pro Ser Glu Gly Ser Ser Ala Trp Asp Ser Ser Asp Arg Ser Trp Ala Ser Thr Val Asp Ser Ser Trp Asp Arg Ala Gly Ser Ser Gly Tyr Leu Ala Glu Lys Gly Pro Gly Gln Gly Pro Gly Gly Asp Gly His Gln Glu Ser Leu Pro Pro Pro Glu Phe Ser Lys Asp Ser G1y Phe Leu Glu Glu Leu Pro Glu Asp Asn Leu Ser Ser Trp A1a Thr Trp Gly Thr Leu Pro Pro Glu Pro Asn Leu Val Pro Gly Gly Pro Pro Val Ser Leu Gln Thr Leu Thr Phe Cys Trp G1u Ser Ser Pro Glu Glu Glu Glu Glu Ala Arg Glu Ser Glu Ile Glu Asp Ser Asp Ala Gly Ser Trp Gly Ala Glu Ser Thr Gln Arg Thr G1u Asp Arg Gly Arg Thr Leu Gly His Tyr Met Ala Arg <210> 12 <211> 531 <212> DNA
<213> Artificial Sequence <220>

<223> SEQID
mature N0:
protein 1, of with 3' Met added <221>
CDS

<222> )...(531) (1 <400>

atggttcct gtcgccagg ctccacggg getctcccg gatgca aggggc 48 MetVa1Pro ValAlaArg LeuHisGly AlaLeuPro AspAla ArgGly tgccacata gcccagttc aagtccctg tctccacag gagctg caggcc 96 CysHisIle AlaGlnPhe LysSerLeu SerProGln GluLeu GlnAla tttaagagg gccaaagat gccttagaa gagtcgctt ctgctg aaggac 144 PheLysArg AlaLysAsp AlaLeuGlu GluSerLeu LeuLeu LysAsp tgcaggtgc cactcccgc ctcttcccc aggacctgg gacctg aggcag 192 CysArgCys HisSerArg LeuPhePro ArgThrTrp AspLeu ArgGln ctgcaggtg agggagcgc cccatgget ttggagget gagctg gccctg 240 LeuGlnVal ArgGluArg ProMetAla LeuGluAla GluLeu AlaLeu acgctgaag gttctggag gccaccget gacactgac ccagcc ctggtg 288:

ThrLeuLys ValLeuGlu AlaThrAla AspThrAsp ProAla LeuVal gacgtcttg gaccagccc cttcacacc ctgcaccat atcctc tcccag 336 AspVa1Leu AspGlnPro LeuHisThr LeuHisHis IleLeu SerGln ttccgggcc tgtatccag cctcagccc acggcaggg cccagg acccgg 384 PheArgAla CysIleGln ProGlnPro ThrAlaGly ProArg ThrArg ggccgcctc caccattgg ctgtaccgg ctccaggag gcccca aaaaag 432 GlyArgLeu HisHisTrp LeuTyrArg LeuGlnGlu AlaPro LysLys gagtcccct ggctgcctc gaggcctct gtcaccttc aacctc ttccgc 480 GluSerPro GlyCysLeu GluAlaSer ValThrPhe AsnLeu PheArg ctcctcacg cgagacctg aattgtgtt gccagtggg gacctg tgtgtc 528 LeuLeuThr ArgAspLeu AsnCysVal AlaSerGly AspLeu CysVal tga 531 <210>

<211>

<212>
PRT

<213>
Artificial Sequence <220>
<223> mature protein of SEQ ID N0: 1, with 3' Met added <400> 13 Met Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys Arg Cys His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr A1a Asp Thr Asp Pro Ala Leu Val Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Phe Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val <210>

<211>

<212>
DNA

<213> Sequence Artificial <220>

<223> tein SEQ ID
mature of N0:
pro 3, with 3' Met added <221>
CDS

<222> (549) (1)...

<400>

atg ggc gtc cccacttcc aagccc accacaact gggaagggc tgc 48 cct Met Gly Val ProThrSer LysPro ThrThrThr GlyLysG1y Cys Pro cac att agg ttcaaatct ctgtca ccacaggag ctagcgagc ttc 96 ggc His Ile Arg PheLysSer LeuSer ProGlnGlu LeuAlaSer Phe Gly aag aag agg gacgccttg gaagag tcactcaag ctgaaaaac tgg 144 gcc Lys Lys Arg AspAlaLeu GluGlu SerLeuLys LeuLysAsn Trp Ala agt tgc tct cctgtcttc cccggg aattgggac ctgaggctt ctc 192 agc Ser Cys Ser ProValPhe ProGly AsnTrpAsp LeuArgLeu Leu Ser cag gtg gag cgccctgtg gccttg gaggetgag ctggccctg acg 240 agg Gln Val Glu ArgProVal AlaLeu GluAlaGlu LeuAlaLeu Thr Arg ctg aag ctg gaggccget getggc ccagccctg gaggacgtc cta 288 gtc Leu Lys Leu GluAlaAla AlaGly ProAlaLeu GluAspVal Leu Val gac cag ctt cacaccctg caccac atcctctcc cagctccag gcc 336 ccc Asp Gln Leu HisThrLeu HisHis IleLeuSer GlnLeuGln Ala Pro tgt atc cct cagcccaca gcaggg cccaggccc cggggccgc ctc 384 cag Cys Ile Pro GlnProThr AlaGly ProArgPro ArgGlyArg Leu Gln cac tggctg caccggctc caggag gcccccaaa aaggagtcc get 432 cac His TrpLeu HisArgLeu GlnG1u AlaProLys LysGluSer Ala His ggc ctggag gcatctgtc accttc aacctcttc cgcctcctc acg 480 tgc Gly LeuGlu AlaSerVal ThrPhe AsnLeuPhe ArgLeuLeu Thr Cys cga ctcaaa tatgtggcc gatggg aacctgtgt ctgagaacg tca 528 gac Arg LeuLys TyrValAla AspGly AsnLeuCys LeuArgThr Ser Asp acc cctgag tccacctga caccccacac tttatg 579 cac ctta cgctgagccc Thr ProG1u SerThr His tactccttcc ttaatttatt ctttatttat ga 621 tcctctcacc <210>

<211> 2 <212>
PRT

<213>
Artificial Sequence <220>

<223> SEQ ID 3' mature N0: Met protein 3, added of with <400>

Met ProVal ProThrSer LysPro ThrThrThr GlyLysGly Cys Gly His GlyArg PheLysSer LeuSer ProGlnG1u LeuAlaSer Phe Ile Lys AlaArg AspAlaLeu GluGlu SerLeuLys LeuLysAsn Trp Lys Ser SerSer ProValPhe ProGly AsnTrpAsp LeuArgLeu Leu Cys Gln ArgGlu ArgProVal AlaLeu GluAlaGlu LeuAlaLeu Thr Val Leu ValLeu GluAlaAla AlaG1y ProAlaLeu GluAspVal Leu Lys Asp ProLeu HisThrLeu HisHis IleLeuSer GlnLeuGln Ala Gln Cys GlnPro GlnProThr AlaGly ProArgPro ArgGlyArg Leu Ile His TrpLeu HisArgLeu GlnGlu AlaProLys LysGluSer Ala His Gly LeuGlu AlaSerVal ThrPhe AsnLeuPhe ArgLeuLeu Thr Cys Arg LeuLys TyrValAla AspGly AsnLeuCys LeuArgThr Ser Asp Thr ProGlu SerThr His <210>

<211> 1 <212>
DNA

<213> icial Artif Sequence <220>
<223> mature protein of SEQ ID NO: 5, with 3' Met added <221> CDS
<222> (1)...(531) <400>

atggtt cctgtc gccaggCtC CgCggg getctcccg gatgcaagg ggc 48 MetVal ProVal AlaArgLeu ArgGly AlaLeuPro AspAlaArg Gly tgccac atagcc cagttcaag tccctg tctccacag gagctgcag gcc 96 CysHis IleAla GlnPheLys SerLeu SerProGln G1uLeuGln Ala tttaag agggcc aaagatgcc ttagaa gagtcgctt ctgctgaag gac 144 PheLys ArgAla LysAspAla LeuGlu GluSerLeu LeuLeuLys Asp tgcaag tgccgc tcccgcctc ttcCcc aggacctgg gacctgagg cag 192 CysLys CysArg SerArgLeu PhePro ArgThrTrp AspLeuArg Gln ctgcag gtgagg gagcgcccc gtgget ttggagget gagctggcc ctg 240 LeuGln ValArg GluArgPro ValAla LeuGluAla GluLeuAla Leu acgctg aaggtt ctggaggcc accget gacactgac ccagccctg ggg 288 ThrLeu LysVal LeuGluAla ThrAla AspThrAsp ProAlaLeu Gly gatgtc ttggac cagcccctt cacacc ctgcaccat atcctctcc cag 336 AspVal LeuAsp GlnProLeu HisThr LeuHisHis IleLeuSer Gln ctccgg gcctgt atccagcct cagccc acggcaggg cccaggacc cgg 384 LeuArg AlaCys IleGlnPro GlnPro ThrAlaGly ProArgThr Arg ggccgc ctccac cattggctg caccgg ctccaggag gccccaaaa aag 432 GlyArg LeuHis HisTrpLeu His.ArgLeuGlnGlu AlaProLys Lys gagtcc cctggc tgCCtCgag gcctct gtcaccttc aacctcttc cgc 480 GluSer ProGly CysLeuGlu AlaSer ValThrPhe AsnLeuPhe Arg ctcctc acgcga gacctgaat tgtgtt gccagcggg gacctgtgt gtc 538 LeuLeu ThrArg AspLeuAsn CysVal AlaSerGly AspLeuCys Val tga 531 <210>

<211>

<212>
PRT

<213> icial Artif Sequence <220>
<223> mature protein of SEQ ID N0: 5, with 3' Met added <400> 17 Met Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys Lys Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg G1n Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Gly Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg G1y Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val <210>

<211>

<212>
DNA

<213>
Artificial Sequence <220>

<223> C48S

mutant <221>
CDS

<222> (528) (1)...

<400>

gtt gtc gccaggctccac ggggetctc ccggatgca aggggc tgc 48 cct Val Val AlaArgLeuHis GlyAlaLeu ProAspAla ArgGly Cys Pro 1 5 l0 15 cac gcc cagttcaagtcc ctgtctcca caggagctg caggcc ttt 96 ata His Ala GlnPheLysSer LeuSerPro GlnGluLeu GlnAla Phe Ile aag gcc aaagatgcctta gaagagtcg cttctgctg aaggac tcc 144 agg Lys Ala LysAspAlaLeu GluGluSer LeuLeuLeu LysAsp Ser Arg agg cac tcccgcctcttc cccaggacc tgggacctg aggcag ctg 192 tgc ' Arg His SerArgLeuPhe ProArgThr TrpAspLeu ArgGln Leu Cys cag agg gagcgccccatg getttggag getgagctg gccctg acg 240 gtg Gln Arg GluArgProMet AlaLeuGlu AlaGluLeu AlaLeu Thr Val ctg gtt ctggaggccacc getgacact gacccagcc ctggtg gac 288 aag Leu Val LeuGluAlaThr AlaAspThr AspProAla LeuVal Asp Lys gtc gac cagccccttcac accctgcac catatcctc tcccag ttc 336 ttg Val Asp GlnProLeuHis ThrLeuHis HisI1eLeu SerGln Phe Leu cgg tgt atccagcctcag cccacggca gggcccagg acccgg ggc 384 gcc Arg Cys IleGlnProGln ProThrAla GlyProArg ThrArg Gly Ala cgc cac cattggctgtac cggctccag gaggcccca aaaaag gag 432 ctc Arg His HisTrpLeuTyr ArgLeuGln GluAlaPro LysLys Glu Leu tcc cct ggc tgc ctc gag gcc tct gtc acc ttc aac ctc ttc cgc ctc 480 Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu ctc acg cga gac ctg aat tgt gtt gcc agt ggg gac ctg tgt gtc tga 528 Leu Thr Arg Asp Leu Asn Cys Val Ala Ser G1y Asp Leu Cys Val <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 His Ile Ala G1n Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Ser 35 40 45 , Arg Cys His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Va1 Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Val Asp Val Leu Asp G1n Pro Leu His Thr Leu His His Ile Leu Ser Gln Phe Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val <210> 20 <211> 531 <212> DNA
<213> Artificial Sequence <220>
<223> met IL-28A mutant C49S
<221> CDS
<222> (1)...(531) <400> 20 atg gtt cct gtc gcc agg ctc cac ggg get ctc ccg gat gca agg ggc 48 Met Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg,Gly tgc cac ata gcc cag ttc aag tcc ctg tct cca cag gag ctg cag gcc 96 Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala 20 25 ~ 30 ttt aag agg gcc aaa gat gcc tta gaa gag tcg ctt ctg ctg aag gac 144 Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp tccaggtgccac tcccgcctc ttCCCC aggacctgg gacctgagg cag 192 SerArgCysHis SerArgLeu PhePro ArgThrTrp AspLeuArg Gln ctgcaggtgagg gagcgcccc atgget ttggagget gagctggcc ctg 240 LeuGlnValArg GluArgPro MetAla LeuGluAla GluLeuAla Leu acgctgaaggtt ctggaggcc accget gacactgac ccagccctg gtg 288 ThrLeuLysVal LeuGluAla ThrAla AspThrAsp ProAlaLeu Val gacgtcttggac cagcccctt cacacc ctgcaccat atcctctcc cag 336 AspValLeuAsp GlnProLeu HisThr LeuHisHis IleLeuSer Gln ttccgggcctgt atccagcct cagccc acggcaggg cccaggacc cgg 384 PheArgAlaCys IleGlnPro GlnPro ThrAlaGly ProArgThr Arg ggccgcctccac cattggctg taccgg ctccaggag gccccaaaa aag 432 GlyArgLeuHis HisTrpLeu TyrArg LeuGlnGlu AlaProLys Lys gagtcccctggc tgCCtCgag gcctct gtcaccttc aacctcttc cgc 480 GluSerProGly CysLeuGlu AlaSer ValThrPhe AsnLeuPhe Arg ctcctcacgcga gacctgaat tgtgtt gccagtggg gacctgtgt gtc 5.28.

LeuLeuThrArg AspLeuAsn CysVal AlaSerGly AspLeuCys Val tga 531 <210>

<211>

<212>
PRT

<213>
Artificial Sequence <220>
<223> met IL-28A mutant C49S
<400> 21 Met Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg G1y Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Ser Arg Cys His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr A1a Asp Thr Asp Pro Ala Leu Val Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Phe Arg Ala Cys Ile Gln Pro G1n Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val <~10> 22 <211> 528 <212> DNA
<213> Artificial Sequence <220>
<~23> IL-28A mutant C50S
<221> CDS
<222> (1)...(528) <400> ~2 gtt cct gtc gcc agg ctc cac ggg get ctc ccg gat gca agg ggc tgc 48 Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly Cys cac ata gcc cag ttc aag tcc ctg tct cca cag gag ctg cag gcc ttt 96 His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe aag agg gcc aaa gat gcc tta gaa gag tcg ctt ctg ctg aag gac tgc 144 Lys Arg A1a Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys agg tcc cac tcc cgc ctc ttc ccc agg acc tgg gac ctg agg cag ctg 192.
Arg Ser His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu cag gtg agg gag cgc ccc atg get ttg gag get gag ctg gcc ctg acg 240 Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu Thr ctg aag gtt ctg gag gcc acc get gac act gac cca gcc ctg gtg gac 288 Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Val Asp gtc ttg gac cag ccc ctt cac acc ctg cac cat atc ctc tcc cag ttc 336 Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Phe cgg gcc tgt atc cag cct cag ccc acg gca ggg ccc agg acc cgg ggc 384 Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly cgc ctc cac cat tgg ctg tac cgg ctc cag gag gcc cca aaa aag gag 432 Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu tcc cct ggc tgc ctc gag gcc tct gtc acc ttc aac ctc ttc cgc ctc 480 Ser Pro G1y Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu ctc acg cga gac ctg aat tgt gtt gcc agt ggg gac ctg tgt gtc tga 528 Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val <210> 23 <211> 175 <212> PRT
<213> Artificial Sequence <220>
<223> IL-28A mutant C50S
<400> 23 Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys Arg Ser His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu A1a Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Va1 Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Phe Arg A1a Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val <210> 24 <211> 531 <212> DNA
<213> Artificial Sequence <220>
<223> met IL-28A mutant C51S
<221>
CDS

<222> )...(531) (1 <400>

atggtt cctgtcgcc aggctc cacgggget ctcccggat gcaaggggc 48 MetVal ProValAla ArgLeu HisGlyAla LeuProAsp AlaArgGly tgccac atagcccag ttcaag tccctgtct ccacaggag ctgcaggcc 96 CysHis IleAlaGln PheLys SerLeuSer ProGlnGlu LeuGlnAla tttaag agggccaaa gatgcc ttagaagag tcgcttctg ctgaaggac 144 PheLys ArgAlaLys AspAla LeuGluGlu SerLeuLeu LeuLysAsp tgcagg tcccactCC CgCCtC ttccccagg acctgggac ctgaggcag 192 CysArg SerHisSer ArgLeu PheProArg ThrTrpAsp LeuArgGln ctgcag gtgagggag cgcccc atggetttg gaggetgag ctggccctg 240 LeuGln ValArgGlu ArgPro MetAlaLeu GluAlaGlu LeuAlaLeu acgctg aaggttctg gaggcc accgetgac actgaccca gccctggtg 288 ThrLeu LysValLeu GluAla ThrAlaAsp ThrAspPro AlaLeuVal gac gtc ttg gac cag CCC Ctt cac acc ctg cac cat atC CtC tcc cag 336 AspValLeuAsp GlnPro LeuHisThr LeuHisHis IleLeuSer Gln ttccgggcctgt atccag cctcagccc acggcaggg cccaggacc cgg 384 PheArgAlaCys IleGln ProGlnPro ThrAlaGly ProArgThr Arg ggccgcctccac cattgg ctgtaccgg ctccaggag gccccaaaa aag 432 GlyArgLeuHis HisTrp LeuTyrArg LeuGlnGlu AlaProLys Lys gagtcccctggc tgcctc gaggcctct gtcaccttc aacctcttc cgc 480 GluSerProGly CysLeu GluAlaSer ValThrPhe AsnLeuPhe Arg ctcctcacgcga gacctg aattgtgtt gccagtggg gacctgtgt gtc 528 LeuLeuThrArg AspLeu AsnCysVal A1aSerGly AspLeuCys Val tga 531 <310> 25 <211> 176 <212> PRT
<213> Artificial Sequence <220>
<223> met IL-28A mutant C51S
<400> 25 Met Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys Arg Ser His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala G1u Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr A1a Asp Thr Asp Pro Ala Leu Val Asp Va1 Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Phe Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Va1 <210> 26 <211> 546 <212> DNA
<213> Artificial Sequence <220>
<223> IL-29 mutant C171S
<221> CDS

<222> (546) (1)...

