HK1133015A - Cytokine protein family - Google Patents

Description

Cytokine protein family

Background

Cell differentiation of multicellular organisms is controlled by hormones and polypeptide growth factors. These diffusible molecules enable cells to communicate with each other and act in concert with each other to form tissues and organs, as well as repair and regenerate damaged tissues. Examples of hormones and growth factors include, inter alia, steroid hormones, parathyroid hormone, follicle stimulating hormone, interferons, interleukins, platelet-derived growth factors, epidermal growth factors, and granulocyte-macrophage colony stimulating factor.

Hormones and growth factors affect cellular metabolism by binding to receptor proteins. Certain receptors are integral membrane proteins that bind to hormones or growth factors extracellularly and are linked to signal transduction pathways, such as second messenger systems, intracellularly. Other types of receptors are soluble intracellular molecules.

Cytokines generally stimulate the proliferation or differentiation of cells of the hematopoietic lineage or participate in the immune and inflammatory response mechanisms of the body. Examples of cytokines that influence hematopoiesis are Erythropoietin (EPO), which stimulates the development of erythrocytes; thrombopoietin (TPO) that stimulates cell development of the megakaryocytic lineage; and granulocyte colony stimulating factor (G-CSF) which stimulates the development of neutrophils. These cytokines are useful for restoring normal blood cell levels in patients with anemia, thrombocytopenia, and neutropenia or in patients undergoing chemotherapy for cancer,

cytokines play important roles in regulating hematopoiesis and the immune response, and can influence the development of lymphocytes. The human class II cytokine family includes the interferon-alpha (IFN-. alpha.) subtypes, interferon-beta (IFN-. beta.), interferon-gamma (IFN-. gamma.), IL-10, IL-19 (U.S. Pat. No. 5,985,614), MDA-7(Jiang et al, Oncogene 11, 2477-type 2486, (1995)), IL-20(Jiang et al, Oncogene 11, 2477-type 2486, (1995)), IL-22(Xie et al, J.biol.chem.275, 31335-type 31339, (2000)) and AK-155(Knappe et al, J.Virol.74, 3881-type 3887 (2000)). Most cytokines transduce signals by binding to class I or class II cytokine receptors. Members of the human class II cytokine receptor family include interferon- α R1(IFN- α R1), interferon- γ -R2(IFN- γ -R2), interferon- γ R1(IFN- γ R1), interferon- γ R2(IFN- γ R2), IL-10R (Liu et al, J.Immunol.152, 1821-1829, (1994)), CRF2-4 (Lutfalana et al, Genomics16, 366-373, (1993)), IL-20R β (Blumberg et al, Cell, 104, 9-19, (2001)) (also known as ztor Cell 7 (US 5,945,511) and CRF2-8(Kotenko et al, Oncogene 19, 2557-2565, (200)), IL-20R β (Blumberg et al, supra, (DIR. RTM.) (DIR S.45 (WO 85S 25-25 (PCT-8222, PCT-22-g et al), j.biol.chem.275, 31335-31339, (2000)), zcytorl (U.S. Pat. No. 5,965,704) and CRF2-9(Kotenko et al, Oncogene, 19, 2557-2565, (2000)), and tissue factor.

Class II cytokine receptors are typically heterodimers, consisting of two distinct receptor chains, the alpha and beta receptor subunits (Stahl et al, Cell, 74, 587-590, (1993)). In general, the alpha subunit is the major cytokine binding protein, while the beta subunit is essential for the formation of high affinity binding sites and for signal transduction. The IL-20 receptor is an exception, in which both subunits are required for IL-20 binding (Blumberg et al, supra (2001)).