<400>

ggtccggttccg acctctaaa ccaaccacc actggt aaaggttgc cac 48 GlyProValPro ThrSerLys ProThrThr ThrGly LysGlyCys His atcggtcgtttc aaatctctg tctccgcag gaactg gettctttc aaa 96 IleGlyArgPhe LysSerLeu SerProG1n GluLeu AlaSerPhe Lys aaagetcgtgac getctggaa gaatctctg aaactg aaaaactgg tct 144 LysAlaArgAsp AlaLeuGlu GluSerLeu LysLeu LysAsnTrp Ser tgctcttctccg gttttcccg ggtaactgg gatctg cgtctgctg cag 192 CysSerSerPro ValPhePro GlyAsnTrp AspLeu ArgLeuLeu Gln gttcgtgaacgt ccggttget ctggaaget gaactg getctgacc ctg 240 ValArgGluArg ProValAla LeuGluAla GluLeu AlaLeuThr Leu aaagttctggaa getgetgca ggtcctget ctggaa gatgttctg gat 288 LysValLeuGlu AlaAlaAla GlyProAla LeuGlu AspValLeu Asp cagccgctgcac actctgCa.CCaCatCCtg tCtcag ctgcagget tgc 336 GlnProLeuHis ThrLeuHis HisIleLeu SerGln LeuGlnAla Cys attcaaccgcaa ccgaccget ggtccgcgt ccgcgt ggtcgtctg cac 384 IleGlnProGln ProThrAla GlyProArg ProArg GlyArgLeu His cactggctgcat cgtctgcag gaagetccg aaaaaa gaatctget ggt 432 HisTrpLeuHis ArgLeuGln GluAlaPro LysLys GluSerAla Gly tgcctggaaget tctgttacc ttcaacctg ttccgt ctgctgacc cgt 480 CysLeuGluAla SerValThr PheAsnLeu PheArg LeuLeuThr Arg 145 150 155 16.0 gatctgaaatac gttgetgat ggtaacctg tctctg cgtacctct acc 528 AspLeuLysTyr ValAlaAsp GlyAsnLeu SerLeu ArgThrSer Thr catccggaatct acctaa 546 HisProGluSer Thr <210> 27 <211> 181 <212> PRT
<213> Artificial Sequence <220>
<223> IL-29 mutant C171S
<400> 27 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln G1u Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala A1a Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Ser Leu Arg Thr Ser Thr 165 x.70 175 His Pro Glu Ser Thr <2l0>

<211> 9 <212>
DNA

<213> tificial Sequence Ar <220>

<223> t mutant me IL-29 C172S

<221> S
CD

<222> )...(549) (1 <400>

atg ccggtt ccgacctct aaaccaacc accactggt aaaggt tgc 48 ggt Met ProVal ProThrSer LysProThr ThrThrGly LysGly Cys Gly cac ggtcgt ttcaaatct ctgtctccg caggaactg gettct ttc 96 atc His GlyArg PheLysSer LeuSerPro GlnG1uLeu AlaSer Phe Ile aaa getcgt gacgetctg gaagaatct ctgaaactg aaaaac tgg 144 aaa Lys AlaArg AspAlaLeu GluGluSer LeuLysLeu LysAsn Trp Lys tct tcttct ccggttttc ccgggtaac tgggatctg cgtctg ctg 192 tgc Ser SerSer ProValPhe ProGlyAsn TrpAspLeu ArgLeu Leu Cys cag cgtgaa cgtccggtt getctggaa getgaactg getctg acc 240 gtt Gln ArgG1u ArgProVal AlaLeuGlu AlaGluLeu AlaLeu Thr Val ctg gttctg gaagetget gcaggtcct getctggaa gatgtt ctg 288 aaa Leu ValLeu GluA1aAla AlaGlyPro AlaLeuGlu AspVal Leu Lys gat ccgctg cacactctg caccacatc ctgtctcag ctgcag get 336 cag Asp ProLeu HisThrLeu HisHisIle LeuSerGln LeuGln Ala Gln 100 l05 110 tgc caaccg caaccgacc getggtccg cgtccgcgt ggtcgt ctg 384 att Cys GlnPro GlnProThr AlaGlyPro ArgProArg GlyArg Leu I1e cac cac tgg ctg cat cgt ctg cag gaa get ccg aaa aaa gaa tct get 432 His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala ggt tgc ctg gaa get tct gtt acc ttc aac ctg ttc cgt ctg ctg acc 480 Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr cgt gat ctg aaa tac gtt get gat ggt aac ctg tct ctg cgt acc tct 528 Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Ser Leu Arg Thr Ser acc cat ccg gaa tct acc taa 549 Thr His Pro Glu Ser Thr <210> 29 <211> 182 <212> PRT
<213> Artificial Sequence <220>
<223> met IL-29 mutant C172S
<400> 29 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile G1y Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro A1a Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu G1n Glu Ala Pro Lys Lys Glu Ser Ala G1y Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Ser Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 30 <211> 525 <212> DNA
<213> Artificial Sequence <220>
<223> degenerate sequence of SEQ ID N0: 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 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 480 ytnacnmgng ayytnaaytg ygtngcnwsn ggngayytnt gygtn 525 <210> 31 <211> 525 <212> DNA
<213> Artificial Sequence <220>
<223> degenerate sequence of SEQ ID N0: 20 <221> misc_feature <222> (1). .(525) <223> n = A,T,C or G
<400> 31 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 480.;
ytnacnmgng ayytnaaytg ygtngcnwsn ggngayytnt gygtn 525 <210> 32 <211> 525 < 212 > DTQA
<213> Artificial Sequence <320>
<223> degenerate sequence of SEQ ID N0: 22 <221> misc_feature <222> (1). .(525) <223> n = A,T,C or G
<400> 32 gtnccngtng cnmgnytnca yggngcnytn ccngaygcnm gnggntgyca yathgcncar 60 ttyaarwsny tnwsnccnca rgarytncar gcnttyaarm gngcnaarga ygcnytngar 120 garwsnytny tnytnaarga ywsnmgritgy 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 480 ytnacnmgng ayytnaaytg ygtngcnwsn ggngayytnt gygtn 525 <210> 33 <211> 525 <212> DNA
<213> Artificial Sequence <220>
<223> degenerate sequence of SEQ ID N0: 24 <221> misc_feature <222> (1). .(525) <223> n = A,T,C or G

<400> 33 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 480 ytnacnmgng ayytnaaytg ygtngcnwsn ggngayytnt gygtn 525 <210> 34 <211>~ 525 <212> DNA
<213> Artificial Sequence <220>
<223> degenerate sequence of SEQ ID N0: 26 <221> misc_feature <222> (1). .(525) <223> n = A,T,C or G
<400> 34 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 480 ytnacnmgng ayytnaaytg ygtngcnwsn ggngayytnt gygtn 525.
<210> 35 <211> 525 <212> DNA
<213> Artificial Sequence <220>
<223> degenerate sequence of SEQ ID NO: 28 <221> misc_feature <222> (1). .(525) <223> n = A,T,C or G
<400> 35 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 480 ytnacnmgng ayytnaaytg ygtngcnwsn ggngayytnt gygtn 525 <210> 36 <211> 175 <212> PRT
<213> Artificial Sequence <220>
<223> IL-28A mutant C48X
<221> VARIANT

<~22> (48)...(48) <2~3> Xaa = Ser, Ala, Thr, Val or Asn <400> 36 Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly Cys His Ile Ala G1n Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Xaa Arg Cys His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu G1n Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Val Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Phe Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro G1y Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val <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 <400> 37 Met Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg G1y Cys His Ile A1a Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Xaa Arg Cys His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Val Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Phe Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln G1u Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu A1a Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser G1y Asp Leu Cys Val <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, Val or Asn <400> 38 Va1 Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys Arg Xaa His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Val Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Phe Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val <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, Val or Asn <400> 39 Met Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys Arg Xaa His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Va1 Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Phe Arg Ala Cys Ile G1n Pro Gln Pro Thr Ala G1y Pro Arg Thr Arg Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln G1u Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val <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, Val or Asn <400> 40 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Va1 Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 41 <211> 182 <212> PRT
<213> Artificial Sequence <220>
<223> met IL-29 mutant C172X
<221> VARIANT
<222> (172)...(172) <223> Xaa = Ser, Ala, Thr, Val or Asn <400> 41 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Va1 Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <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 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 50 <210> 45 <211> 42 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC29741 <400> 45 tctgatttaa tctgtatcag gctgaaaatc ttatctcatc cg 42 <210> 46 <211> 62 <212> DNA
<313> Artificial Sequence <220>
<223> oligonucleotide primer ZC29736 <400> 46 gtggaattgt gagcggataa caatttcaca cagaattcat taaagaggag aaattaactc 60 cc 62 <210> 47 <211> 63 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC29738 <400> 47 gctgaaaatc ttatctcatc cgccaaaaca cccgggagtt aatttctcct ctttaatgaa 60 ttc 63 <210> 48 <211> 78 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC44566 <400> 48 tcttccagag cgtcacgagc ttttttgaaa gaagccagtt cctgcggaga cagagatttg 60 aaacgaccga tgtggcaa 78 <210> 49 <211> 84 <212> DNA
<213> Artificial Sequence <220>
<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 <400> 50 aacagaagct tccaggcaac cagcagattc ttttttcgga gcttcctgca gacgatgcag 60 ccagtggtgc a 71 <210> 51 <211> 73 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC44563 <400> 51 aactggctct gaccctgaaa gttctggaag ctgctgcagg tcctgctctg gaagatgttc 60 tggatcagcc get 73 <210> 52 ' <211> 74 <312> 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 <220>
<223> oligonucleotide primer ZC44561 <400> 53 tcagctgcag gcttgcattc aaccgcaacc gaccgctggt ccgcgtccgc gtggtcgtct 60 gcaccactgg ctgcat ' 76 <210> 54 <211> 60 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC44560 <400> 54 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 tt 62 <210> 56 <211> 65 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC44558 <400> 56 ttaggtagat tccggatggg tagaggtacg caggcacagg ttaccatcag caacgtattt 60 cagat 65 <210> 57 <211> 69 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC44557 <400> 57 tgcctggaag cttctgttac cttcaacctg ttccgtctgc tgacccgtga tctgaaatac 60 gttgctgat <210> 58 <211> 41 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC44340 <400> 58 cgttgctgat ggtaacctgt ctctgcgtac ctctacccat c 41 <210> 59 , <211> 41 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC44341 <400> 59 gatgggtaga ggtacgcaga gacaggttac catcagcaac g 41 <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 tctgtatcag gctgaaaatc ttatctcatc cgccaaaaca ttaggtggac tcagggtggg 60 ttgacgt 67 <210> 62 <211> 65 <212> DNA
<213> Artificial Sequence <320>
<223> oligonucleotide primer ZC43431 <400> 62 ctagaaataa ttttgtttaa ctttaagaag gagatatata tatggttcct gtcgccaggc 60 tccac 65 <210> 63 <211> 67 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC43437 <400> 63 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 39 <210> 65 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC44328 <400> 65 gtgggtt.gac gttctcaggg acaggttccc atcggccac 39 <210> 66 <211> 83 <212> DNA
<213> Artificial Sequence <230> .
<223> oligonucleotide primer ZC45399 <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
<222> (1)...(531) <400> 67 atg gtt cct gtc gcc agg ctc cac ggg get ctc ccg gat gca agg ggc 48 Met Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly tgc cac ata gcc cag ttc aag tcc ctg tct cca cag gag ctg cag gcc 96 Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala tttaagagggccaaa gatgcc ttagaagag tcgctt ctgctgaag gac 144 PheLysArgAlaLys AspAla LeuGluGlu SerLeu LeuLeuLys Asp tgcaggtgccactcc cgcctc ttccccagg acctgg gacctgagg cag 192 CysArgCysHisSer ArgLeu PheProArg ThrTrp AspLeuArg Gln ctgcaggtgagggag cgcccc atggetttg gagget gagctggcc ctg 240 LeuGlnValArgGlu ArgPro MetA1aLeu G1uAla GluLeuAla Leu acgctgaaggttCtg gaggCC aCCgetgac actgac ccagccctg gtg 288 ThrLeuLysValLeu GluAla ThrAlaAsp ThrAsp ProA1aLeu Val gacgtcttggaccag CCCCtt CaCaCCCtg caccat atcCtCtcc cag 336 AspValLeuAspGln ProLeu HisThrLeu HisHis IleLeuSer Gln ttccgggcctgtatc cagcct cagcccacg gcaggg cccaggacc cgg 384 PheArgAlaCysIle GlnPro GlnProThr AlaGly ProArgThr Arg ggccgcctccaccat tggctg taccggctc caggag gccccaaaa aag 432 GlyArgLeuHisHis TrpLeu TyrArgLeu GlnGlu AlaProLys Lys gagtcccctggctgc ctcgag gcctctgtc accttc aacctcttc cgc 480 GluSerProGlyCys LeuG1u AlaSerVal ThrPhe AsnLeuPhe Arg ctcctcacgcgagac ctgaat tgtgttgcc agtggg gacctgtgt gtc 528 LeuLeuThrArgAsp LeuAsn CysVa1Ala SerGly AspLeuCys Val tga 531 <210> 68 <211> 83 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC45398 <400> 68 ggccaaagat gccttagaag agtcgcttct gctgaaggac tCCaggtgCC aCtCCCgCCt 60 cttccccagg acctgggacc tga 83 <210> 69 <211> 83 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC45397 <400> 69 gctgcctcag gtcccaggtc ctggggaaga ggcgggagtg ggacctgcag tccttcagca 60 gaagcgactc ttctaaggca tct 83 <210> 70 <211> 83 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC45396 <400> 70 agatgcctta gaagagtcgc ttctgctgaa ggaCtgCagg tCCCaCtCCC gCCtCttCCC 60 caggacctgg gacctgaggc agc 83 <210> 71 <211> 1013 <212> DNA
<213> Homo Sapiens <220>
<221> CDS
<222> (14)...(991) <400> 71 ccagcgtccg tcc atg gcg tgg agc ctt ggg agc tgg ctg ggt ggc tgc 49 Met Ala Trp Ser Leu Gly Ser Trp Leu Gly Gly Cys ctg ctg gtg tca gca ttg gga atg gta cca cct ccc gaa aat gtc aga 97 Leu Leu Val Ser Ala Leu Gly Met Val Pro Pro Pro Glu Asn Val Arg atg aat tct gtt aat ttc aag aac att cta cag tgg gag tca cct get 145-.
Met Asn Ser Val Asn Phe Lys Asn Ile Leu Gln Trp G1u Ser Pro Ala ttt gcc aaa ggg aac ctg act ttc aca get cag tac cta agt tat agg 193 Phe Ala Lys Gly Asn Leu Thr Phe Thr Ala Gln Tyr Leu Ser Tyr Arg ata ttc caa gat aaa tgc atg aat act acc ttg acg gaa tgt gat ttc 241 Ile Phe G1n Asp Lys Cys Met Asn Thr Thr Leu Thr Glu Cys Asp Phe tca agt ctt tcc aag tat ggt gac cac acc ttg aga gtc agg get gaa 289 Ser Ser Leu Ser Lys Tyr Gly Asp His Thr Leu Arg Va1 Arg Ala Glu ttt gca gat gag cat tca gac tgg gta aac atc acc ttc tgt cct gtg 337 Phe Ala Asp Glu His Ser Asp Trp Val Asn Ile Thr Phe Cys Pro Val gat gac acc att att gga ccc cct gga atg caa gta gaa gta ctt get 385 Asp Asp Thr Ile Ile Gly Pro Pro Gly Met Gln Val Glu Val Leu Ala gat tct tta cat atg cgt ttc tta gCC CCt aaa att gag aat gaa tac 433 Asp Ser Leu His Met Arg Phe Leu Ala Pro Lys Ile Glu Asn Glu Tyr gaa act tgg act atg aag aat gtg tat aac tca tgg act tat aat gtg 481 Glu Thr Trp Thr Met Lys Asn Val Tyr Asn Ser Trp Thr Tyr Asn Val caa tac tgg aaa aac ggt act gat gaa aag ttt caa att act ccc cag 529 Gln Tyr Trp Lys Asn Gly Thr Asp Glu Lys Phe Gln Ile Thr Pro Gln tatgactttgag gtcctcaga aacctg gagccatgg acaacttat tgt 577 TyrAspPheGlu ValLeuArg AsnLeu GluProTrp ThrThrTyr Cys gttcaagttcga gggtttctt cctgat cggaacaaa getggggaa tgg 625 ValGlnValArg GlyPheLeu ProAsp ArgAsnLys AlaGlyGlu Trp agtgagcctgtc tgtgagcaa acaacc catgacgaa acggtcccc tcc 673 SerGluProVal CysGluGln ThrThr HisAspGlu ThrValPro Ser tggatggtggcc gtcatcctc atggcc tcggtcttc atggtctgc ctg 721 TrpMetValA1a ValIleLeu MetAla SerValPhe MetValCys Leu gcactcctcggc tgcttctcc ttgctg tggtgcgtt tacaagaag aca 769 AlaLeuLeuGly CysPheSer LeuLeu TrpCysVal TyrLysLys Thr aagtacgccttc tCCCCtagg aattct cttccacag cacctgaaa gag 817 LysTyrAlaPhe SerProArg AsnSer LeuProGln HisLeuLys Glu tttttgggccat cctcatcat aacaca cttctgttt ttctccttt cca 865 PheLeuGlyHis ProHisHis AsnThr LeuLeuPhe PheSerPhe Pro ttgtcggatgag aatgatgtt tttgac aagctaagt gtcattgca gaa 913 LeuSerAspG1u AsnAspVal PheAsp LysLeuSer ValIleAla Glu gactctgagagc ggcaagcag aatcct ggtgacagc tgcagcctc ggg 961 AspSerGluSer GlyLysGln AsnPro GlyAspSer Cy~SerLeu Gly accccgcctggg caggggccc caaagc taggctctgagaa ggaaacacac 1011 ThrProProGly GlnGlyPro GlnSer tc 1013 <210> 72 <211> 49 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC40922 <400> 72 tccagggaat tcatataggc cggccaccat ggctgcagct tggaccgtg 49 <210> 73 ' <211> 71 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide primer ZC43153 <400> 73 ggggtgggta caaccccaga gctgttttaa ggcgcgcctc tagactattt ttaggtggac 60 tcagggtggg t 71 <210> 74 <211> 546 <212> DNA