Class II cytokine receptors can be identified by a conserved cytokine binding domain of approximately 200 amino acids (D200) in the extracellular portion of the receptor. The cytokine binding domain consists of two fibronectin type III (FnIII) domains (approximately 100 amino acids each) (BazanJ.F., Proc. Natl.Acad.Sci.USA 87, 6934-6938 (1990); Thoreau et al, FEBSLett.282, 16-31 (1991)). Each FnIII domain contains conserved Cys, Pro and Trp residues that determine the characteristic folding pattern of the 7 β chains, similar to the constant region of an immunoglobulin (Uze et al, j. interferon Cytokin res.15, 3-26 (1995)). Conserved structural elements of the class II cytokine receptor family allow the identification of new members of this family based on primary amino acid sequence homology. Using this approach, we have previously successfully identified two new members of the class II cytokine receptor family, zcytor7 (U.S. Pat. No. 5,945,511) (also known as IL-22 Ra (Blumberg et al, supra (2001)) and zcytor1l (U.S. Pat. No. 5,965,704) (also known as IL-22R (Blumberg et al, supra (2001)) because of the important role of cytokines in the regulation of biological responses, it is of interest to identify other new members of the class II cytokine receptor family.

IL-22, also known as IL-TIF (inducible factor derived from IL-10-associated T cells) (Dumoutier et al, J.immunology 164, 1814-one 1819, (2000)), is a newly described IL-10 homolog. IL-22 in mice was originally identified as an IL-9-induced gene in T cells and mast cells in vitro (Dumoutier et al, J.immunology, 164, 1814-1819, (2000)). Acute phase reactant-inducing activity was observed in mouse livers after IL-22 injection and IL-22 expression was rapidly induced after Lipopolysaccharide (LPS) injection, suggesting that IL-22 is involved in inflammatory responses in vivo (Dumoutier et al, Proc. Natl. Acad. Sci. USA, 97, 10144-.

Interleukins are a family of cytokines that mediate immunological responses, including inflammation. Interleukins mediate a variety of inflammatory pathologies. T cells are the central part of the immune response, which produces many cytokines and acquired immunity against antigens. Cytokines produced by T cells are classified into type 1 and type 2 (Kelso, A., Immun. cell biol. 76: 300-317, 1998). Type 1 cytokines include IL-2, IFN- γ, LT- α, which are involved in inflammatory responses, viral immunity, intracellular parasite immunity, and allograft rejection. Type 2 cytokines include IL-4, IL-5, IL-10 and IL-13, which are involved in humoral responses, helminth immunity and allergic reactions. Cytokines common to both types 1 and 2 include IL-3, GM-CSF, and TNF- α. There is some evidence that T cell populations that give rise to types 1 and 2 prefer to migrate into different types of inflamed tissue.

Interferons are of particular interest from a therapeutic point of view (for reviews on interferons see DeMaeyer and De Maeyer-Guignard, "interferons"; the Handbook of cytokines (the cytokine Handbook), 3 rd edition, Thompson (eds.), pages 491-. Interferons exhibit a variety of biological activities and are useful in the treatment of certain autoimmune diseases, particularly cancer, and in enhancing immune responses to infectious agents, including viruses, bacteria, fungi, and protists. To date, 6 forms of interferon have been identified, which fall into two large groups. The so-called "type I" interferons include interferon alpha, interferon beta, interferon omega, interferon delta, interferon tau. Currently, a subset of interferons gamma and alpha are the only type II interferons.

Type I interferons are thought to be derived from the same ancestral gene, retaining sufficiently similar structure to function through the same cell surface receptor. The alpha chain of the human interferon alpha/beta receptor comprises an N-terminal extracellular domain, which has the characteristics of a class II cytokine receptor. Interferon gamma has no significant homology to type I interferons or type II interferon alpha subtypes, but has many of the same biological activities as type I interferons.

For humans, at least 16 non-alleles encode different subtypes of interferon alpha, whereas interferons beta and omega are encoded by only a single gene. The type I interferon genes are clustered in the short arm of chromosome 9. Unlike the typical structural genes of humans, interferon alpha, interferon beta and interferon omega lack introns. A single gene encoding human interferon gamma is located on chromosome 12 and contains 3 introns. To date, interferon tau has only been described in cattle and sheep, whereas interferon delta has only been described in pigs.

Clinicians use this protein to treat a variety of diseases with the help of a variety of activities of interferon. For example, one form of interferon alpha has been approved by over 50 countries for the treatment of medical conditions such as hairy cell leukemia, renal cell carcinoma, basal cell carcinoma, malignant melanoma, AIDS-related kaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia, non-hodgkin's lymphoma, laryngeal papilloma, mycosis fungoides, condyloma acuminatum, chronic hepatitis b, hepatitis c, chronic hepatitis d, chronic non-a, non-b/c hepatitis. The U.S. drug and food administration has approved the use of interferon beta for the treatment of multiple sclerosis, a chronic disease of the nervous system. Interferon gamma is used to treat chronic granulomatous disease, where it enhances the immune response of the patient to destroy infectious bacterial, fungal and protozoan pathogens. Clinical studies have also indicated that interferon gamma may be used in the treatment of AIDS, leishmaniasis and leprosy.

The demonstrated in vivo activity of the cytokine family suggests great clinical potential and demand for other cytokines, cytokine agonists and cytokine antagonists. The present invention fills these needs by providing novel cytokines and related compositions and methods that stimulate cells of the hematopoietic cell lineage.

Detailed Description

Before setting forth the invention in detail, the following terms are defined to possibly aid in understanding the invention.

The term "affinity tag" is used herein to refer to a polypeptide fragment that can bind to a second polypeptide to facilitate purification or detection of the second polypeptide or to provide a site for binding of the second polypeptide to a substrate. In principle, 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 polyhistidine stretches, protein A (Nilsson et al, EMBO J.4: 1075, 1985; Nilsson et al, Methods enzymol.198: 3, 1991), glutathione S-transferase (Smith and Johnson et al, Gene 67: 31, 1988), Glu-Glu affinity tag (Grussenmeyer et al, Proc. Natl.Acad.Sci.USA 82: 7952-4, 1985), substance P, Flag^TMPeptides (Hopp et al, Biotechnology 6; 1204-10, 1988), streptavidin binding peptides, or other antigenic epitopes or binding domains. See generally Ford et al, Protein Expression and Purification 2: 95-107, 1991. DNA encoding an affinity tag is available from a supplier (e.g., Pharmacia Biotech, Piscataway, NJ).

The term "allelic variant" is used herein to refer to any of two or more different gene forms that occupy the same chromosomal locus. Allelic variation can arise naturally through mutation and can lead to phenotypic polymorphism within a population. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides with altered amino acid sequences. The term allelic variant is also used herein to refer to the protein encoded by the allelic variant.

The terms "amino terminus" and "carboxy terminus" are used herein to refer to a position within a polypeptide. Where the context permits, reference to a particular sequence or portion of a polypeptide is used to refer to adjacent or relative positions. For example, a sequence that is located at the carboxy-terminus of a reference sequence in a polypeptide refers to a position of the sequence that is proximal to the carboxy-terminus of the reference sequence, but not necessarily at the carboxy-terminus of the entire polypeptide.

The term "complement/anti-complement pair" refers to moieties that are not identical and, under the appropriate conditions, will form a stable pair that is linked by non-covalent means. For example, biotin and avidin (or streptavidin) are typical members of the complement/anti-complement pair. Other examples of complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. When it is desired to subsequently dissociate the complement/anti-complement pair, it is preferred that the complement/anti-complement pair have a size of less than 10 ⁹M^-1Binding affinity of (4).

The term "complementary molecule of a polynucleotide molecule" refers to a polynucleotide molecule having a complementary base sequence and an opposite orientation as compared to a reference sequence. For example, the sequence 5 'ATGCACGGG 3' is complementary to 5 'CCCGTGCAT 3'.

The term "degenerate nucleotide sequence" refers to a nucleotide sequence that includes one or more degenerate codons (as compared to a reference polynucleotide molecule encoding a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (e.g., GAU and GAC triplets both encode Asp).