<213> ArtificialSequence <220>

<223> IL29 15X,Asn169 mutant C

<221> CDS

<222> (1)...(546) <221> variation <222> (44)...(45) <223> n G, T, or = A, C

<400> 74 ggc cct cccacttcc aagcccacc acaactggg aagggc dnncac 48 gtc Gly Pro ProThrSer LysProThr ThrThrGly LysGly XaaHis Val att ggc ttcaaatct ctgtcacca caggagcta gcgagc ttcaag 96 agg Ile Gly PheLysSer LeuSerPro GlnGluLeu AlaSer PheLys Arg aag gcc gacgccttg gaagagtca ctcaagctg aaaaac tggagt 144 agg Lys Ala AspAlaLeu GluGluSer LeuLysLeu LysAsn TrpSer Arg tgc agc cctgtcttC CCCgggaat tgggacctg aggctt ctccag 19'2.:
tct Cys Ser ProValPhe ProGlyAsn TrpAspLeu ArgLeu LeuGln Ser gtg agg cgccctgtg gccttggag getgagctg gccctg acgctg 240 gag Val Arg ArgProVa1 AlaLeuGlu AlaGluLeu AlaLeu ThrLeu Glu aag gtc~ctggaggccget getggccca gccctggag gacgtc ctagac 288 Lys Val GluAlaAla AlaGlyPro AlaLeuGlu AspVal LeuAsp Leu Cag CCC Ca.CaCCCtg Ca.CCaCatC CtCtcccag CtCCag gCCtgt 336 Ctt Gln Pro HisThrLeu HisHisIle LeuSerGln LeuGln AlaCys Leu atc cag cagcccaca gcagggccc aggCCCCgg ggCCg'CCtCCa.C384 cct Ile Gln GlnProThr AlaGlyPro ArgProArg GlyArg LeuHis Pro cac tgg caccggctc caggaggcc cccaaaaag gagtcc getggc 432 ctg His Trp HisArgLeu GlnGluAla ProLysLys GluSer AlaGly Leu tgc ctg gcatctgtc accttcaac ctcttccgc ctcctc acgcga 480 gag Cys Leu AlaSerVal ThrPheAsn LeuPheArg LeuLeu ThrArg Glu gac ctc tatgtggcc gatgggaay ctgtgtctg agaacg tcaacc 528 aaa Asp Leu TyrValAla AspGlyAsn LeuCysLeu ArgThr SerThr Lys CdC CCt tCCaCCtga 546 gag His Pro SerThr Glu <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, Asn169 <400> 75 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu A1a Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 76 <211> 549 <212> DNA
<213> Artificial Sequence <220>
<223> Met IL29 mutant C16X, Asn170 <221> CDS
<222> (1)...(549) <221> variation <222> (47)...(48) <223> n = A, T, G, or C
<400> 76 atg ggc cct gtc ccc act tcc aag ccc acc aca act ggg aag ggc dnn 48 Met Gly Pro Va1 Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa cac att ggc agg ttc aaa tct ctg tca cca cag gag cta gcg agc ttc 96 His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe aag aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg 144 Lys Lys Ala Arg Asp A1a Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp agttgcagctct cctgtc ttccccggg aattgggac ctgagg cttctc 192 SerCysSerSer ProVal PheProGly AsnTrpAsp LeuArg LeuLeu caggtgagggag cgcect gtggcettg gaggetgag ctggcc ctgacg 240 GlnValArgGlu ArgPro ValAlaLeu GluAlaGlu LeuAla LeuThr ctgaaggtcctg gaggcc getgetggc ccagccctg gaggac gtccta 288 LeuLysValLeu GluAla AlaAlaGly ProAlaLeu GluAsp ValLeu gaccagcccctt cacacc ctgcaccac atcctctcc cagctc caggcc 336 AspGlnProLeu HisThr LeuHisHis IleLeuSer GlnLeu GlnAla tgtatccagcct cagccc acagcaggg cccaggccc cggggc cgcctc 384 CysIleGlnPro GlnPro ThrAlaGly ProArgPro ArgGly ArgLeu caccactggctg caccgg ctccaggag gcccccaaa aaggag tccget 432 HisHisTrpLeu HisArg LeuGlnGlu AlaProLys LysGlu SerAla ggctgcctggag gcatct gtcaccttc aacctcttc cgcctc ctcacg 480 GlyCysLeuGlu AlaSer ValThrPhe AsnLeuPhe ArgLeu LeuThr cgagacctcaaa tatgtg gccgatggg aayctgtgt ctgaga acgtca 52'8.

ArgAspLeuLys TyrVal AlaAspGly AsnLeuCys LeuArg ThrSer acccaccctgag tccacc tga 549' ThrHisProGlu SerThr <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, Asn170 <400> 77 Met Gly Pro Va1 Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg G1u Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His TrpLeu HisArg Gln Glu Ala LysLys Glu Ala Leu Pro Ser G1y Cys LeuGlu AlaSer Thr Phe Asn PheArg Leu Thr Val Leu Leu Arg Asp LeuLys TyrVal Asp Gly Asn CysLeu Arg Ser Ala Leu Thr Thr His ProGlu SerThr <210> 78 <211> 546 <213> DNA

<213> ArtificialSequence <220>

<223> IL29 5X,Asp169 mutant <221> CDS

<222> (1)...(546) <221> variation <222> (44)...(45) <223> n T, G, = A, or C

<400> 78 ggc cct ccc acttccaag cccaccaca actgggaag ggcdnncac 48 gtc Gly Pro Pro ThrSerLys ProThrThr ThrGlyLys GlyXaaHis Val att.ggc ttc aaatctctg tcaccacag gagctagcg agcttcaag 96 agg Ile Gly Phe LysSerLeu SerProGln GluLeuAla SerPheLys Arg aag gcc gac gccttggaa gagtcactc aagctgaaa aactggagt 144 agg ~

Lys Ala Asp AlaLeuGlu GluSerLeu LysLeuLys AsnTrpSer Arg tgc agc cct gtcttcccc gggaattgg gacctgagg cttctccag 192 tct Cys Ser Pro ValPhePro GlyAsnTrp AspLeuArg LeuLeuGln Ser gtg agg cgc cctgtggcc ttggagget gagctggcc ctgacgctg 240 gag Val Arg Arg ProValAla LeuGluAla GluLeuAla LeuThrLeu Glu aag gtc gag gccgetget ggcccagcc ctggaggac gtcctagac 288 ctg Lys Val Glu AlaAlaAla GlyProAla LeuGluAsp ValLeuAsp Leu Cag CCC cac accctgcac cacatcctc tcccagCtC CaggCCtgt 336 Ctt Gln Pro His ThrLeuHis HisIleLeu SerGlnLeu GlnA1aCys Leu atc cag cag cccacagca gggcccagg CCCCggggC CgCCtCC1C 384 cct Ile Gln Gln ProThrAla GlyProArg ProArgG1y ArgLeuHis Pro cac tgg cac cggctccag gaggccccc aaaaaggag tccgetggc 432 ctg His Trp His ArgLeuGln G1uAlaPro LysLysGlu SerAlaGly Leu tgc ctg gca tctgtcacc ttcaacctc ttccgcctc ctcacgcga 480 gag Cys Leu Ala SerValThr PheAsnLeu PheArgLeu LeuThrArg Glu gac ctc aaa tat gtg gcc gat ggg gay ctg tgt ctg aga acg tca acc 528 Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg Thr Ser Thr cac cct gag tcc acc tga 546 His Pro Glu Ser Thr <210> 79 <211> 181 <212> PRT
<213> Artificial Sequence <220>
<223> IL29 mutant C15X, Asp169 <221> VARIANT
<222> (15)...(15) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 79 G1y Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa His I1e Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser G1n Leu Gln Ala Cys Ile Gln Pro G1n Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu A1a Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val A1a Asp Gly Asp Leu Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 80 <211> 549 <212> DNA
<213> Artificial Sequence <220>
<~23> Met IL29 mutant C16X, Asp170 <~21> CDS
<222> (1)...(549) <221> variation <222> (47)...(48) <223> n = A, T, G, or C
<400> 80 atg ggc cct gtc ccc act tcc aag ccc acc aca act ggg aag ggc dnn 48 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa cacatt ggcaggttc aaatct ctgtcacca caggagcta gcgagcttc 96 HisIle GlyArgPhe LysSer LeuSerPro GlnGluLeu AlaSerPhe aagaag gccagggac gccttg gaagagtca ctcaagctg aaaaactgg 144 LysLys AlaArgAsp AlaLeu GluGluSer LeuLysLeu LysAsnTrp agttgc agctctcct gtcttc cccgggaat tgggacctg aggcttctc 192 SerCys SerSerPro ValPhe ProGlyAsn TrpAspLeu ArgLeuLeu caggtg agggagcgc cctgtg gccttggag getgagctg gccctgacg 240 GlnVal ArgGluArg ProVal AlaLeuGlu AlaGluLeu AlaLeuThr ctgaag gtcctggag gccget getggccca gccctggag gacgtccta 288 LeuLys ValLeuGlu AlaAla AlaGlyPro AlaLeuGlu AspValLeu gaccag CCCCttCaC aCCCtg Ca.CCaCatC CtCtcccag CtCCaggCC 336 AspGln ProLeuHis ThrLeu HisHisIle LeuSerGln LeuGlnA1a tgtatc cagcctcag cccaca gcagggccc aggccccgg ggccgcctc 384 CysIle GlnProGln ProThr AlaGlyPro ArgProArg GlyArgLeu caccac tggctgcac cggctc caggaggcc cccaaaaag gag,~tccget 432 HisHis TrpLeuHis ArgLeu GlnGluAla ProLysLys GluSerAla ggctgc ctggaggca tctgtc accttcaac ctcttccgc ctcctcacg 480 G1yCys LeuGluAla SerVa1 ThrPheAsn LeuPheArg LeuLeuThr cgagac ctcaaatat gtggcc gatggggay ctgtgtctg agaacgtca 528 ArgAsp LeuLysTyr ValA1a AspGlyAsp LeuCysLeu ArgThrSer acccac cctgagtcc acctga 549 ThrHis ProGluSer Thr <210> 81 <211> 182 <212> PRT
<213> Artificial Sequence <~20>
<223> Met IL29 mutant C16X, Asp170 <221> VARIANT
<222> (16)...(16) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 81 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu A1a Ser Phe Lys Lys Ala Arg Asp A1a Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu G1n Val Arg Glu Arg Pro Val Ala Leu Glu Ala G1u Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 . 120 125 His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr _ 180 <210> 82 <211> 546 <212> DNA

<213> ArtificialSequence <220> ' <223> IL29 mutant Asp169, <221> CDS

<222> (1)...(546) <221> variation <222> (512)...(513) <E23> n T, G, = A, or C

<400> 82 ggc cct ccc acttccaag cccaccaca actgggaag ggctgc cac 48 gtc Gly Pro Pro ThrSerLys ProThrThr ThrGlyLys GlyCys His Val att ggc ttc aaatctctg tcaccacag gagctagcg agcttc aag 96 agg Ile G1y Phe LysSerLeu SerProGln GluLeuAla SerPhe Lys Arg aag gcc gac gccttggaa gagtcactc aagctgaaa aactgg agt 144 agg Lys Ala Asp AlaLeuGlu GluSerLeu LysLeuLys AsnTrp Ser Arg tgc agc cct gtcttcccc gggaattgg gacctgagg cttctc cag 192 tct Cys Ser Pro ValPhePro GlyAsnTrp AspLeuArg LeuLeu Gln Ser gtg agg cgc cctgtggcc ttggagget gagctggcc ctgacg ctg 240 gag Val Arg Arg ProValAla LeuGluAla GluLeuA1a LeuThr Leu Glu aag gtc gag gccgCtgCt ggCCCagcc ctggaggac gtccta gac 288 ctg Lys Val Glu AlaAlaAla GlyProAla LeuGluAsp ValLeu Asp Leu Cag CCC CaC aCCCtgCaC CaCatCCtC tcccagctc caggcc tgt 336 Ctt Gln Pro His ThrLeuHis HisIleLeu SerGlnLeu GlnAla Cys Leu atc cag cag cccacagca gggcccagg ccccggggc cgcctc cac 384 cct Ile Gln Gln ProThrAla GlyProArg ProArgGly ArgLeu His Pro cactgg ctgcaccgg ctccag gaggccccc aaaaaggag tccgetggc 432 HisTrp LeuHisArg LeuGln GluAlaPro LysLysGlu SerAlaGly tgcctg gaggcatct gtcacc ttcaacctc ttccgcctc ctcacgcga 480 CysLeu GluAlaSer ValThr PheAsnLeu PheArgLeu LeuThrArg gacctc aaatatgtg gccgat ggggayctg dnnctgaga acgtcaacc 528 AspLeu LysTyrVal AlaAsp GlyAspLeu XaaLeuArg ThrSerThr caccct gagtccacc tga 546 HisPro GluSerThr <210> 83 <211> 181 <212> PRT
<213> Artificial Sequence <220>
<223> IL29 mutant Asp169, C171X
<221> VARIANT
<222> (171)...(171) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 83 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu G1n Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp G1y Asp Leu Xaa Leu Arg Thr Ser Thr His Pro G1u Ser Thr <210> 84 <211> 549 <212> DNA
<213> Artificial Sequence <220>
<223> Met IL29 mutant Asp170, C172X

<~21>
CDS

<222> )...(549) (1 <221> ion variat <222> 15)...(516) (5 <223> = T, G, n A, or C

<400>

atg ggccctgtc cccacttcc aagccc accacaactggg aagggc tgc 48 Met GlyProVal ProThrSer LysPro ThrThrThrGly LysGly Cys cac attggcagg ttcaaatct ctgtca ccacaggagcta gcgagc ttc 96 His IleGlyArg PheLysSer LeuSer ProGlnGluLeu AlaSer Phe aag aaggccagg gacgccttg gaagag tcactcaagctg aaaaac tgg 144 Lys LysA1aArg AspAlaLeu GluGlu SerLeuLysLeu LysAsn Trp agt tgcagctct CCtgtCttC CCCggg aattgggacctg aggctt ctc 192 Ser CysSerSer ProValPhe ProGly AsnTrpAspLeu ArgLeu Leu cag gtgagggag cgccctgtg gccttg gaggetgagctg gccctg acg 240 Gln ValArgGlu ArgProVa1 AlaLeu GluAlaGluLeu AlaLeu Thr ctg aaggtcctg gaggccget getggc ccagccctggag gacgtc cta 288 Leu LysValLeu GluAlaAla AlaGly ProAlaLeuGlu AspVal Leu gac cagcccctt cacaccctg caccac atcctctcccag ctccag gcc 336 Asp GlnProLeu HisThrLeu HisHis IleLeuSerGln LeuGln Ala tgt atccagcct cagcccaca gcaggg cccaggccccgg ggccgc ctc 384 Cys IleGlnPro GlnProThr AlaGly ProArgProArg GlyArg Leu cac cactggctg caccggctc caggag gcccccaaaaag gagtcc get 432 His HisTrpLeu HisArgLeu GlnGlu AlaProLysLys GluSer Ala ggc tgcctggag gcatctgtc accttc aacctcttccgc ctcctc acg 480 Gly CysLeuGlu AlaSerVal ThrPhe AsnLeuPheArg LeuLeu Thr cga gacctcaaa tatgtggcc gatggg gayctgdnnctg agaacg tca 528 Arg AspLeuLys TyrValAla AspGly AspLeuXaaLeu ArgThr Ser acc caccctgag tccacctga 549 Thr HisProGlu SerThr <210>

<211>

<212>
PRT

<213>
Artificial Sequence <220>
<223> Met IL29 mutant Asp170, C172X
<221> VARIANT

<222> (172)...(172) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 85 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr G1y Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu G1n Val Arg Glu Arg Pro Val Ala Leu Glu A1a Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln G1u Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu .Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210>

<211>

<212>
DNA

<213> cialSequence Artifi <220>

<223> t Asn169, C171X
IL29 T10P, mutan <221>
CDS

<222> (546) (1)...

<221>
variation <222> ..(513) (512).

<223> T, G, n = A, or C

<400>

ggc cct ccc acttccaag cccacc ccnactggg aagggctgc cac 48 gtc Gly Pro Pro ThrSerLys ProThr ProThrGly LysGlyCys His Val att ggc ttc aaatctctg tcacca caggagcta gcgagcttc aag 96 agg Ile Gly Phe LysSerLeu SerPro GlnGluLeu AlaSerPhe Lys Arg aag gcc gac gccttggaa gagtca ctcaagctg aaaaactgg agt 144 agg Lys Ala Asp AlaLeuGlu GluSer LeuLysLeu LysAsnTrp Ser Arg tgc agc cct gtcttcccc gggaat tgggacctg aggcttctc cag 192 tct Cys Ser Pro ValPhePro GlyAsn TrpAspLeu ArgLeuLeu Gln Ser gtg agg cgc cctgtggcc ttggag getgagctg gccctgacg ctg 240 gag Val Arg Arg ProVa1Ala LeuG1u AlaGluLeu AlaLeuThr Leu Glu aaggtc ctggaggcc getgetggc ccagccctg gaggac gtcctagac 288 LysVal LeuGluAla A1aA1aG1y ProAlaLeu GluAsp Va1LeuAsp cagccc cttcacacc ctgcaccac atcctctcc cagctc caggcctgt 336 GlnPro LeuHisThr LeuHisHis I1eLeuSer GlnLeu GlnAlaCys atCCag CCtcagCCC aCagcaggg CCCaggccc cggggc cgcctCCaC 384 IleGln ProGlnPro ThrA1aGly ProArgPro ArgGly ArgLeuHis cactgg ctgcaecgg ctccaggag gcccccaaa aaggag tccgetggc 432 HisTrp LeuHisArg LeuGlnGlu AlaProLys LysGlu SerAlaGly tgCCtg gaggcatCt gtCaCCttC aaCCtCttC Cg'CCtC CtCacgcga 480 CysLeu GluAlaSer ValThrPhe AsnLeuPhe ArgLeu LeuThrArg gacctc aaatatgtg gccgatggg aacctgdnn ctgaga acgtcaacc 5~8 AspLeu LysTyrVal AlaAspGly AsnLeuXaa LeuArg ThrSerThr caccct gagtccacc tga 546 HisPro GluSerThr <210> 87 <211> 181 <212> PRT
<213> Artificial Sequence <220>
<223> IL29 mutant T10P, Asn169, C171X
<221> VARIANT
<222> (171)...(171) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 87 Gly Pro Va1 Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala A1a Ala Gly Pro Ala Leu Glu Asp Val Leu Asp GIn Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro G1u Ser Thr <210>