The term "expression vector" is used to refer to a linear or circular DNA molecule comprising a segment encoding a polypeptide of interest operably linked in the DNA molecule to other segments that facilitate its transcription. These other segments include promoter and termination sequences, and may also include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

The term "isolated" when applied to a polynucleotide means that the polynucleotide has left its natural genetic environment and is therefore free of other unrelated or unwanted coding sequences, and that it is present in a form suitable for use in a genetically engineered protein production system. The isolated molecules are those molecules that are isolated from their natural environment, including cDNA and genomic clones. The isolated DNA molecules of the present invention do not contain other genes to which they are originally linked, but may include naturally occurring 5 'and 3' untranslated regions, such as promoters and terminators. The identification of the regions of attachment will be apparent to one of ordinary skill in the art (see, e.g., Dynan and Tijan, Nature 316: 774-78, 1985).

An "isolated" polypeptide or protein is one that is present under conditions other than its native environment, e.g., away from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. Preferably, the polypeptide is provided in a highly purified form, i.e., greater than 95% pure, more preferably greater than 99% pure. In this context, the term "isolated" does not exclude the presence of other possible physical forms (such as dimers) or other possible glycosylated or derivatized forms of the same polypeptide.

The term "neoplasm", when referring to a cell, means that the cell is undergoing a new abnormal proliferation, particularly in tissues where proliferation is uncontrolled and progressive, resulting in the production of a neoplasm. Neoplastic cells can be malignant, e.g., invasive and metastatic, or benign.

The term "operably linked" when referring to a segment of DNA indicates that the segments are arranged in a manner such that they act in concert for their intended purposes, e.g., to initiate transcription in a promoter and proceed through the coding segment to a terminator.

The term "ortholog" (ortholog) refers to a polypeptide or protein obtained from one species that is a functional counterpart of a polypeptide or protein from a different species. Sequence differences between orthologs are the result of speciation.

"paralogs" (paralogs) are distinct but structurally related proteins produced by an organism. Paralogs are thought to originate from gene repeats. For example, alphaglobin, betaglobin, and myoglobin are paralogs of each other.

"Polynucleotide" refers to 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 in vitro, or prepared by a combination of natural and synthetic molecules. The size of a polynucleotide is expressed as base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). The latter two terms may describe a polynucleotide that is either single-stranded or double-stranded, where the context permits. When the term is applied to double-stranded molecules, it is used to refer to the entire length and should be understood as equivalent to the term "base pair". It is understood by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that their ends may be staggered by enzymatic cleavage; thus, it is possible that not all nucleotides in a double-stranded polynucleotide molecule are paired.

"Polypeptides" refers to polymers of amino acid residues joined by peptide bonds, which may be naturally occurring or synthetically produced. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides".

The term "promoter" is used herein to denote its art-recognized meaning and refers to the portion of a gene that contains a DNA sequence that provides conditions for RNA polymerase binding and transcription initiation. Promoter sequences are typically, but not always, present in the 5' non-coding region of a gene.

"protein" refers to a macromolecule comprising one or more polypeptide chains. The protein may also comprise non-peptide components, such as sugar groups. Sugars and other non-peptide substituents may be added to the protein by the cell that produces the protein, and they will vary from cell type to cell type. Proteins are defined herein in terms of their amino acid backbone structure; substituents such as glycosyl groups and the like are generally not specified, but may be present.

The term "receptor" refers to a cell-associated protein that binds to a biologically active molecule (i.e., a ligand) and mediates the action of the ligand on the cell. Membrane-bound receptors are characterized by a structure of multiple peptides that contain an extracellular ligand-binding domain and an intracellular effector domain (typically involved in signal transduction). Binding of the ligand to the receptor results in a conformational change in the receptor, causing interactions between the effector domain and other molecules within the cell. This interaction in turn leads to alterations in cellular metabolism. Metabolic events linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increased production of cyclic AMP, cellular calcium mobilization, membrane lipid mobilization, cell adhesion, hydrolysis of inositol lipids, and hydrolysis of phospholipids. Receptors may be membrane-bound, cytoplasmic or nuclear in general; monomeric (e.g., thyroid stimulating hormone receptor, beta adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor, and IL-6 receptor).