<211> 9 <212>
DNA

<213> tificialSequenc e Ar <220>

<223> t 1P, Asn170, C172X
Me IL29 mutant <221> S
CD

<222> )...(549) (1 <221> riation va <222> 15)...(516) (5 <223> = T, G, n A, or C

<400>

atg cctgtc cccacttcc aagccc accccnact gggaagggc tgc 48 ggc Met ProVal ProThrSer LysPro ThrProThr GlyLysGly Cys Gly cac ggcagg ttcaaatct ctgtca ccacaggag ctagcgagc ttc 96 att His GlyArg PheLysSer LeuSer ProGlnGlu LeuAlaSer Phe Ile aag gccagg gacgccttg gaagag tcactcaag ctgaaaaac tgg 144 aag Lys AlaArg AspAlaLeu GluGlu SerLeuLys LeuLysAsn Trp Lys agt agctct CCtgtCttc cccggg aattgggac ctgaggctt ctc 192 tgc Ser SerSer ProValPhe ProGly AsnTrpAsp LeuArgLeu Leu Cys cag agggag CgCCCtgtg gCCttg gaggetgag ctggccctg acg 240 gtg Gln ArgGlu ArgProVal AlaLeu GluAlaGlu LeuAlaLeu Thr Val ctg gtcctg gaggccget getggc ccagccctg gaggacgtc cta 288 aag Leu ValLeu GluAlaAla A1aGly ProAlaLeu GluAspVal Leu Lys gac cccctt cacaccctg caccac atcctctcc cagctccag gcc 336 cag Asp ProLeu HisThrLeu HisHis IleLeuSer GlnLeuGln Ala Gln tgt cagCCt CagCCCaCa gCaggg cccaggCCC CggggCCgC CtC 384 atc Cys GlnPro GlnProThr AlaGly ProArgPro ArgGlyArg Leu 21e cac tggctg caccggctc caggag gcccccaaa aaggagtcc get 432 cac His TrpLeu HisArgLeu GlnG1u AlaProLys LysGluSer Ala His ggc ctggag gcatctgtc accttc aacctcttc cgcctcctc acg 480 tgc Gly LeuGlu AlaSerVal ThrPhe AsnLeuPhe ArgLeuLeu Thr Cys cga ctcaaa tatgtggcc gatggg aacctgdnn ctgagaacg tca 528 gac Arg LeuLys TyrValAla AspGly AsnLeuXaa LeuArgThr Ser Asp aCC CCtgag tCCaCCtga 549 Ca.C

Thr ProGlu SerThr His <210> 89 <211> 182 <212> PRT
<213> Artificial Sequence <220>
<223> Met IL29 mutant T11P, Asn170, C172X
<221> VARIANT
<222> (172)...(172) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 89 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro G1y Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro A1a Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu , His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 90 <211> 546 <212> DNA
<213> Artificial Sequence <220>
<223> IL29 mutant T10P, C15X, Asn169 <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 CCC aCC CCn act ggg aag ggc dnn CdC 48 Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Xaa His att ggc agg ttc aaa tct ctg tca cca cag gag cta gcg agc ttc aag 96 Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln G1u Leu A1a Ser Phe Lys aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg agt 144 Lys Ala Arg Asp A1a Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser tgcagc tctcctgtc ttcccc gggaattgg gacctgagg cttctc cag 192 CysSer SerProVal PhePro GlyAsnTrp AspLeuArg LeuLeu Gln gtgagg gagcgccct gtggcc ttggagget gagctggcc ctgacg ctg 240 ValArg GluArgPro ValAla LeuGluAla GluLeuAla LeuThr Leu aaggtc ctggaggcc getget ggcccagcc ctggaggac gtccta gac 288 LysVal LeuGluAla AlaAla GlyProAla LeuG1uAsp ValLeu Asp CagCCC CttCc'LCaCC CtgC3C Ca.CatCCtC tCCCagCt CaggCC tgt 336 C

GlnPro LeuHisThr LeuHis HisIleLeu SerGlnLeu GlnAla Cys atccag cctcagccc acagca 'gggcccagg ccccggggc cgcctc cac 384 I1eGln ProGlnPro ThrAla GlyProArg ProArgGly ArgLeu His cactgg ctgcaccgg ctccag gaggccccc aaaaaggag tccget ggc 432 HisTrp LeuHisArg LeuGln GluAlaPro LysLysGlu SerAla Gly tgcctg gaggcatct gtcacc ttcaacctc ttccgcctc ctcacg cga 480 CysLeu GluAlaSer ValThr PheAsnLeu PheArgLeu LeuThr Arg gacctc aaatatgtg gccgat gggaayctg tgtctgaga acgtca acc 528 AspLeu LysTyrVal AlaAsp GlyAsnLeu CysLeuArg ThrSer Thr CaCCCt gagtCCaCC tga 546.

HisPro GluSerThr <210> 91 <211> 181 <212> PRT
<213> Artificial Sequence <220>
<223> IL29 mutant T10P, C15X, Asn169 <221> VARIANT
<222> (15)...(15) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 91 Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Xaa His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu A1a Leu Thr Leu Lys Val Leu Glu Ala Ala A1a Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210>

<211> 9 <212>
DNA

<213> tificialSequence Ar <220>

<223> t 29 1P,C16X, Me IL mutant Asn170 <221> S
CD

<222> )...(549) (1 <221> riation va <222> , , <223> = T, G, n A, or C

<400>

atg Cctgtcccc acttccaag cccacc ccnactggg aagggcdnn ~-8 ggc ;

Met ProValPro ThrSerLys ProThr ProThrGly LysGlyXaa Gly cac ggcaggttc aaatctctg tcacca caggagcta gcgagcttc 96 att His GlyArgPhe LysSerLeu SerPro GlnGluLeu AlaSerPhe Ile aag gccagggac gccttggaa gagtca ctcaagctg aaaaactgg 144' aag Lys AlaArgAsp AlaLeuGlu GluSer LeuLysLeu LysAsnTrp Lys agt agctctcct gtcttcccc gggaat tgggacctg aggcttctc 192 tgc Ser SerSerPro ValPhePro GlyAsn TrpAspLeu ArgLeuLeu Cys cag agggagcgc cctgtggcc ttggag getgagctg gccctgacg 240 gtg Gln ArgGluArg ProValAla LeuGlu AlaGluLeu AlaLeuThr Val ctg gtcctggag gccgetget ggccca gccctggag gacgtccta 288 aag Leu ValLeuGlu AlaAlaAla GlyPro AlaLeuGlu AspValLeu Lys gac ccccttcac accctgcac cacatc ctctcccag ctccaggcc 336 cag Asp ProLeuHis ThrLeuHis HisI1e LeuSerGln LeuGlnAla Gln tgt cagcctcag cccacagca gggccc aggCccCgg ggccgcctc 384 atc Cys G1nProGln ProThrAla G1yPro ArgProArg GlyArgLeu Ile cac tggctgcac cggctccag gaggcc cccaaaaag gagtccget 432 cac His TrpLeuHis ArgLeuGln GluAla ProLysLys GluSerAla His ggc ctggaggca tctgtcacc ttcaac ctcttccgc ctcctcacg 480 tgc Gly LeuGluAla SerValThr PheAsn LeuPheArg LeuLeuThr Cys cga gac ctc aaa tat gtg gcc gat ggg aay ctg tgt ctg aga acg tca 528 Arg Asp Leu Lys Tyr Va1 Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser acc cac cct gag tcc acc tga 549 Thr His Pro Glu Ser Thr <210> 93 <211> 182 <212> PRT
<213> Artificial Sequence <220>
<223> Met IL29 mutant T11P, C16X, Asn170 <221> VARIANT
<222> (16)...(16) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 93 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Xaa His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala P.la Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys~Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 94 <211> 546 <212> DNA
<213> Artificial Sequence <220>
<223> IL29 mutant T10P, Asp169, C171X
<221> CDS
<222> (1)...(546) <221> variation <222> 30, 512, 513 <223> n = A, T, G, or C
<400> 94 ggccct gtccccact tccaag cccaccccn actgggaag ggctgc cac 48 GlyPro ValProThr SerLys ProThrPro ThrGlyLys GlyCys His attggc aggttcaaa tctctg tcaccacag gagctagcg agcttc aag 96 IleGly ArgPheLys SerLeu SerProGln G1uLeuAla SerPhe Lys aaggcc agggacgcc ttggaa gagtcactc aagctgaaa aactgg agt 144 LysAla ArgAspAla LeuGlu GluSerLeu LysLeuLys AsnTrp Ser tgcagc tctcctgtc ttcccc gggaattgg gacctgagg cttctc cag 192 CysSer SerProVal PhePro GlyAsnTrp AspLeuArg LeuLeu G1n gtgagg gagcgccct gtggcc ttggagget gagctggcc ctgacg ctg 240 ValArg GluArgPro ValA1a LeuGluAla GluLeuAla LeuThr Leu aaggtc ctggaggcc getget ggcccagcc ctggaggac gtccta gac 288 LysVal LeuGluAla AlaAla GlyProAla LeuGluAsp ValLeu Asp CagCCC CttCaCaCC CtgCc'~.CCaCatCCtC tcccagCtC CaggCC tgt 336 GlnPro LeuHisThr LeuHis HisIleLeu SerGlnLeu GlnAla Cys atccag cctcagccc acagca gggcccagg ccccggggc cgcctc cac 3,84, I1eG1n ProGlnPro ThrAla GlyProArg ProArgGly ArgLeu His cactgg ctgcaccgg ctccag gaggccccc aaaaaggag tccget ggc 432 HisTrp LeuHisArg LeuG1n GluAlaPro LysLysGlu SerAla Gly 130 1.35 140 tgcctg gaggcatct gtcacc ttcaacctc ttccgcctc ctcacg cga 480.

CysLeu GluAlaSer ValThr PheAsnLeu PheArgLeu LeuThr Arg gacctc aaatatgtg gccgat ggggayctg dnnctgaga acgtca acc 528 AspLeu LysTyrVal AlaAsp GlyAspLeu XaaLeuArg ThrSer Thr caccct gagtccacc tga 546 HisPro G1uSerThr <210> 95 <211> 181 <212> PRT
<213> Artificial Sequence <220>
<223> IL29 mutant T10P, Asp169, C171X
<221> VARIANT
<222> (171)...(171) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 95 Gly Pro Va1 Pro Thr Ser Lys Pro Thr Pro Thr G1y Lys G1y Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu A1a Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 96 <211> 549 ' <21~> DNA
<213> Artificial Sequence <220>
<223> Met IL29 mutant T11P, Asp170, C172X
<221>
CDS

<222> )...(549) (1 <221> riation va <222> , 5, <223> = T, G, n A, or C

<400>

atgggc cctgtcccc acttcc aagcccacc ccnactggg aagggctgc 48 MetGly ProValPro ThrSer LysProThr ProThrGly LysGlyCys cacatt ggcaggttc aaatct ctgtcacca caggagcta gcgagcttc 96 HisIle GlyArgPhe LysSer LeuSerPro GlnGluLeu AlaSerPhe aagaag gccagggac gccttg gaagagtca ctcaagctg aaaaactgg 144 LysLys AlaArgAsp AlaLeu GluGluSer LeuLysLeu LysAsnTrp agttgc agctctcct gtcttc cccgggaat tgggacctg aggcttctc 192 SerCys SerSerPro ValPhe ProG1yAsn TrpAspLeu ArgLeuLeu caggtg agggagcgc cctgtg gccttggag getgagctg gccctgacg 240 GlnVal ArgGluArg ProVal AlaLeuGlu AlaGluLeu AlaLeuThr ctgaag gtcctggag gccget getggccca gccctggag gacgtccta 288 LeuLys ValLeuGlu AlaAla AlaG1yPro AlaLeuGlu AspValLeu gaccag CCCCttcac accctg caccacatc ctCtcccag CtCCaggCC 336 AspGln ProLeuHis ThrLeu HisHisIle LeuSerGln LeuGlnAla tgtatc cagcctcag cccaca gcagggccc aggccccgg ggccgcctc 384 CysIle GlnProGln ProThr AlaGlyPro ArgProArg GlyArgLeu caccac tggctgcac cggctc caggaggcc cccaaaaag gagtccget 432 HisHis TrpLeuHis ArgLeu GlnGluAla ProLysLys GluSerAla ggctgc ctggaggca tctgtc accttcaac ctcttccgc ctcctcacg 480 GlyCys LeuGluAla SerVa1 ThrPheAsn LeuPheArg LeuLeuThr cgagac ctcaaatat gtggcc gatggggay ctgdnnctg agaacgtca 528 ArgAsp LeuLysTyr ValAla AspGlyAsp LeuXaaLeu ArgThrSer acccac cctgagtcc acctga 549 ThrHis ProGluSer Thr <210> 97 <211> 182 <212> PRT
<213> Artificial Sequence <220>
<223> Met IL29 mutant T11P, Asp170~, C172X
<221> VARIANT
<222> (172)...(172) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 97 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu A1a Ser Phe Lys Lys Ala Arg Asp Ala Leu G1u Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Va1 Ala Leu Glu Ala Glu Leu A1a Leu Thr Leu Lys Val Leu Glu A1a Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu G1n Ala Cys I1e Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 98 <211> 546 <212> DNA
<213> Artificial Sequence <220>

<223> 29 t C15X, IL mutan T10P, Asp169 <221>
CDS

<222> )...(546) (1 <221> ion variat <222> , , <223> = T, G, n A, or C

<400>

ggc cctgtc cccacttccaag cccacc ccnactggg aagggcdnn cac 48 Gly ProVal ProThrSerLys ProThr ProThrGly LysGlyXaa His att ggcagg ttcaaatctctg tcacca caggagcta gcgagcttc aag 96 Ile GlyArg PheLysSerLeu SerPro GlnGluLeu AlaSerPhe Lys aag gccagg gacgccttggaa gagtca ctcaagctg aaaaactgg agt 144 Lys AlaArg AspAlaLeuGlu GluSer LeuLysLeu LysAsnTrp Ser tgc agctct CCtgtCttcccc gggaat tgggacctg aggcttctc cag 192 Cys SerSer ProValPhePro GlyAsn TrpAspLeu ArgLeuLeu Gln gtg agggag cgccctgtggcc ttggag getgagctg gccctgacg ctg 240' Val ArgGlu ArgProValAla LeuGlu AlaGluLeu AlaLeuThr Leu aag gtcctg gaggccgetget ggccca gccctggag gacgtccta gac 288 Lys ValLeu GluAlaAlaAla GlyPro AlaLeuGlu Asp'ValLeu Asp Cag CCCCtt CdCaCCCtgCaC CaCatC CtCtCCCag ctccaggcc tgt 336 Gln ProLeu HisThrLeuHis HisIle LeuSerGln LeuGlnAla Cys atc cagcct cagcccacagca gggccc aggCCCCgg ggCCgCCtC CaC 384 Ile GlnPro GlnProThrAla GlyPro ArgProArg GlyArgLeu His cac tggctg caccggctccag gaggcc cccaaaaag gagtccget ggc 432 His TrpLeu HisArgLeuGln GluAla ProLysLys GluSerAla Gly tgC Ctggag gcatCtgtCaCC ttCaaC CtCttCCgC CtCCtCacg cga 480 Cys LeuGlu AlaSerValThr PheAsn LeuPheArg LeuLeuThr Arg gac ctcaaa tatgtggccgat ggggay ctgtgtctg agaacgtca.acc 528 Asp LeuLys TyrValAlaAsp G1yAsp LeuCysLeu ArgThrSer Thr cac cctgag tccacctga 546 His ProGlu SerThr <210>

<211>

<212>
PRT

<213>
Artificial Sequence <220>
<223> IL29 mutant T10P, C15X, Asp169 <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 G1y Xaa His Ile Gly Arg Phe Lys Ser Leu Ser Pro G1n Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln 50 55 ~ 60 Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 100 <211> 549 <312> DNA

<~13> ArtificialSequence <220>

<223> Met 1P, C16X, IL29 mutant Asp170 <221> CDS

<222> (1)...(549) , <~21> variation <222> 33, 47, 48 <223> n T, G, = A, or C

<400> 100 atg ggc gtc cccacttcc aagcccacc ccnactggg aagggcdnn 48 cct Met Gly Va1 ProThrSer LysProThr ProThrGly LysGlyXaa Pro cac att agg ttcaaatct ctgtcacca caggagcta gcgagcttc 96 ggc His Ile Arg PheLysSer LeuSerPro G1nGluLeu AlaSerPhe Gly aag aag agg gacgccttg gaagagtca ctcaagctg aaaaactgg 144 gcc Lys Lys Arg AspAlaLeu GluGluSer LeuLysLeu LysAsnTrp Ala agt tgc tct cctgtcttc cccgggaat tgggacctg aggcttctc 192 agc Ser Cys Ser ProValPhe ProGlyAsn TrpAspLeu ArgLeuLeu Ser cag gtg gag cgccctgtg gccttggag getgagctg gccctgacg 240 agg Gln Val Glu ArgProVa1 AlaLeuGlu AlaGluLeu AlaLeuThr Arg etgaag gtcctggag gccgetget ggcecagcc ctggaggac gtccta 288 LeuLys ValLeuGlu AlaAlaAla GlyProAla LeuGluAsp ValLeu gaccag ccccttcac accctgcac cacatcctc tcccagctc caggcc 336 AspGln ProLeuHis ThrLeuHis HisIleLeu SerGlnLeu GlnAla tgtatc cagcctcag cccacagca gggcccagg ccccggggc cgcctc 384 CysIle GlnProGln ProThrAla GlyProArg ProArgGly ArgLeu ~-15 220 125 caccac tggctgcac cggctccag gaggccecc aaaaaggag tccget 432 HisHis TrpLeuHis ArgLeuGln GluAlaPro LysLysGIu SerAla ggctgc ctggaggca tctgtcacc ttcaacctc ttccgcctc ctcacg 480 GlyCys LeuGluAla SerValThr PheAsnLeu PheArgLeu LeuThr 145 150 l55 160 cgagac ctcaaatat gtggccgat ggggayctg tgtctgaga acgtca 528 ' ArgAsp LeuLysTyr ValAlaAsp GlyAspLeu CysLeuArg ThrSer acccac cctgagtcc acctga ThrHis ProGluSer Thr <210> l01 <211> 182 <212> PRT
<213> Artificial Sequence <220>
<223> Met IL29 mutant T11P, C16X, Asp170 <221> VARIANT
<222> (16)...(16) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 101 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Xaa His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu'Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro A1a Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala 100 105 l10 Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu G1n Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210>

<211>

<212>
DNA

<213> cial Artifi Sequence <220>

<223> Asn169, C171X

mutant G18D, <221>
CDS

<222> (546) (1)...

<221>
variation <222> ..(513) (512).

<223> = T, G,~
n A, or C

<400>

ggc gtc cccacttccaag cccacc acaactggg aagggctgc cac 48 cct Gly Val ProThrSerLys ProThr ThrThrGly LysGlyCys His Pro att agg ttcaaatctctg tcacca caggagcta gcgagcttc aag 96 gay Ile Arg PheLysSerLeu SerPro GlnG1uLeu AlaSerPhe Lys Asp aag agg gacgccttggaa gagtca ctcaagctg aaaaactgg agt l44 gcc Lys Arg AspAlaLeuGlu GluSer LeuLysLeu LysAsnTrp Ser Ala tgc tct cctgtcttcccc gggaat tgggacctg aggcttctc Cag 192' agc Cys Ser ProValPhePro G1yAsn TrpAspLeu ArgLeuLeu Gln Ser gtg gag CgCCCtgtggCC ttggag getgagctg gccctgacg ctg 240 agg Val Glu ArgProValAla LeuGlu AlaGluLeu AlaLeuThr Leu Arg aag ctg gaggccgetget ggccca gccctggag gacgtccta gac 288 gtc Lys Leu GluAlaAlaAla GlyPro AlaLeuGlu AspValLeu Asp Val cag Ctt cacaccctgcac cacatc ctctcccag ctccaggcc tgt 336 CCC

Gln Leu HisThrLeuHis HisIle LeuSerGln LeuGlnAla Cys Pro atC CCt CagCCCaCagCa gggccc aggCCCCgg ggCCgCCtC CaC 384 Cag Ile Pro GlnProThrAla GlyPro ArgProArg GlyArgLeu His Gln cac ctg caccggctccag gaggcc cccaaaaag gagtccget ggc 432 tgg His Leu HisArgLeuGln GluA1a ProLysLys GluSerAla G1y Trp tgc gag gcatctgtcacc ttCaaC CtCttCCgC CtCCtCaCg Cga 480 ctg Cys Glu A1aSerValThr PheAsn LeuPheArg LeuLeuThr Arg Leu gac aaa tatgtggccgat gggaac ctgdnnctg agaacgtca acc 528 ctc Asp Lys TyrValAlaAsp GlyAsn LeuXaaLeu ArgThrSer Thr Leu cac gag tccacctga cct 546 His Glu SerThr Pro <210> 103 <211> 181 <~12> PRT
<213> Artificial Sequence <220>
<223> IL29 mutant G18D, Asn169, C171X
<221> VARIANT
<222> (171)...(171) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 103 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His, Ile Asp Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp A1a Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala G~.u Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 104 <211> 549 <212> DNA
<213> Artificial Sequence <220>
<223> Met IL29 mutant G19D, Asn170, C172X
<221> CDS
<222> (1)...(549) <221> variation <222> (515)...(516) <223> n = A, T, G, or C
<400> 104 atg ggc cct gtc ccc act tcc aag ccc acc aca act ggg aag ggc tgc 48 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys cac att gay agg ttc aaa tct ctg tca cca cag gag cta gcg agc ttc 96 His I1e Asp Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe aagaag gccagggac gccttg gaagagtca ctcaagctg aaaaactgg 144 LysLys AlaArgAsp AlaLeu GluGluSer LeuLysLeu LysAsnTrp agttgc agctctcct gtcttC CCCgggaat tgggacctg aggcttctc 192 SerCys SerSerPro ValPhe ProGlyAsn TrpAspLeu ArgLeuLeu caggtg agggagcgc cctgtg gccttggag getgagctg gccctgacg 240 GlnVal ArgGluArg ProVal AlaLeuGlu AlaGluLeu AlaLeuThr ctgaag gtcctggag gccgCt gCtggCCCa gccctggag gacgtccta 288 LeuLys ValLeuGlu AlaAla AlaGlyPro AlaLeuGlu AspValLeu gaccag CCCCttcac accctg caccacatc ctctcccag ctccaggcc 336 AspGln ProLeuHis ThrLeu HisHisIle LeuSerGln LeuGlnAla tgtatc cagcctcag cccaca gcagggccc aggccccgg ggccgcctc 384 CysIle GlnProGln ProThr AlaGlyPro ArgProArg GlyArgLeu caccac tggctgcac cggctc caggaggcc cccaaaaag gagtccget 432 HisHis TrpLeuHis ArgLeu GlnGluAla ProLysLys GluSerAla ggctgc ctggaggca tctgtc accttcaac ctcttCCgC CtCCtCaCg 4,80;-.

GlyCys LeuGluAla SerVal ThrPheAsn LeuPheArg LeuLeuThr cgagac ctcaaatat gtggcc gatgggaac ctgdnnctg agaacgtca 528' ArgAsp LeuLysTyr Va1Ala AspGlyAsn LeuXaaLeu ArgThrSer acccac cctgagtcc acctga 549 ThrHis ProGluSer Thr <210> 105 <211> 182 <212> PRT
<213> Artificial Sequence <220>
<223> Met IL29 mutant G19D, Asn170, C172X
<221> VARIANT
<~22> (172)...(172) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 105 Met Gly Pro Va1 Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Asp Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Va1 Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Va1 Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala G1y Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120 , 125 His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Va1 Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 106 <211> 546 <212> DNA

<213> ArtificialSequence <220>

<223> IL29 5X,G18D, mutant Asn169 <221> CDS

<222> (1)...(546) <221> variation <222> (44)...(45) <223> n T, G, = A, or C

<400> 106 ggC CCt CCC aCttCCaagCCC aCCaCa aCtgggaag ggcdnriCaC 48 gtC

Gly Pro Pro ThrSerLysPro ThrThr ThrGlyLys GlyXaaHis Val att gay ttc aaatctctgtca ccacag gagctagcg agcttcaag 96 agg Ile Asp Phe LysSerLeuSer ProGln GluLeuAla SerPheLys Arg aag gcc gac gccttggaagag tcactc aagctgaaa aactggagt 144 agg Lys Ala Asp AlaLeuGluGlu SerLeu LysLeuLys AsnTrpSer Arg 35 ~ 40 45 tgc agc cct gtcttCCCCggg aattgg gacctgagg cttctccag 192 tct Cys Ser Pro ValPheProGly AsnTrp AspLeuArg LeuLeuGln Ser gtg agg cgc cctgtggccttg gagget gagctggcc ctgacgctg 240 gag Val Arg Arg ProValAlaLeu GluAla GluLeuAla LeuThrLeu Glu 65 ~ 70 75 80 aag gtc gag gccgetgetggc ccagcc ctggaggac gtcctagac 288 ctg Lys Val Glu AlaAlaAlaG1y ProA1a LeuGluAsp ValLeuAsp Leu Cag CCC CaC aCCCtgCaCCaC atCCtC tcccagctc caggcctgt 336 Ctt Gln Pro His ThrLeuHisHis I1eLeu SerGlnLeu GlnAlaCys Leu atc cag cag cccacagcaggg cccagg ccccggggc cgcctccac 384 cct Ile Gln Gln ProThrA1aGly ProArg ProArgGly ArgLeuHis Pro cac tgg cac cggctccaggag gccccc aaaaaggag tccgetggc 432 ctg His Trp His ArgLeuGlnGlu AlaPro LysLysGlu SerA1aGly Leu tgc ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc ctc acg cga 480 Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg gac ctc aaa tat gtg gcc gat ggg aay ctg tgt ctg aga acg tca acc 528 Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr cac cct gag tcc acc tga 546 His Pro Glu Ser Thr <210> 107 <211> 181 <212> PRT
<213> Artificial Sequence <220>
<223> IL29 mutant C15X, G18D, Asn169 <221> VARIANT
<222> (15)...(15) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 107 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa His Ile Asp Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu G1u Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 108 <211> 549 <212> DNA
<213> Artificial Sequence <220>
<223> Met IL29 mutant C16X, G19D, Asn170 <221> CDS
<222> (1)...(549) <221> variation <222> (47)...(48) <223> n = A, T, G, or C

<400>

atgggccctgtc cccact tccaagccc accacaact gggaag ggcdnn 48 MetGlyProVal ProThr SerLysPro ThrThrThr GlyLys GlyXaa cacattgayagg ttcaaa tctctgtca ccacaggag ctagcg agcttc 96 HisIleAspArg PheLys SerLeuSer ProGlnG1u LeuAla SerPhe aagaaggccagg gacgcc ttggaagag tcactcaag ctgaaa aactgg 144 LysLysAlaArg AspAla LeuGluGlu SerLeuLys LeuLys AsnTrp agttgcagctct cctgtc ttccccggg aattgggac ctgagg cttctc 192 SerCysSerSer ProVa1 PheProGly AsnTrpAsp LeuArg LeuLeu caggtgagggag cgccct gtggccttg gaggetgag ctggcc ctgacg 240 GlnValArgGlu ArgPro ValAlaLeu GluAlaGlu LeuAla LeuThr ctgaaggtcctg gaggcc getgetggc ccagccctg gaggac gtccta 288 LeuLysValLeu GluAla AlaAlaGly ProAlaLeu GluAsp ValLeu gaccagcccctt cacacc ctgcaccac atcctctcc cagctc caggcc 336 AspGlnProLeu HisThr LeuHisHis IleLeuSer GlnLeu GlnAla tgtatccagcct cagccc acagcaggg cccaggccc cggggc cgcctc 384 CysIleGlnPro GlnPro ThrAlaGly ProArgPro ArgGly ArgLeu caccactggctg caccgg ctccaggag gcccccaaa aaggag tccget 432 HisHisTrpLeu HisArg LeuGlnGlu AlaProLys LysGlu SerAla ggctgcctggag gcatct gtcaccttc aacctcttc cgcctc ctcacg 480 GlyCysLeuGlu AlaSer ValThrPhe AsnLeuPhe ArgLeu LeuThr cgagacctcaaa tatgtg gccgatggg aayctgtgt ctgaga acgtca 528 ArgAspLeuLys TyrVal AlaAspGly AsnLeuCys LeuArg ThrSer acccaccctgag tccacc tga 549 ThrHisProGlu SerThr <210> 109 <211> 182 <212> PRT
<213> Artificial Sequence <220>
<223> Met IL29 mutant C16X, G19D, Asn170 <221> VARIANT
<222> (16)...(16) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 109 Met G1y Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa His I12 Asp Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu G1u Ala G1u Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Sex Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 110 <211> 546 <212> DNA

<213> qArtificial equence S

<220> , <223> IL29mutant 18D,Asp169,C171X
G

<221> CDS

<222> (2)...(546) <221> variation <222> (512)...(513) <323> n G, or = A, T, C

<400> 110 ggc cct cccacttcc aagccc accacaact gggaagggc tgccac 48 gtc Gly Pro ProThrSer LysPro ThrThrThr GlyLysGly CysHis Val att gay ttcaaatct ctgtca ccacaggag ctagcgagc ttcaag 96 agg Ile Asp PheLysSer LeuSer ProGlnGlu LeuAlaSer PheLys Arg aag gcc gacgccttg gaagag tcactcaag ctgaaaaac tggagt 144 agg Lys A1a AspAlaLeu G1uGlu SerLeuLys LeuLysAsn TrpSer Arg tgc agc cctgtcttC CCCggg aattgggac ctgaggctt ctccag 192 tct Cys Ser ProVa1Phe ProGly AsnTrpAsp LeuArgLeu LeuGln Ser 50 , 55 60 gtg agg cgccctgtg gccttg gaggetgag ctggccctg acgctg 240 gag Val Arg ArgProVal AlaLeu GluAlaGlu LeuAlaLeu ThrLeu Glu aag gtc gaggccget getggc ccagccctg gaggacgtc ctagac 288 ctg Lys Val GluAlaAla AlaGly ProAlaLeu GluAspVal LeuAsp Leu Cag CCC CaCaCCCtg CaCCaC atCCtCtcc cagCtCCag gCCtgt 336 Ctt Gln Pro HisThrLeu HisHis IleLeuSer GlnLeu.Gln AlaCys Leu atc cag cct cag ccc aca gca ggg ccc agg ccc cgg ggc cgc ctc cac 384 Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His cactgg ctgcaccgg ctccag gaggccccc aaaaaggag tccgetggc 432 HisTrp LeuHisArg LeuGln GluAlaPro LysLysGlu SerAlaGly tgCCtg gaggcatCt gtCaCC ttcaacctC ttccgcctc ctcacgcga 480 CysLeu GluAlaSer ValThr PheAsnLeu PheArgLeu LeuThrArg gacctc aaatatgtg gccgat ggggayctg dnnctgaga acgtcaacc 528 AspLeu LysTyrVal AlaAsp GlyAspLeu XaaLeuArg ThrSerThr caccct gagtccacc tga 546 HisPro GluSerThr <210> 111 <211> 181 <212> PRT
<213> Artificial Sequence <220>
<223> IL29 mutant G18D, Asp169, C171X
<221> VARIANT
<~22> (171)...(171) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 111 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Asp Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Xaa Leu Arg Thr Ser Thr His Pro G1u Ser Thr <210> 112 <211> 549 <212> DNA
<213> Artificial Sequence <220>

<223> 9D,Asp170, C172X
Met mutant <221>
CDS

<222> (549) (1)...

<221>
variation <222> ..(516) (515).

<223> = T, G, n A, or C

<400>

atg cctgtc cccacttccaag cccacc acaactggg aagggctgc 48 ggc Met ProVal ProThrSerLys ProThr ThrThrGly LysGlyCys Gly cac gayagg ttcaaatctctg tcacca caggagcta gcgagcttc 96 att His AspArg PheLysSerLeu SerPro GlnGluLeu AlaSerPhe Ile aag gccagg gacgccttggaa gagtca ctcaagctg aaaaactgg 144 aag Lys AlaArg AspAlaLeuGlu GluSer LeuLysLeu LysAsnTrp Lys agt agctct CCtgtCttcccc gggaat tgggacctg aggcttctc 192 tgc Ser SerSer ProValPhePro GlyAsn TrpAspLeu ArgLeuLeu Cys cag agggag cgccctgtggcc ttggag getgagctg gccctgacg 240' gtg Gln ArgGlu ArgProValAla LeuGlu AlaGluLeu A1aLeuThr Val ctg gtcctg gaggccgetget ggccca gccctggag gacgtccta 288.
aag Leu Va1Leu GluAlaAlaAla GlyPro AlaLeuGlu AspValLeu Lys gac CCCCtt cacaccctgcac cacatc ctctcccag Ct CaggCC 336.
cag C

Asp ProLeu HisThrLeuHis HisIle LeuSerGln LeuGlnAla Gln tgt cagcct cagcccacagca gggccc aggccccgg ggccgcctc 384 atc Cys GlnPro GlnProThrAla GlyPro ArgProArg GlyArgLeu Ile cac tggctg caccggctccag gaggcc cccaaaaag gagtccget 432 cac His TrpLeu HisArgLeuGln GluAla ProLysLys GluSerAla His ggc ctggag gcatCtgtCaCC ttcaac ctCttCCgC CtCCtCaCg 48O
tgc Gly LeuGlu AlaSerValThr PheAsn LeuPheArg LeuLeuThr Cys cga ctcaaa tatgtggccgat ggggay ctgdnnctg agaacgtca 528 gac Arg LeuLys TyrValAlaAsp GlyAsp LeuXaaLeu ArgThrSer Asp acc cctgag tccacctga 549 cac Thr ProGlu SerThr His <210> 13 <211> 82 <212>
PRT

<213> icial Artif Sequence <220>
<223> Met IL29 mutant G19D, Asp170, C172X
<221> VARIANT
<222> (172)...(172) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 113 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Asp Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 114 <211> 546 <212> DNA

<213> ArtificialSequence <220>

<223> IL29 G18D, mutant Asp169 C15X, <221> CDS

<222> (1)...(546) <221> variation <222> (44)...(45) <223> n T, G, or = A, C

<400> 114 ggc cct cccacttccaag cccacc acaactggg aagggcdnn cac 48 gtc Gly Pro ProThrSerLys ProThr ThrThrGly LysGlyXaa His Val att gay ttcaaatctctg tcacca caggagcta gcgagcttc aag 96 agg Ile Asp PheLysSerLeu SerPro GlnGluLeu AlaSerPhe Lys Arg aag gcc gacgccttggaa gagtca ctcaagctg aaaaactgg agt 144 agg Lys Ala AspAlaLeuGlu GluSer LeuLysLeu LysAsnTrp Ser Arg tgc agc cctgtcttcccc gggaat tgggacctg aggcttctc cag 192 tct Cys Ser ProValPhePro GlyAsn TrpAspLeu ArgLeuLeu Gln Ser gtgagg gagcgc cctgtggcc ttggag getgagctg gccctgacg ctg 240 ValArg GluArg ProValAla LeuGlu A1aGluLeu AlaLeuThr Leu aaggtc ctggag gccgetget ggccca gccctggag gacgtccta gac 288 LysVal LeuGlu AlaAlaAla GlyPro A1aLeuGlu AspValLeu Asp cagccc cttcac accctgcac cacatc ctctcccag ctccaggcc tgt 336 GlnPro LeuHis ThrLeuHis HisIle LeuSerGln LeuGlnAla Cys atccag cctcag cccacagca gggccc aggccccgg ggccgcctc cac 384 IleGln ProG1n ProThrAla GlyPro ArgProArg GlyArgLeu His cactgg ctgcac cggctccag gaggcc cccaaaaag gagtccget ggc 432 HisTrp LeuHis ArgLeuGln GluAla ProLysLys GluSerAla Gly tgcctg gaggca tctgtcacc ttcaac ctcttccgc ctcctcacg cga 480 CysLeu GluAla SerVa1Thr PheAsn LeuPheArg LeuLeuThr Arg gacctc aaatat gtggccgat ggggay ctgtgtctg agaacgtca acc 528 AspLeu LysTyr ValAlaAsp GlyAsp LeuCysLeu ArgThrSer Thr caccct gagtcc acctga 546;

HisPro GluSer Thr <210> 115 <211> 181 <212> PRT
<213> Artificial Sequence <220>
<223> IL29 mutant C15X, G18D, Asp169 <221> VARIANT
<322> (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 Ile Asp Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys A1a Arg Asp A1a Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu A1a Leu Thr Leu Lys Val Leu Glu A1a A1a Ala Gly Pro Ala Leu Glu Asp Va1 Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Tyr ValAlaAsp GlyAspLeu CysLeuArg ThrSerThr Lys His Pro Ser Thr Glu <210> 116 <211> 549 <212> DNA