The term "secretory signal sequence" refers to a DNA sequence that encodes a polypeptide ("secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through the secretory pathway of a cell in which it is synthesized. The larger polypeptide is typically cleaved during transport through the secretory pathway to remove the secretory peptide.

The term "splice variant" is used herein to refer to a variety of alternative forms of RNA produced by transcription of a gene. Splice variants arise naturally by using multiple alternative splice sites present in the transcribed RNA molecule, or in less cases between separately transcribed RNA molecules, and can result in the transcription of several mrnas from the same gene. Splice variants can encode polypeptides having altered amino acid sequences. The term splice variant is also used herein to refer to the protein encoded by the splice variant of mRNA produced by transcription of the gene.

It is understood that the molecular weights and lengths of the polymers determined by crude analytical methods such as gel electrophoresis are approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X is understood to be accurate to. + -. 10%.

All references cited herein are incorporated by reference in their entirety.

The present invention includes a class of polynucleotide and polypeptide molecules having functional and structural similarities to interferon. This new family includes molecules designated zcyto20(SEQ ID NOS: 1 and 2), zcyto21(SEQ ID NOS: 4 and 5), zcyto22(SEQ ID NOS: 6 and 7), zcyto24(SEQ ID NOS: 8 and 9), zcyto25(SEQ ID NOS: 10 and 11), where zcyto20, 21 and 22 are human sequences and zcyto24 and 25 are mouse sequences. Table 1 shows the homology at the nucleotide and amino acid levels within this family, which is about 72% to 98% at the nucleotide level and about 51% to 97% at the amino acid level.

TABLE 1

Nucleosidase sequence identity

Table 2 shows the sequence identity of zcyto20, zcyto21, zcyto22, IFN α, IFN β, IFN γ and IL-10 at the amino acid level.

TABLE 2

Amino acid sequence identity

All members of this family were shown to bind to the same class II cytokine receptor, designated zcytor19 receptor. Furthermore, it has been demonstrated that all members of the family exhibit certain biological activities. These activities include, for example, antiviral activity and increasing the level of circulating myeloid cells. While not wishing to be bound by theory, these molecules appear to all signal along the same pathway through the zcytor19 receptor.

The zcyto20 gene encodes a 205 amino acid polypeptide shown in SEQ ID NO: 2 in (c). The signal sequence of Zcyto20 can be predicted to comprise SEQ ID NO: 2 from amino acid residue 1 (Met) to amino acid residue 21 (Ala). The mature peptide of Zcyto20 begins at amino acid residue 22 (Val).

The Zcyto21 gene encodes a 200 amino acid polypeptide shown in SEQ ID NO: 5 in (c). The signal sequence of Zcyto21 can be predicted to comprise SEQ ID NO: 5 amino acid residue 1 (Met) to amino acid residue 19 (Ala). The mature peptide of Zcyto21 begins at amino acid residue 20 (Gly). Zcyto21 is described in PCT application WO 02/02627.

The Zcyto22 gene encodes a 205 amino acid polypeptide shown in SEQ ID NO: 7 (c). The signal sequence of Zcyto22 can be predicted to comprise SEQ ID NO: 7 amino acid residue 1 (Met) to amino acid residue 21 (Ala). The mature peptide of Zcyto22 begins at amino acid residue 22 (Val).

The Zcyto24 gene encodes a 202 amino acid polypeptide shown in SEQ ID NO: 9 (c). The secretion signal sequence of Zcyto24 comprises SEQ ID NO: 9 amino acid residue 1 (Met) to amino acid residue 28 (Ala). Another alternative cleavage site for the secretion signal sequence can be found at amino acid residue 24 (Thr). The mature polypeptide comprises amino acid residues 29 (Asp) to 202 (Val).