<213> ArtificialSequence <220>

<223> Met 29 G19D, IL mutant Asp170 C16X, <221> CDS

<222> (1)...(549) <221> variation <222> (47)...(48) <223> n T, G.
= A, or C

<400> 116 atg ggc gtc cccacttcc aagcccacc acaactggg aagggcdnn 48 cct Met Gly Val ProThrSer LysProThr ThrThrGly LysGlyXaa Pro cac att agg ttcaaatct ctgtcacca caggagcta gcgagcttc 96 gay His Ile Arg PheLysSer LeuSerPro GlnGluLeu AlaSerPhe Asp aag aag agg gacgccttg gaagagtca ctcaagctg aaaaactgg 144 gcc Lys Lys Arg AspAlaLeu GluGluSer Leu.LysLeu LysAsnTrp Ala agt tgc tct cctgtcttc cccgggaat tgggacctg aggcttctc 192 agc Ser Cys Ser ProValPhe ProG1yAsn TrpAspLeu ArgLeuLeu Ser cag gtg gag cgccctgtg gccttggag getgagctg gccctgacg 240 agg Gln Val Glu ArgProVal AlaLeuGlu AlaGluLeu AlaLeuThr Arg ctg aag ctg gaggccget getggccca gccctggag gacgtccta 288 gtc Leu Lys Leu GluAlaAla AlaGlyPro AlaLeuGlu AspValLeu Val gac cag Ctt CaCaCCCtg caccacatC CtCtcccag CtCCaggCC 336 CCC

Asp Gln Leu HisThrLeu HisHisIle LeuSerGln LeuGlnAla Pro tgt atc cct cagcccaca gcagggccc aggccccgg ggccgcctc 384 cag Cys Ile Pro GlnProThr AlaGlyPro ArgProArg GlyArgLeu Gln cac cac ctg caccggctc caggaggcc cccaaaaag gagtccget 432 tgg His His Leu HisArgLeu GlnGluAla ProLysLys GluSerA1a Trp ggc tgc gag gcatctgtc accttcaac ctcttCCgC CtCCtCaCg 4HO
ctg Gly Cys G1u AlaSerVal ThrPheAsn LeuPheArg LeuLeuThr Leu cga gac aaa tatgtggcc gatggggay ctgtgtctg agaacgtca 528 ctc Arg Asp Lys TyrValAla AspG1yAsp LeuCysLeu ArgThrSer Leu acc cac cct gag tcc acc tga 549 Thr His Pro Glu Ser Thr <210> 117 <211> 182 <312> PRT
<213> Artificial Sequence <220>
<223> Met IL29 mutant C16X, G19D, Asp170 <221> VARIANT
<222> (16)...(16) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 117 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys G1y Xaa His Ile Asp Arg Phe Lys Ser Leu Ser Pro G1n Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Va1 Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln A1a Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 118 <211> 57 <212> DNA
<213> Artificial Sequence <220>
<223> Signal sequence <221> CDS
<222> (1)...(57) <400> 118 atg get gca get tgg acc gtg gtg ctg gtg act ttg gtg cta ggc ttg 48 Met Ala Ala Ala Trp Thr Val Val Leu Val Thr Leu Val Leu Gly Leu gcc gtg gca 57 Ala Val Ala <210> 119 <211> 19 <212> PRT
<213> Artificial Sequence <220>
<223> Signal sequence <400> 119 Met Ala Ala Ala Trp Thr Val Val Leu Val Thr Leu Val Leu Gly Leu Ala Val Ala <210> 120 <211> 66 <212> DNA
<213> Artificial Sequence <220>
<223> Signal sequence <221> CDS
<222> (1)...(66) <400> 120 atg gtg ccc acc aca ttg get tgg acc gtg gtg ctg gtg act ttg gtg 48 Met Val Pro Thr Thr Leu A1a Trp Thr Val Val Leu Val Thr Leu Val cta ggc ttg gcc gtg gca 66 Leu G1y Leu Ala Val Ala <210> 121 <211> 22 <212> PRT
<213> Artificial Sequence <220>
<223> Signal sequence <400> 121 Met Val Pro Thr Thr Leu Ala Trp Thr Val Val Leu Val Thr Leu Val Leu Gly Leu Ala Val Ala <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 gttcct gtcgccagg ctccgc ggggetctc ccggat gcaaggggc tgc 48 ValPro ValA1aArg LeuArg GlyAlaLeu ProAsp AlaArgGly Cys cacata gcccagttc aagtcc ctgtctcca caggag ctgcaggcc ttt 96 HisI1e AlaGlnPhe LysSer LeuSerPro GlnGlu LeuGlnAla Phe aagagg gccaaagat gcctta gaagagtcg cttctg ctgaaggac dnn 144 LysArg AlaLysAsp A1aLeu GluGluSer LeuLeu LeuLysAsp Xaa aagtgc cgctcccgc ctcttc cccaggacc tgggac ctgaggcag ctg 192 LysCys ArgSerArg LeuPhe ProArgThr TrpAsp LeuArgGln Leu caggtg agggagcgc cccgtg getttggag getgag ctggccctg acg 240 G1nVa1 ArgGluArg ProVa1 AlaLeuGlu AlaGlu LeuAlaLeu Thr ctgaag gttctggag gccacc getgacact gaccca gccctgggg gat 288 LeuLys ValLeuGlu AlaThr AlaAspThr AspPro AlaLeuGly Asp gtcttg gaccagCCC Cttcac accctgcac catatC CtCtCCCag CtC 336 ValLeu AspGlnPro LeuHis ThrLeuHis HisIle LeuSerGln Leu cgggcc tgtatccag cctcag cccacggca gggccc aggacccgg ggc 384 ArgAla CysIleGln ProGln ProThrAla G7.yPro ArgThrArg Gly CgCCtC CaCCattgg ctgcac cggctccag gaggcc ccaaaaaag gag 432 ArgLeu HisHisTrp LeuHis ArgLeuGln GluAla ProLysLys Glu tcccct ggctgcctc gaggcc tctgtcacc ttcaac ctcttccgc ctc 480 SerPro GlyCysLeu GluAla SerValThr PheAsn LeuPheArg Leu ctcacg cgagacctg aattgt gttgccagc ggggac ctgtgtgtc tga 528 LeuThr ArgAspLeu AsnCys Va1A1aSer GlyAsp LeuCysVal <210> 123 <211> 175 <212> PRT
<213> Artificial Sequence <220>
<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 G1y Cys His I1e Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg A1a Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Xaa Lys Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu G1y Asp Va1 Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu His Arg Leu G1n Glu Ala Pro Lys Lys Glu Ser Pro G1y Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser G1y Asp Leu Cys Val <210> 124 <211> 531 <212> DNA

<213> ArtificialSequenc e <220>

<223> Met -28BC49 S
IL

<221> CDS

<222> (1)...(531) ' <221> variation <222> (146)...(147) <223> n ~T,G, = A, or C

<400> l24 atg gtt gtCgcc aggctccgc gggget ctcccggat gcaaggggc 48 CCt Met Val ValAla ArgLeuArg GlyAla LeuProAsp AlaArgGly Pro tgc cac gcccag ttcaagtcc ctgtct ccacaggag ctgcaggcc 96 ata Cys His AlaGln PheLysSer LeuSer ProGlnGlu LeuGlnAla I1e ttt aag gccaaa gatgcctta gaagag tcgcttctg ctgaaggac 144 agg Phe Lys AlaLys AspAlaLeu GluGlu SerLeuLeu LeuLysAsp Arg dnn aag cgctcc cgcctcttc cccagg acctgggac ctgaggcag 192 tgc Xaa Lys ArgSer ArgLeuPhe ProArg ThrTrpAsp LeuArgGln Cys ctg cag agggag cgccccgtg getttg gaggetgag ctggccctg 240 gtg Leu Gln ArgGlu ArgProVal AlaLeu GluAlaGlu LeuAlaLeu Val acg ctg gttctg gaggccacc getgac actgaccca gccctgggg 288 aag Thr Leu ValLeu GluAlaThr AlaAsp ThrAspPro AlaLeuGly Lys gat gtc gaccag ccccttcac accctg caccatatc ctctcccag 336 ttg Asp Val AspGln ProLeuHis ThrLeu HisHisIle LeuSerGln Leu ctc cgg tgtatc cagcctcag cccacg gcagggccc aggacccgg 384 gcc Leu Arg CysIle GlnProGln ProThr AlaGlyPro ArgThrArg Ala ggc cgc caccat tggctgcac cggctc caggaggcc ccaaaaaag 432 ctc Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys gag tcc CCt ggC tgC CtC gag gcc tCt gtc aCC ttc aac ctc ttC cgc 480 Glu Ser Pro G1y Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg ctc ctc acg cga gac ctg aat tgt gtt gcc agc ggg gac ctg tgt gtc 528 Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val tga 531 <210> 125 <211> 176 <212> PRT
<213> Artificial Sequence <220>
<221> VARIANT
<222> (49)...(49) <323> Xaa = Ser, Ala, Thr, Val, or Asn <223> Met TL-28B C49S
<400> 125 Met Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln G1u Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Xaa Lys Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Va1 Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Gly Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg 145 150 155 . 160 Leu Leu Thr Arg Asp Leu Asn Cys Val A1a Ser Gly Asp Leu Cys Val <210> 126 <211> 528 <212> DNA
<213> Artificial Sequence <220>
<223> IL-28B C50S
<221> CDS
<222> (1)...(528) <221> variation <222> (149)...(150) <223> = T, G, n A, or C

<400>

gttcct gtcgccagg ctccgcggg getctc ccggatgca aggggctgc 48 ValPro ValAlaArg LeuArgGly AlaLeu ProAspAla ArgGlyCys cacata gcccagttc aagtccctg tctcca caggagctg caggccttt 96 HisIle AlaGlnPhe LysSerLeu SerPro GlnGluLeu GlnAlaPhe aagagg gccaaagat gccttagaa gagtcg cttctgctg aaggactgc 144 LysArg AlaLysAsp AlaLeuGlu GluSer LeuLeuLeu LysAspCys aagdnn cgctcccgc ctcttcccc aggacc tgggacctg aggcagctg 192 LysXaa ArgSerArg LeuPhePro ArgThr TrpAspLeu ArgGlnLeu caggtg agggagCgC CCCgtgget ttggag getgagctg gccctgacg 240 GlnVal ArgGluArg ProValAla LeuGlu AlaGluLeu AlaLeuThr ctgaag gttctggag gccaccget gacact gacccagcc ctgggggat 288 LeuLys ValLeuGlu AlaThrAla AspThr AspProAla LeuGlyAsp gtcttg gaccagccc cttcacacc ctgcac catatcctc tcccagctc 336 ValLeu AspGlnPro LeuHisThr LeuHis HisIleLeu SerGlnLeu cgggcc tgtatccag cctcagccc acggca gggcccagg acccggggc 384 ArgAla CysIleGln ProGlnPro ThrAla GlyProArg ThrArgGly cgcctc caccattgg ctgcaccgg ctccag gaggcccca aaaaaggag 432 ArgLeu HisHisTrp LeuHisArg LeuGln GluAlaPro LysLysGlu tCCCCt ggctgCCtC gaggcctCt gtCaCC ttCaaCCtC ttCCgCCtC 480 SerPro GlyCysLeu GluAlaSer ValThr PheAsnLeu PheArgLeu ctcacg cgagacctg aattgtgtt gccagc ggggacctg tgtgtctga 528 LeuThr ArgAspLeu AsnCysVal AlaSer GlyAspLeu CysVal <210> 127 <~11> 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 Va1 Ala Arg Leu Arg Gly A1a Leu Pro Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp A1a Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys Lys Xaa Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Gly Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val <210> 128 ) <211> 531 <212> DNA

<213> ArtificialSequence <~20>

<223> Met C51S

<221> CDS

<222> (1)...(531) <221> variation <222> (152)...(153) <223> n T, G, = A, or C

<400> 128 atg gtt gtc gccaggctc cgcgggget ctcccg gatgcaagg ggc 48 cct Met Val Val AlaArgLeu ArgGlyAla LeuPro AspAlaArg Gly Pro tgc cac gcc cagttcaag tccctgtct ccacag gagctgcag gcc 96 ata Cys His Ala GlnPheLys SerLeuSer ProGln GluLeuG1n Ala Ile ttt aag gcc aaagatgcc ttagaagag tcgctt ctgctgaag gac 144 agg Phe Lys Ala LysAspAla LeuGluG1u SerLeu LeuLeuLys Asp Arg tgc aag cgc tcccgcctc ttccccagg acctgg gacctgagg cag 192 dnn Cys Lys Arg SerArgLeu PheProArg ThrTrp AspLeuArg Gln Xaa ctg cag agg gagCgCCCC gtggetttg gagget gagctggcc ctg 240 gtg Leu Gln Arg GluArgPro ValAlaLeu GluAla GluLeuAla Leu Va1 acg ctg gtt ctggaggcc accgetgac actgac ccagccctg ggg 288 aag Thr Leu Val LeuGluAla ThrAlaAsp ThrAsp ProAlaLeu Gly Lys gat gtc gac cagcccctt cacaccctg caccat atcctctcc cag 336 ttg Asp Val Asp G1nProLeu HisThrLeu HisHis IleLeuSer Gln Leu ctc cgg tgt atccagcct cagcccacg gcaggg cccaggacc cgg 384 gcc Leu Arg Cys IleGlnPro GlnProThr AlaGly ProArgThr Arg Ala ggccgc ctccaccat tggctg caccggctc caggaggcc ccaaaaaag 432 GlyArg LeuHisHis TrpLeu HisArgLeu GlnGluAla ProLysLys gagtcc cctggctgc ctcgag gcctctgtc accttcaac ctcttccgc 480 GluSer ProG1yCys LeuGlu AlaSerVal ThrPheAsn LeuPheArg ctcctc acgcgagac ctgaat tgtgttgcc agcggggac ctgtgtgtc 528 LeuLeu ThrArgAsp LeuAsn CysValAla SerGlyAsp LeuCysVal tga 531 <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 Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln G1u Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys Lys Xaa Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Gly Asp Va1 Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Va1 Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val <210> 130 <211> 528 <212> DNA
<213> Artificial Sequence <220>
<223> IL-28B C48S T87S H135Y
<221> CDS
<222> (1)...(528) <221>
variation <222> 44,261 143, <223> = T, G, n A, or C

<400>

gtt cctgtcgccagg ctccgc ggggetctc ccggatgca aggggc tgc 48 Va1 ProValAlaArg LeuArg GlyAlaLeu ProAspAla ArgGly Cys cac atagcccagttc aagtcc ctgtctcca caggagctg caggcc ttt 96 His IleAlaGlnPhe LysSer LeuSerPro GlnGluLeu GlnA1a Phe aag agggccaaagat gcctta gaagagtcg cttctgctg aaggac dnn 144 Lys ArgAlaLysAsp AlaLeu GluGluSer LeuLeuLeu LysAsp Xaa aag tgccgctcccgc ctcttc cccaggacc tgggacctg aggcag ctg 192 Lys CysArgSerArg LeuPhe ProArgThr TrpAspLeu ArgGln Leu cag gtgagggagcgc cccgtg getttggag getgagctg gccctg acg 240 Gln ValArgGluArg ProVal AlaLeuG1u AlaGluLeu AlaLeu Thr ctg aaggttctggag gccwsn getgacact gacccagcc ctgggg gat 288 Leu LysValLeuGlu AlaXaa AlaAspThr AspProAla LeuGly Asp gtc ttggaccagccc cttcac accctgcac catatcctc tcccag ctc 336 Val LeuAspG1nPro LeuHis ThrLeuHis HisIleLeu SerGln Leu cgg gcctgtatccag cctcag cccacggca gggcccagg acccgg ggc 384 Arg AlaCysIleGln ProGln ProThrAla GlyProArg ThrArg Gly cgc ctccaccattgg ctgtay cggctccag gaggcccca aaaaag gag 432 Arg LeuHisHisTrp LeuTyr ArgLeuGln GluAlaPro LysLys Glu tCC CCtggctgCCtC gaggcc tCtgtCaCC ttcaacctC ttCCgC CtC 480 Ser ProGlyCysLeu GluAla SerValThr PheAsnLeu PheArg Leu ctc acgcgagacctg aattgt gttgccagc ggggacctg tgtgtc tga 528 Leu ThrArgAspLeu AsnCys ValAlaSer GlyAspLeu CysVal <210> 31 <211> 75 <212> RT
P

<213> l Artificia 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 Ala Leu Pro Asp Ala Arg G1y Cys His Ile Ala G1n Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln A1a Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Xaa Lys Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu A1a Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Xaa Ala Asp Thr Asp Pro Ala Leu Gly Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Arg Ala Cys Ile Gln Pro G1n Pro Thr Ala G1y Pro Arg Thr Arg Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln G1u Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser G1y Asp Leu Cys Val <210> 132 <211> 531 <212> DNA

<213> Artificial Sequence <220>

<223> Met C49S

<221> CDS

<222> (1)...(531) <221> variation <222> 146, 264 147, <223> n T, G, = A, or C

<400> 132 atg gtt gtc gccaggctccgc gggget ctcccggat gcaaggggc 48 cct Met Val Val AlaArgLeuArg GlyA1a LeuProAsp AlaArgGly Pro tgc cac gcc cagttcaagtcc ctgtct ccacaggag ctgcaggcc 96 ata Cys His Ala G1nPheLysSer LeuSer ProGlnGlu LeuGlnAla Ile ttt aag gcc aaagatgcctta gaagag tcgcttctg ctgaaggac 144 agg Phe Lys Ala LysAspA1aLeu GluGlu SerLeuLeu LeuLysAsp Arg clnn aag cgc tcccgcctcttc cccagg acctgggac ctgaggcag 192 tgc Xaa Lys Arg SerArgLeuPhe ProArg ThrTrpAsp LeuArgGln Cys ctg cag agg gagcgccccgtg getttg gaggetgag ctggccctg 240 gtg Leu Gln Arg GluArgProVal AlaLeu GluAlaGlu LeuAlaLeu Val acg ctg gtt ctggaggccwsn getgac actgaccca gccctgggg 288 aag Thr Leu Val LeuGluAlaXaa AlaAsp ThrAspPro AlaLeuGly Lys gatgtc ttggaccag ccccttcac accctgcac catatc ctctcccag 336 AspVal LeuAspGln ProLeuHis ThrLeuHis HisIle LeuSerGln ctccgg gcctgtatc cagcctcag cccacggca gggccc aggacccgg 384 LeuArg AlaCysIle GlnProGln ProThrAla G1yPro ArgThrArg ggccgc ctccaccat tggctgtay cggctccag gaggcc ccaaaaaag 432 GlyArg LeuHisHis TrpLeuTyr ArgLeuGln GluAla ProLysLys gagtcc cctggctgc ctcgaggcc tctgtcacc ttcaac ctcttccgc 480 GluSer ProGlyCys LeuGluAla SerValThr PheAsn LeuPheArg ctcctc acgcgagac ctgaattgt gttgccagc ggggac ctgtgtgtc 528 LeuLeu ThrArgAsp LeuAsnCys ValAlaSer GlyAsp LeuCysVal tga~ 531 <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 Gly Ala Leu Pro Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Xaa Lys Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu A1a Xaa Ala Asp Thr Asp Pro Ala Leu G1y Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Arg Ala Cys Ile Gln Pro G1n Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val <210> 134 <211> 528 <212> DNA

<213> ArtificialSequence <220>

<223> IL-28B S 7S

<221> CDS

<222> (1)...(528) <221> variation <222> 149, 261 150, <223> n T, G, = A, or C

<400> 134 gtt cct gcc aggCtCCgC ggggetctc ccggat gcaaggggc tgc 48 gtc Val Pro Ala ArgLeuArg GlyAlaLeu ProAsp AlaArgGly Cys Val 1 5 l0 15 cac ata cag ttcaagtcc ctgtctcca caggag ctgcaggcc ttt 96 gcc His Ile G1n PheLysSer LeuSerPro GlnGlu LeuGlnAla Phe Ala aag agg aaa gatgcctta gaagagtcg cttctg ctgaaggac tgc 144 gcc Lys Arg Lys AspAlaLeu GluGluSer LeuLeu LeuLysAsp Cys Ala aag dnn tcc cgcctcttc cccaggacc tgggac ctgaggcag ctg 192 cgc Lys Xaa Ser ArgLeuPhe ProArgThr TrpAsp LeuArgGln Leu Arg cag gtg gag cgccccgtg getttggag getgag ctggccctg acg 240.
agg Gln Val Glu ArgProVal AlaLeuGlu AlaGlu LeuAlaLeu Thr Arg ctg aag ctg gaggccwsn getgacact gaccca gccctgggg gat 288 gtt Leu Lys Leu GluAlaXaa AlaAspThr AspPro AlaLeuGly Asp Val gtc ttg cag CCCCttCaC ~accctgcac catatC CtCtCCCag CtC 336 gac Val Leu Gln ProLeuHis ThrLeuHis HisIle LeuSerGln Leu Asp cgg gcc atc cagcctcag cccacggca gggccc aggacccgg ggc 384 tgt Arg Ala Ile GlnProGln ProThrAla GlyPro ArgThrArg Gly Cys cgc ctc cat tggctgtay cggctccag gaggcc ccaaaaaag gag 432 cac Arg Leu His TrpLeuTyr ArgLeuGln GluAla ProLysLys Glu His tcc cct tgC CtCgaggcc tCtgtcacc ttcaac ctCttCCgC CtC 480 ggc Ser Pro Cys LeuGluAla SerValThr PheASn LeuPheArg Leu Gly ctc acg gac ctgaattgt gttgccagc ggggac ctgtgtgtc tga 528 cga Leu Thr Asp LeuAsnCys ValAlaSer GlyAsp LeuCysVal Arg <210> 135 <211> 175 <212> PRT

<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
<400> 135 Val Pro Val A1a Arg Leu Arg Gly A1a Leu Pro Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln A1a Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys Lys Xaa Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Xaa Ala Asp Thr Asp Pro Ala Leu Gly Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val <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
<400> 136 atg gtt cct gtc gcc agg ctc cgc ggg get ctc ccg gat gca agg ggc 48 Met Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly tgc cac ata gcc cag ttc aag tcc ctg tct cca cag gag ctg cag gcc 96 Cys His I1e Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala ttt aag agg gcc aaa gat gcc tta gaa gag tcg ctt ctg ctg aag gac 144 Phe Lys Arg Ala Lys Asp Ala Leu Glu G1u Ser Leu Leu Leu Lys Asp tgcaag dnncgctcc cgcctc ttccccagg acctgggac ctgaggcag 192 CysLys XaaArgSer ArgLeu PheProArg ThrTrpAsp LeuArgGln ctgcag gtgagggag CgCCCC gtggetttg gaggetgag ctggccctg 240 LeuGln ValArgGlu ArgPro ValAlaLeu GluAlaGlu LeuAlaLeu acgctg aaggttctg gaggcc wsngetgac actgaccca gccctgggg 288 ThrLeu LysValLeu GluAla XaaAlaAsp ThrAspPro AlaLeuGly g5 90 95 gatgtc ttggaccag cccctt cacaccctg caccatatc ctctcccag 336 AspVal LeuAspGln ProLeu HisThrLeu HisHisIle LeuSerG1n ctccgg gcctgtatc cagcct cagcccacg gcagggccc aggacccgg 384 LeuArg AlaCysIle GlnPro GlnProThr AlaGlyPro ArgThrArg ggccgc ctccacoat tggctg taycggctc caggaggcc ccaaaaaag 432 GlyArg LeuHisHis TrpLeu TyrArgLeu GlnGluAla ProLysLys gagtcc cctggctgc ctcgag gcctctgtc accttcaac ctcttccgc 480 GluSer ProGlyCys LeuGlu AlaSerVal ThrPheAsn LeuPheArg ctcctc acgcgagac ctgaat tgtgttgcc agcggggac ctgtgtgtc 528 LeuLeu ThrArgAsp LeuAsn CysValAla SerGlyAsp LeuCysVal tga 531.

<210> 137 <211> 176 <212> PRT
<~13> 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
<400> 137 Met Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys Lys Xaa Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Val Ala Leu G1u Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Xaa Ala Asp Thr Asp Pro Ala Leu Gly Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu Tyr Arg Leu G1n Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val <210> 138 <211> 543 <212> DNA

<213> ArtificialSequence <220>

<223> IL-29C170X, truncated after N-terminal Methionine and Gl ycine <221> variation <222> (509)...(510) <223> n T, G, = A, or C

<221> CDS

<222> (1)...(543) <400> 138 cct gtc act tccaagccc accacaact gggaagggc tgccac att 48 ccc Pro Val Thr SerLysPro ThrThrThr GlyLysGly CysHis Ile Pro ggc agg aaa tctctgtca ccacaggag ctagcgagc ttcaag aag 96 ttc Gly Arg Lys SerLeuSer ProGlnGlu LeuAlaSer PheLys Lys Phe gcc agg gcc ttggaagag tcactcaag ctgaaaaac tggagt tgc 144 gac Ala Arg Ala LeuGluGlu SerLeuLys LeuLysAsn TrpSer Cys Asp agc tct gtC ttccccggg aattgggac ctgaggctt ctccag gtg 192 CCt Ser Ser Val PheProGly AsnTrpAsp LeuArgLeu LeuGln Val Pro agg gag cct gtggccttg gaggetgag ctggccctg acgctg aag 240 cgc Arg Glu Pro ValAlaLeu G1uAlaGlu LeuAlaLeu ThrLeu Lys Arg gtc ctg gcc getgetggc ccagccctg gaggacgtc ctagac.cag 288 gag Va1 Leu Ala AlaAlaGly ProA1aLeu GluAspVal LeuAsp Gln Glu CCC Ctt acc ctgcaccac atcctCtCC Cagctccag gcctgt atC 336 cac Pro Leu Thr LeuHisHis I1eLeuSer GlnLeuGln A1aCys Ile His cag cct ccc acagcaggg cccaggccc cggggccgc ctccac cac 384 cag Gln Pro Pro ThrA1aGly ProArgPro ArgGlyArg LeuHis His Gln tgg ctg cgg ctccaggag gcccccaaa aaggagtcc getggc tgc 432 cac Trp Leu Arg LeuGlnGlu AlaProLys LysGluSer A1aGly Cys His ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc ctc acg cga gac 480 Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp ctc aaa tat gtg gcc gat ggg aac ctg dnn ctg aga acg tca acc cac 528 Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His cct gag tcc acc tga Pro Glu Ser Thr <210> 139 <211> 180 <212> PRT
<213> Artificial Sequence <~20>
<221> VARIANT
<222> (170)...(170) <323> Xaa = Ser, Ala, Thr, Val, or Asn <223> IL-29 C170X, truncated after N-terminal Methionine and Glycine <400> 139 Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Sex Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Th.r His Pro Glu Ser Thr <210> 140 <211> 540 <212> DNA
<213> Artificial Sequence <220>
<223> IL-29 C169X, truncated after N-terminal Methionine, Glycine, and Proline <221> variation <222> (506)...(507) <223> n = A, T, G, or C
<221> CDS

<222> (540) (1)...

<400>

gtcCcc acttccaag cccacc acaactggg aagggctgc cacattggc 48 Va1Pro ThrSerLys ProThr ThrThrGly LysGlyCys HisIleGly aggttc aaatctctg tcacca caggagcta gcgagcttc aagaaggcc 96 ArgPhe LysSerLeu SerPro GlnGluLeu AlaSerPhe LysLysAla agggac gccttggaa gagtca ctcaagctg aaaaactgg agttgcagc 144 ArgAsp AlaLeuGlu GluSer LeuLysLeu LysAsnTrp SerCysSer tctcct gtcttcccc gggaat tgggacctg aggcttctc caggtgagg 192 SerPro ValPhePro GlyAsn TrpAspLeu ArgLeuLeu GlnValArg gagcgc cctgtggcc ttggag getgagctg gccctgacg ctgaaggtc 240 G1uArg ProValAla LeuGlu AlaGluLeu AlaLeuThr LeuLysVal ctggag gccgetget ggccca gccctggag gacgtccta gaccagccc 288 LeuGlu AlaAlaAla GlyPro AlaLeuGlu AspValLeu AspGlnPro cttcac accctgcac cacatc ctctcccag ctccaggcc tgtatccag 336 LeuHis ThrLeuHis HisIle LeuSerGln LeuGlnAla CysIleGln cctcag cccacagca gggccc aggccccgg ggccgcctc caccactgg 384.

ProGln ProThrAla GlyPro ArgProArg GlyArgLeu HisHisTrp 115 1~0 125 ctgcac cggctccag gaggccccc aaaaaggag tccget ggctgcctg 432 LeuHis ArgLeuGln GluAlaPro LysLysGlu SerAla GlyCysLeu gaggca tctgtcacc ttcaacctc ttccgcctc ctcacg cgagacctc 480 GluAla SerValThr PheAsnLeu PheArgLeu LeuThr ArgAspLeu aaatat gtggccgat gggaacctg dnnctgaga acgtca acccaccct 528 LysTyr ValAlaAsp GlyAsnLeu XaaLeuArg ThrSer ThrHisPro gagtcc acctga 540 GluSer 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 Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp G1n Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu G1u Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210>

<211>

<212>
DNA

<213> Sequence Artificial <220>

<223> C168X, after IL-29 truncated N-terminal Methionine , roline, and Valine Glycine, P

<221>
variation <222> ..(504) (503).

<223> T, G, n = A, or C

<221>
CDS

<222> (537) (1)...

<400>

ccc act aag cccaccaca actgggaag ggctgccac attggc agg 48 tcc Pro Thr Lys ProThrThr ThrGlyLys GlyCysHis IleGly Arg Ser ttc aaa ctg tcaccacag gagctagcg agcttcaag aaggcc agg 96 tct Phe Lys Leu SerProGln GluLeuAla SerPheLys LysAla Arg Ser gac gcc gaa gagtcactc aagctgaaa aactggagt tgcagc tct 144 ttg Asp Ala Glu GluSerLeu LysLeuLys AsnTrpSer CysSer Ser Leu cct gtc CCC gggaattgg gacctgagg cttctccag gtgagg gag 192 ttC

Pro Val Pro GlyAsnTrp AspLeuArg LeuLeuGln ValArg Glu Phe cgc cct gcc ttggagget gagctggcc ctgacgctg aaggtc ctg 240 gtg Arg Pro Ala LeuGluAla GluLeuA1a LeuThrLeu LysVal Leu Val gag gcc get ggcccagcc ctggaggac gtcctagac cagccc ctt 288 get Glu Ala Ala GlyProAla LeuGluAsp ValLeuAsp GlnPro Leu Ala cacaccctg caccac atcctctcc cagctccag gcctgtatc eagcct 336 HisThrLeu HisHis IleLeuSer GlnLeuGln AlaCysIle GlnPro cagcccaca gcaggg cccaggCCC CggggCCgC CtCCaCCa.Ctggctg 384 GlnProThr AlaGly ProArgPro ArgG1yArg LeuHisHis TrpLeu caccggctc caggag gcccccaaa aaggagtcc getggctgc ctggag 432 HisArgLeu GlnGlu AlaProLys LysGluSer AlaGlyCys LeuGlu gcatctgtc accttc aacctcttc cgcctcctc acgcgagac ctcaaa 480 AlaSerVal ThrPhe AsnLeuPhe ArgLeuLeu ThrArgAsp LeuLys tatgtggcc gatggg aacctgdnn ctgagaacg tcaacccac cctgag 528 TyrValAla AspGly AsnLeuXaa LeuArgThr SerThrHis ProG1u tccacctga 537 SerThr <210> 143 <211> 178 <212> PRT
<213> Artificial Sequence <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, or Asn <400> 143 Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro G1n Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu G1u Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His I1e Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala G1y Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro G1u Ser Thr <210> 144 <211> 534 <212> DNA

<213> ArtificialSequenc e <220>

<223> IL-29C167X, after truncated N-terminal Methionine , roline, Valine, and Proline Glycine, P

<221> variation <222> (500)...(501) <223> n T, G, = A, or C

<221> CDS

<222> (1)...(534) <400> 144 act tcc cccaccaca actgggaag ggctgccac attggc aggttc 48 aag Thr Ser ProThrThr ThrGlyLys GlyCysHis IleG1y ArgPhe Lys aaa tct tcaccacag gagctagcg agcttcaag aaggcc agggac 96 ctg Lys Ser SerProGln GluLeuAla SerPheLys LysAla ArgAsp Leu 20 ~ 25 30 gcc ttg gagtcactc aagctgaaa aactggagt tgcagc tctcct 144 gaa Ala Leu GluSerLeu LysLeuLys AsnTrpSer CysSer SerPro Glu gtc ttc gggaattgg gacctgagg cttctccag gtgagg gagcgc 192 ccc Val Phe GlyAsnTrp AspLeuArg LeuLeuGln ValArg GluArg Pro cct gtg ttggagget gagctggcc ctgacgctg aaggtc ctggag 240 gcc Pro Val LeuGluA.laGluLeuAla LeuThrLeu LysVal LeuGlu Ala gcc get ggcccagcc ctggaggac gtcctagac cagCCC Cttcac 288' get Ala A1a GlyProAla LeuGluAsp ValLeuAsp GlnPro LeuHis Ala aCC ctg cacatcctc tcccagCtC CaggCCtgt atccag CCtcag 336 cac Thr Leu HisI1eLeu SerGlnLeu GlnAlaCys IleGln ProGln His ccc aca gggcccagg ccccggggc cgcctccac cactgg ctgcac 384 gca Pro Thr G1yProArg ProArgGly ArgLeuHis HisTrp LeuHis Ala cgg ctc gaggccccc aaaaaggag tccgetggc tgcctg gaggca 432 cag Arg Leu GluA1aPro LysLysGlu SerAlaG1y CysLeu GluA1a Gln tct gtc ttcaacctc ttccgcctc ctcacgcga gacctc aaatat 480 acc Ser Val PheAsnLeu PheArgLeu LeuThrArg AspLeu LysTyr Thr gtg gcc gggaacctg dnnctgaga acgtcaacc caccct gagtcc 528 gat Val Ala GlyAsnLeu XaaLeuArg ThrSerThr HisPro GluSer Asp acc tga 534 Thr <210> 145 <211> 177 <212> PRT
<213> Artificial Sequence <2~0>
<223> IL-29 C167X, truncated after N-terminal Methionine, Glycine, Proline, Valine, and Proline <221> VARIANT
<222> (167)...(167) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 145 Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Va1 Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln G1u Ala Pro Lys Lys G1u Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 146 <211> 531 <212> DNA
<213> Artificial Sequence <220>
<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
<222> (1)...(531) <400> 146 tcc aag ccc acc aca act ggg aag ggc tgc cac att ggc agg ttc aaa 48 Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys tct ctg tca cca cag gag cta gcg agc ttc aag aag gcc agg gac gcc 96 Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala ttg gaa gag tca ctc aag ctg aaa aac tgg agt tgc agc tct cct gtc 144 Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Va1 ttc ccc ggg aat tgg gac ctg agg ctt ctc cag gtg agg gag cgc cct 192 Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro gtg gcc ttg gag get gag ctg gcc ctg acg ctg aag gtc ctg gag gcc 240 Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala get get gge cca gcc ctg gag gac gtc cta gac cag ccc ctt cac acc 288 Ala Ala Gly Pro Ala Leu G1u Asp Val Leu Asp Gln Pro Leu His Thr ctg cac cac atc ctc tcc cag ctc cag gcc tgt atc cag cct cag ccc 336 Leu His His Ile Leu Ser Gln Leu Gln A1a Cys Ile Gln Pro Gln Pro aca gca ggg ccc agg CCC Cgg ggC CgC CtC CdC CaC tgg ctg cac cgg 384 Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg ctc cag gag gcc ccc aaa aag gag tcc get ggc tgc ctg gag gca tct 432 Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser gtc acc ttc aac ctc ttc cgc ctc ctc acg cga gac ctc aaa tat gtg 480 Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val gcc gat ggg aac ctg dnn ctg aga acg tca acc cac cct gag tcc acc 528 Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr 165 170 l75 tga 531 <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, Val, or Asn <400> 147 Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 148 <211> 528 <212> DNA

<213> ArtificialSequenc e <220>

<223> IL-29C165X, runcatedafter t N-terminal Methio nine, ycine, ne,Valine,Proline, Gl Proli Threon ine,andSerine <221> variation <222> (494)...(495) <223> n T, G, r = A, o C