The Zcyto25 gene encodes a 202 amino acid polypeptide shown in SEQ ID NO: 11 in (b). The secretion signal sequence of Zcyto25 comprises SEQ ID NO: 11 from amino acid residue 1 (Met) to amino acid residue 28 (Ala). Another alternative cleavage site for the secretion signal sequence can be found at amino acid residue 24 (Thr). The mature polypeptide comprises amino acid residues 29 (Asp) to 202 (Val).

The Zcyto20, Zcyto21, and Zcyto22 genes have been mapped to human chromosome 19q 13.13. Based on the findings of these genes, the region of chromosome 19 was identified as containing a cluster of interferon-like genes. Another evidence that this is a novel gene family is the identification of a cluster of homologous genes on mouse chromosome 7, zcyto24(SEQ ID NO: 8) and zcyto25(SEQ ID NO: 10).

As follows, the present invention provides a polypeptide having an amino acid sequence substantially similar to SEQ ID NO: 2 or amino acid residues 22 to 205 of SEQ ID NO: 2, at least 70%, at least 80%, or at least 90%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1 to 205, or some fragment thereof. The invention also includes a polypeptide further comprising a signal secretion sequence located at an amino acid position of the first amino acid sequence, wherein the signal secretion sequence comprises SEQ ID NO: 2 amino acid sequence 1 to21 amino acid residues.

In another embodiment, the present invention provides a polypeptide having an amino acid sequence substantially identical to SEQ ID NO: 7, amino acid residues 22 to 205 or SEQ ID NO: 7, amino acid residues 1 to 205, at least 70%, at least 80%, or at least 90%, 95%, 96%, 97%, 98%, or 99%. The invention also includes a polypeptide further comprising a signal secretion sequence located at an amino acid position of the first amino acid sequence, wherein the signal secretion sequence comprises seq id NO: 7 amino acid residues 1 to21 of the amino acid sequence.

In general, cytokines such as Erythropoietin (EPO) are predicted to have a 4 alpha-helical structure (of which helices A, C and D are most important in the interaction of ligand and receptor) and are more highly conserved among members of this family. However, Interferons (IFNs), particularly interferon alpha and interferon tau, are characterized by 6 helical bundles. EPO helix a corresponds to helix a of zcyto 20; EPO helix B corresponds to helix C of zcyto 20; EPO helix C corresponds to helix D of zcyto 20; EPO helix D corresponds to helix F of zcyto 20. Thus, the loop between the AB loop and the CD loop of EPO is expanded in zcyto20 to contain the short helices B and E of zcyto 20. The helix structures of zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 are similar to the 6-helix structures found in interferons. Boundaries of secondary structures in proteins are generally determined from 3-dimensional models of proteins according to a series of PHI and PSI angles of the protein backbone. The model can be constructed from, for example, x-ray crystallography or NMR data, or homology modeling based on the structure already elucidated. The boundaries of these secondary structures may vary slightly depending on the technique used, including the conditions used for crystal formation and for flexible NMR solution structure determination. Thus, it will be apparent to those skilled in the art that the helical boundaries and in general secondary structure may be shifted by as many as 2, 3, 4 or more residues depending on the circumstances, but the helical regions are substantially as described below. (see Brandon and toaste, Introduction to Protein Structure, Garland Publishing Co, inc. New York, 1991; Anderson et al, Structure, 10 (2): 175-84, 2002.)

The predicted helices of Zcyto20 are as follows: helix a is defined by amino acid residues 52(Ala) to 66 (Leu); helix B is amino acid residues 78(Arg) to 87 (Val); helix C is amino acid residues 91(Pro) to 108 (Thr); helix D is amino acid residues 116(Val) to 138 (Ser); helix E is the amino acid residue from position 151(Thr) to 172 (Lys); and helix F is amino acid residue number 177(Gly) to 197 (Cys); see SEQ ID NO: 2. the 4 cysteine residues are conserved in Zcyto20, Zcyto21 and IFN- α. In addition, zcyto20 also has 3 other cysteines. The cysteine at amino acid residue position 204 may form an intermolecular disulfide bond, particularly as a homodimer with other zcyto20 molecules. Further analysis of zcyto20 based on multiple alignments predicts that amino acid residues 37 and 136; positions 69 and 197; and cysteines at positions 71 and 178 (shown in SEQ ID NO: 2) will form intramolecular disulfide bonds. The corresponding polynucleotides encoding the Zcyto20 polypeptide regions, domains, motifs, residues and sequences described herein are shown in SEQ ID NO: 1 in (c).

The predicted helices of zcyto21 are as follows: helix a is defined by amino acid residues 49(Ser) to 63 (Leu); helix B is amino acid residues 76(Asn) to 84 (Val); helix C is amino acid residues 89(Val) to 104 (Ala); helix D is amino acid residues 111(Glu) to 133 (Gln); helix E is amino acid residue 137(Thr) to 158 (Lys); and helix F is amino acid residue 163(Gly) to 189 (Leu); as shown in SEQ ID NO: 5, respectively. These cysteine residues are conserved in zcyto21, zcyto21 and IFN- α and may form intermolecular disulfide bonds, particularly homodimers with other zcyto21 molecules. Further analysis of zcyto21 based on multiple alignments predicts that cysteines at amino acid residues 34 and 131, and 68 and 164 will form intramolecular disulfide bonds. Cysteine 190 is free and may form intermolecular disulfide bonds. The corresponding polynucleotides encoding the regions, domains, motifs, residues and sequences of the zcyto21 polypeptide described herein are shown in SEQ ID NO: 4 in (b).

The predicted helices of zcyto22 are as follows: helix a is defined by amino acid residues 52(Ala) to 66 (Leu); helix B is amino acid residues 78(Arg) to 87 (Val); helix C is amino acid residues 91(Pro) to 108 (Thr); helix D is amino acid residues 116(Val) to 138 (Ser); helix E is the amino acid residue from position 151(Thr) to 172 (Lys); and helix F is amino acid residue number 177(Gly) to 197 (Cys); as shown in SEQ ID NO: shown at 7. The 4 cysteine residues are conserved in zcyto22, zcyto21 and IFN- α. In addition, zcyto22 also has 3 other cysteines. The cysteine at amino acid residue position 204 may form an intermolecular disulfide bond, particularly as a homodimer with other zcyto22 molecules. Further analysis of zcyto22 based on multiple alignments predicts that amino acid residues 37 and 136; positions 69 and 197; and cysteines at positions 71 and 178 (shown in SEQ ID NO: 7) will form intramolecular disulfide bonds. The corresponding polynucleotides encoding the regions, domains, motifs, residues and sequences of the zcyto22 polypeptide described herein are shown in seq id NO: 6 in (A).

The conserved cysteine of Zcyto24 was confirmed in SEQ ID NO: residues 44, 78, 141 and 175 of 9. Further analysis of zcyto24 based on multiple alignments predicts that amino acid residues 44 and 141; disulfide bonds will form between positions 78 and 175 (shown in SEQ ID NO: 9). The corresponding polynucleotides encoding the regions, domains, motifs, residues and sequences of the zcyto24 polypeptide described herein are shown in SEQ ID NO: 9 (c). The predicted helix in Zcyto24 (shown in SEQ ID NO: 9) is: residues 59-73 (helix A); residues 85-94 (helix B); residues 98-115 (residue C); residue 121-143 (helix D); residue 147-169 (helix E); residue 174-194 (helix F).