<221> CDS

<222> (1)...(528) <400> 148 aag ccc aca actgggaag ggctgccac attggcagg ttcaaa tct 48 acc Lys Pro Thr ThrGlyLys GlyCysHis IleGlyArg PheLys Ser Thr ctg tca cag gagctagcg agcttcaag aaggccagg gacgcc ttg 96 cca Leu Ser Gln GluLeuAla SerPheLys LysAlaArg AspAla Leu Pro gaa gag ctc aagctgaaa aactggagt tgcagctct CCtgtC ttc 144 tca Glu Glu Leu LysLeuLys AsnTrpSer CysSerSer ProVa1 Phe Ser ccc ggg tgg gacctgagg cttctccag gtgagggag cgccct gtg 192 aat Pro Gly Trp AspLeuArg LeuLeuGln ValArgGlu ArgPro Val Asn gcc ttg get gagctggcc ctgacgctg aaggtcctg gaggcc get 240 gag Ala Leu Ala GluLeuAla LeuThrLeu LysValLeu GluAla A1a Glu get ggc gcc ctggaggac gtcctagac cagcccctt CaCaCC Ctg 288 cca Ala Gly Ala LeuGluAsp ValLeuAsp GlnProLeu HisThr Leu Pro cac cac ctc tcccagctc caggcctgt atccagcct cagccc aca 336 atc His His Leu SerGlnLeu GlnAlaCys IleGlnPro GlnPro Thr I1e gca ggg agg ccccggggc cgcctccac cactggctg caccgg ctc 384 ccc Ala Gly Arg ProArgGly ArgLeuHis HisTrpLeu HisArg Leu Pro cag gag ccc aaaaaggag tccgetggc tgcctggag gcatct gtc 432 gcc Gln Glu Pro LysLysGlu SerAlaGly CysLeuGlu AlaSer Val Ala acc ttc aac ctc ttc cgc ctc ctc acg cga gac ctc aaa tat gtg gcc 480 Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala gat ggg aac ctg dnn ctg aga acg tca acc cac cct gag tcc acc tga 528 Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 149 <211> 175 <212> PRT
<~13> Artificial Sequence <220> ' <223> IL-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 <400> 149 Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu G1u Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val A1a Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 150 <211> 552 <212> DNA
<213> Artificial Sequence <220>
<223> IL-29 Leu insert after N-terminal Met, C173X
<221> variation <222> (518)...(519) <223> n = A, T, G, or C
<221> CDS
<222> (1)...(552) <400> 150 atg ytn ggc cct gtc ccc act tcc aag ccc acc aca act ggg aag ggc 48 Met Leu Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys G1y tgc cac att ggc agg ttc aaa tct ctg tca cca cag gag cta gcg agc 96 Cys His Ile G1y Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser ttc aag aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg aaa aac 144 Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn tgg agt tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg ctt 192 Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu ctc cag gtg agg gag cgc ect gtg gcc ttg gag get gag ctg gcc etg 240 Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu acg ctg aag gtc ctg gag gcc get get ggc cca gcc ctg gag gac gtc 288 Thr Leu Lys Val Leu Glu Ala A1a Ala Gly Pro Ala Leu Glu Asp Val cta gac cag ccc ctt cac acc ctg cac cac atc ctc tcc cag ctc cag 336 Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln gcc tgt atc cag cct cag ccc aca gca ggg ccc agg ccc cgg ggc cgc 384.
Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Ar,g ctc cac cac tgg ctg cac cgg ctc cag gag gcc ccc aaa aag gag tcc 432' Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser get ggc tgc ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc ctc 480 Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu acg cga gac ctc aaa tat gtg gcc gat ggg aac ctg dnn ctg aga acg 528 Thr Arg Asp Leu Lys Tyx Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr tCa aCC Cc'~.C CCt gag tCC aCC tga 55~
Ser Thr His Pro Glu Ser Thr <210> 151 <211> 183 <212> PRT
<213> Artificial Sequence <220>
<223> IL-29 Leu insert after N-terminal Met, C173X
<221> VARIANT
<222> (173)..,(173) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 151 Met Leu Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro G1y Asn Trp Asp Leu Arg Leu Leu Gln Val Arg G1u Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 152 <211> 549 <212> DNA

<213> ArtificialSequence <220>

<223> IL-29G2LC172X

<22l> variation <222> (515)...(516) <223> n T, G, = A, or C

<221> CDS

<222> (1)...(549) <400> 152 atg ytn gtccccacttcc aagccc accacaact gggaagggc tgc 48 cct Met Leu ValProThrSer LysPro ThrThrThr GlyLysGly Cys Pro cac att aggttcaaatct ctgtca ccacaggag ctagcgagc ttc 96 ggc His Ile ArgPheLysSer LeuSer ProGlnGlu LeuAlaSer Phe Gly aag aag agggacgccttg gaagag tcactcaag ctgaaaaac tgg 144 gcc Lys Lys ArgAspAlaLeu GluGlu SerLeuLys LeuLysAsn Trp Ala agt tgc tctcctgtcttc cccggg aattgggac ctgaggctt ctc 192 agc Ser Cys SerProValPhe ProGly AsnTrpAsp LeuArgLeu Leu Ser cag gtg gagcgccctgtg gccttg gaggetgag ctggccctg acg 240 agg Gln Val GluArgProVal AlaLeu GluAlaGlu LeuAlaLeu Thr Arg ctg aag ctggaggccget getggc ccagccctg gaggacgtc cta 288 gtc Leu Lys LeuGluAlaA1a AlaGly ProAlaLeu GluAspVal Leu Val gac cag cttcacaccctg caccac atcctctcc cagctccag gcc 336 ccc Asp Gln LeuHisThrLeu HisHis IleLeuSer GlnLeuGln Ala Pro tgtatccagcct cagcccaca gcagggccc aggccc cggggccgc ctc 384 CysIleGlnPro GlnProThr A1aGlyPro ArgPro ArgGlyArg Leu caccactggctg caccggctc caggaggcc cccaaa aaggagtcc get 432 HisHisTrpLeu HisArgLeu GlnGluAla ProLys LysGluSer Ala ggctgcctggag gcatctgtc accttcaac ctcttC CgCCtCCtC aCg 48O

GlyCysLeuG1u AlaSerVal ThrPheAsn LeuPhe ArgLeuLeu Thr cgagacctcaaa tatgtggcc gatgggaac ctgdnn ctgagaacg tca 528 ArgAspLeuLys TyrValAla AspGlyAsn LeuXaa LeuArgThr Ser acccaccctgag tccacctga 549 ThrHisProGlu SerThr <210> 153 <211> 182 <212> PRT
<213> Artificial Sequence <220>
<223> IL-29 G2L C172X
<221> VARIANT
<222> (172)...(172) <223> Xaa = Ser, Ala, Thr, Val, or Asn <400> 153 Met Leu Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 154 <211> 552 <212> DNA
<213> Artificial Sequence <220>

<223> IL-29Ileinsertafter Met,C173X
N-terminal <221> variation <222> (518)...(519) <223> n T, G, or = A, C

<221> CDS

<222> (1)...(552) <400> 154 atg ath cctg'tccccact tccaag cccaccaca actgggaag ggc 48 ggc Met Ile ProValProThr SerLys ProThrThr ThrGlyLys Gly Gly tgc cac ggcaggttcaaa tctctg tcaccacag gagctagcg agc 96 att Cys His GlyArgPheLys SerLeu SerProGln GluLeuAla Ser Ile ttc aag gccagggacgcc ttggaa gagtcactc aagctgaaa aac 144 aag Phe Lys AlaArgAspAla LeuGlu GluSerLeu LysLeuLys Asn Lys tgg agt agctctcctgtc ttcccc gggaattgg gacctgagg ctt 192 tgc Trp Ser SerSerProVal PhePro GlyAsnTrp AspLeuArg Leu Cys ctc cag agggagcgccct gtggcc ttggagget gagctggcc ctg 240 gtg Leu Gln ArgGluArgPro ValAla LeuGluAla GluLeuAla Leu Val acg ctg gtcctggaggcc getget ggcccagcc ctggaggac gtc 288, aag Thr Leu ValLeuGluAla AlaAla GlyProAla LeuGluAsp Val Lys cta gac ccccttcacacc ctgcac cacatcctc tcccagctc cag 336 cag Leu Asp ProLeuHisThr LeuHis HisIleLeu SerGlnLeu Gln Gln gcc tgt cagcctcagccc acagca gggcccagg ccccggggc cgc 384 atc Ala Cys GlnProGlnPro ThrAla GlyProArg ProArgGly Arg Ile ctc cac tggctgcaccgg ctccag gaggccccc aaaaaggag tcc 432 cac Leu His TrpLeuHisArg LeuGln GluAlaPro LysLysGlu Ser His get ggc ctggaggcatct gtcacc ttcaacctc ttccgcctc ctc 480 tgc Ala Gly LeuGluAlaSer ValThr PheAsnLeu PheArgLeu Leu Cys acg cga ctcaaatatgtg gccgat gggaacctg dnnctgaga acg 528 gac Thr Arg LeuLysTyrVal AlaAsp GlyAsnLeu XaaLeuArg Thr Asp tca acc cctgagtccacc tga 552 cac Ser Thr ProGluSerThr His <210> 155 <211> 183 <212> PRT

<213> ArtificialSequence <220>

<~23> IL-29 Ile insert after N-terminal Met, C173X
<221> VARIANT
<22~> (173)...(173) <~23> Xaa = Ser, Ala, Thr, Val, or Asn <400> 155 Met Ile Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro G1n Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu A1a Pro Lys Lys Glu Ser A1a Gly Cys Leu Glu A1a Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 156 <211> 549 <212> DNA

<213> Artificial Sequence <220> ' <223> IL-29G2IC172X

<221> variation <222> (515)...(516) <223> n T, G, = A, or C

<221> CDS

<222> (1)...(549) <400> 156 atg ath gtccccacttcc aagcccacc acaactggg aagggc tgc 48 cct Met Ile ValProThrSer LysProThr ThrThrGly LysGly Cys Pro cac att aggttcaaatct ctgtcacca caggagcta gcgagc ttc 96 ggc His Ile ArgPheLysSer LeuSerPro GlnGluLeu AlaSer Phe Gly aag aag agggacgccttg gaagagtca ctcaagctg aaaaac tgg 144 gcc Lys Lys ArgAspA1aLeu G1uGluSer LeuLysLeu LysAsn Trp Ala agt tgc tctcctgtcttc cccgggaat tgggacctg aggctt ctc 192 agc Ser Cys SerProVa1Phe ProGlyAsn TrpAspLeu ArgLeu Leu Ser cag gtg agg gag cgc cct gtg gcc ttg gag get gag ctg gcc ctg acg 240 GlnVal ArgGluArg ProValAla LeuG1u AlaGluLeu AlaLeuThr ctgaag gtcctggag gccgetget ggccca gccctggag gacgtccta 288 LeuLys ValLeuGlu AlaAlaAla GlyPro AlaLeuGlu AspValLeu gaccag ccccttcac accctgcac cacatc ctctcccag ctccaggcc 336 AspGln ProLeuHis ThrLeuHis HisIle LeuSerGln LeuGlnAla tgtatc cagcctcag cccacagca gggccc aggccccgg ggccgcctc 384 CysIle GlnProGln ProThrAla GlyPro ArgProArg GlyArgLeu caccac tggctgcac cggctccag gaggcc cccaaaaag gagtccget 432 HisHis TrpLeuHis ArgLeuGln GluA1a ProLysLys GluSerAla ggctgc ctggaggca tctgtcacc ttcaac ctcttccgc ctcctcacg 480 GlyCys LeuGluAla SerValThr PheAsn LeuPheArg LeuLeuThr cgagac ctcaaatat gtggccgat gggaac ctgdnnctg agaacgtca 528 ArgAsp LeuLysTyr Va1AlaAsp GlyAsn LeuXaaLeu ArgThrSer acccac cctgagtcc acctga 549 ThrHis ProGluSer Thr <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 Ile Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile G1y Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu.Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp G1y Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <210> 158 <211> 531 <~12> DNA

<213> Artificial quence Se <220>

<223> IL-29after -terminal acidresidues N Met 2-7 amino deleted, C166X

<221> variation <222> (497)...(498) <223> n T, G, or = A, C

<221> CDS

<222> (1)...(531) <400> 158 atg aag accacaactggg aagggc tgccacatt ggcaggttc aaa 48 ccc Met Lys ThrThrThrGly LysGly CysHisIle GlyArgPhe Lys Pro tct ctg ccacaggagcta gcgagc ttcaagaag gccagggac gcc 96 tca Ser Leu ProGlnGluLeu AlaSer PheLysLys AlaArgAsp Ala Ser ttg gaa tcactcaagctg aaaaac tggagttgc agctctcct gtc 144 gag Leu G1u SerLeuLysLeu LysAsn TrpSerCys SerSerPro Val Glu ttC CCC aattgggacctg aggctt ctccaggtg agggagcgc cct 192 ggg Phe Pro AsnTrpAspLeu ArgLeu LeuGlnVal ArgGluArg Pro Gly gtg gcc gaggetgagctg gccctg acgctgaag gtcctggag gcc 240 ttg Val Ala GluAlaGluLeu AlaLeu ThrLeuLys ValLeuGlu Ala Leu get get ccagccctggag gacgtc ctagaccag ccccttcac acc 288 ggc Ala Ala ProA1aLeuGlu AspVa1 LeuAspGln ProLeuHis Thr Gly ctg cac atcctctcccag ctccag gcctgtatc cagcctcag ccc 336 cac Leu His IleLeuSerGln LeuGln AlaCysIle GlnProGln Pro His aca gca cccaggCCCCgg ggCCgC CtCCaCCaC tggctgcac cgg 384 ggg Thr Ala ProArgProArg GlyArg LeuHisHis TrpLeuHis Arg Gly ctc cag gcccccaaaaag gagtcc getggctgc ctggaggca tct 432 gag Leu Gln A1aProLysLys GluSer AlaG1yCys LeuGluAla Ser Glu gtc acc aacctcttccgc ctcctc acgcgagac ctcaaatat gtg 480 ttc Val Thr AsnLeuPheArg LeuLeu ThrArgAsp LeuLysTyr Val Phe gcc gat aacctgdnnctg agaacg tcaacccac cctgagtcc acc 528 ggg A1a Asp AsnLeuXaaLeu ArgThr SerThrHis ProGluSer Thr Gly tga 531 <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) <323> Xaa~= Ser, Ala, Thr, Val, or Asn <400> 159 Met Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Va7. Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys I1e Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr <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 atg gar gcn gar ggc cct gtc ccc act tcc aag ccc acc aca act ggg 48 Met Glu Ala Glu Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly aag ggc tgc cac att ggc agg ttc aaa tct ctg tca cca cag gag cta 96 Lys G1y Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu gcg agc ttc aag aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg 144 Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu aaa aac tgg agt tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg 193 Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu agg ctt ctc cag gtg agg gag cgc cct gtg gcc ttg gag get gag ctg 240 Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu gcc ctg acg ctg aag gtc ctg gag gcc get get ggc cca gcc ctg gag 288 Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu gac gtc cta gac cag ccc ctt cac acc ctg cac cac atc ctc tcc cag 336 Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln ctc cag gcc tgt atc cag cct cag ccc aca gca ggg ccc agg ccc cgg 384 Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg ggc cgc ctc cac cac tgg ctg cac cgg ctc cag gag gcc ccc aaa aag 432 Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys gag tcc get ggc tgc ctg gag gca tct gtc acc ttc aac ctc ttc cgc 480 Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg ctc ctc acg cga gac ctc aaa tat gtg gcc gat ggg aac ctg dnn ctg 528 Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu aga acg tca acc cac cct gag tcc acc tga 558 Arg Thr Ser Thr His Pro Glu Ser Thr <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 Met Glu Ala Glu Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Va1 Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Va1 Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro G1n Pro Thr A1a Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr

Claims (34)

1. An isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity to a sequence selected from the group consisting 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; wherein the polypeptide has anti-viral activity.

2. The polypeptide of claim 1 wherein the polypeptide comprises a sequence having at least 95% sequence identity to a sequence selected from the group consisting 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.

3. The polypeptide of claim 1 wherein the polypeptide has anti-hepatitis activity.

4. The polypeptide of claim 1 wherein the polypeptide has anti-hepatitis B
activity.

5. The polypeptide of claim 1 wherein the polypeptide has anti-hepatitis C activity.

6. The polypeptide of claim 1 wherein the polypeptide further comprises a polyethylene glycol moiety covalently attached N-terminally to the polpeptide.

7. The polypeptide of claim 6 wherein the polyethylene glycol moiety is mPEG propionaldehyde.

8. The polypeptide of claim 7 wherein the mPEG propionaldehyde has a molecular weight of about 20kD or 30kD.

9. The polypeptide of claim 7 wherein the mPEG propionaldehyde is linear.

10. An isolated polypeptide comprising an amino acid sequence selected from the group consisting 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.

11. An isolated polynucleotide encoding a polypeptide wherein the encoded polypeptide comprises an amino acid sequence having at least 90%
sequence identity to a sequence selected from the group consisting 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; wherein the polypeptide has anti-viral activity.

12. The isolated polynucleotide of claim 11 wherein the encoded polypeptide comprises a sequence having at least 95% sequence identity to a sequence selected from the group consisting 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.

13. The isolated polynucleotide of claim 11 wherein the encoded polypeptide has anti-hepatitis B activity.

14. The isolated polynucleotide of claim 11 wherein the encoded polypeptide has anti-hepatitis C activity.

15. An isolated polynucleotide capable of hybridizing to a nucleotide sequence selected from the group consisting 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 a complement thereof, under hybridization conditions of 50% formamide, 5xSSC
(1xSSC: 0.15 M sodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH
7.6), 5x Denhardt's solution, and 2% (w/v) bovine serum albumin, 10% dextran sulfate, and 20 mg/ml denatured, sheared salmon sperm DNA at about 42°C to about 70°C, wherein the polynucleotide encodes a polypeptide having anti-viral activity.

16. The isolated polynucleotide of claim 15 wherein the encoded polypeptide has anti-hepatitis B activity.

17. The isolated polynucleotide of claim 15 wherein the encoded polypeptide has anti-hepatitis C activity.

18. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting 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.

19. An expression vector comprising the following operably linked elements:
a transcription promoter;
a DNA segment encoding a polypeptide of claim 1; and a transcription terminator.

20. A cultured cell into which has been introduced an expression vector of claim 19, wherein the cell expresses the polypeptide encoded by the DNA
segment.

21. A method of producing a polypeptide comprising:
culturing a cell into which has been introduced an expression vector of claim 19, wherein the cell expresses the polypeptide encoded by the DNA
segment; and recovering the expressed polypeptide.

22. An antibody or antibody fragment that specifically binds to a polypeptide of claim 1.

23. The antibody of claim 22, wherein the antibody is selected from the group consisting of a polyclonal antibody, a murine monoclonal antibody, a humanized antibody derived from a murine monoclonal antibody, an antibody fragment, neutralizing antibody, and a human monoclonal antibody.

24. The antibody fragment of claim 22, wherein the antibody fragment is selected from the group consisting of F(ab'), F(ab), Fab', Fab, Fv, scFv, and minimal recognition unit.

25. An anti-idiotype antibody comprising an anti-idiotype antibody that specifically binds to the antibody of claim 22.

26. A fusion protein comprising a polypeptide that comprises a sequence of amino acid residues selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37, 38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, and 161; and a polyalkyl oxide moiety.

27. The fusion protein of claim 26 wherein the polyalkyl oxide moiety is polyethylene glycol.

28. The fusion protein of claim 27 wherein the polyethylene glycol is N-terminally or C-terminally attached to the polypeptide.

29. The fusion protein of claim 27 wherein the polyethylene glycol is mPEG propionaldehyde.

30. The fusion protein of claim 27 wherein the polyethylene glycol is branched or linear.

31. The fusion protein of claim 27 wherein the polyethylene glycol has a molecular weight of about 20kD or 30kD.

32. 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 selected from the group consisting 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; and wherein the second portion comprises a polyethylene glycol.

33. A formulation comprising:
an isolated polypeptide selected from the group consisting 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; and a pharmaceutically acceptable vehicle.

34. A kit comprising the formulation of claim 33.