The conserved cysteine of Zcyto25 was confirmed in SEQ ID NO: 11 at residues 44, 78, 141 and 175. Further analysis of zcyto25 based on multiple alignments predicts that amino acid residues 44 and 141; disulfide bonds will form between positions 78 and 175 (shown in SEQ ID NO: 11). The corresponding polynucleotides encoding the regions, domains, motifs, residues and sequences of the zcyto25 polypeptide described herein are shown in SEQ ID NO: 11 in (b). The predicted helix in Zcyto25 (shown in SEQ ID NO: 11) is: residues 59-73 (helix A); residues 85-94 (helix B); residues 98-115 (residue C); residue 121-143 (helix D); residue 147-169 (helix E); residue 174-194 (helix F).

Detailed mutational analysis of murine IL-2 (Zurawski et al, EMBO J. 12: 5113-5119, 1993) showed that residues in helices A and C are important for binding to IL-2R β; the critical residue being Asp₃₄、Asn₉₉And Asn₁₀₃. Multiple residues in the A/B loop and helix B of murine IL-2 are important for binding of IL-2R α, while only one residue in helix D, Gln₁₄₁Is crucial for binding to IL-2R α. Similarly, helices A and C are sites of interaction between IL-4 and IL-4R α (structurally similar to IL-2R α), and residues in helix D are critical for interaction with IL-2R α. (Wang et al, Proc. Natl. Acad. Sci. USA94: 1657-1662, 1997; Kruse et al, EMBO J.11: 3237-3244, 1992). In particular, Tyr in human IL-4 ₁₂₄Mutation to Asp results in an antagonist which binds to IL-4R α but not to IL-2R α and therefore does not produce a signal (Kruse et al, supra, 1992).

Cytokines of the 4-helix bundle are also grouped by the length of their constituent helices. Cytokines in the form of "long helices" typically consist of a helix of 24-30 residues, including IL-6, ciliary neurotrophic factor (CNTF), Leukemia Inhibitory Factor (LIF), and human growth hormone (hGH). Cytokines in the form of "short helices" typically consist of a helix of 18-21 residues, including IL-2, IL-4, and GM-CSF. Studies using CNTF and IL-6 demonstrated that the CNTF helix can be replaced by an equivalent helix in IL-6, thus giving the chimera CTNF binding properties. Thus, it appears that the functional domains of 4-helix cytokines can be determined based on structural homology, regardless of sequence identity, and that these domains are capable of maintaining functional integrity in chimeras (Kallen et al, J.biol.chem.274: 11859-11867, 1999). Thus, the helical domains of zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 are useful for making chimeric fusion molecules (especially with other interferons) to determine and modulate the binding specificity of the receptor. Of particular interest are fusion proteins that combine the helical and loop regions from interferons and cytokines such as IFN- α, IL-10, human growth hormone.

It has been demonstrated that zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 can form a complex with an orphan receptor known as zcytor 19. Zcytor19 is described in commonly assigned patent application PCT/US 01/44808. It has been demonstrated that Zcyto22, Zcyto21, and Zcyto24 also bind to or signal through zcytor19, which further supports Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 to be members of the same cytokine family. The Zcytor19 receptor is a class II cytokine receptor. Class II cytokine receptors typically bind to 4 helix bundle cytokines. For example, interleukin 10 and interferon bind to receptors in this class (e.g., the alpha and beta chains of the interferon gamma receptor, the alpha and beta chains of the interferon alpha/beta receptor).

Class II cytokine receptors are characterized by the presence of one or more Cytokine Receptor Modules (CRM) in their extracellular domain. Other class II cytokine receptors include zcytor11 (commonly owned U.S. Pat. No. 5,965,704), CRF2-4(Genbank accession No. Z17227), IL-10R (Genbank accession Nos. U00672 and NM-001558), DIRS1, zcytor7 (commonly owned U.S. Pat. No. 5,945,511), and tissue factor. Zcytor19 has only a single class II CRM in its extracellular domain, as do all known class II receptors except the alpha chain of the interferon alpha/beta receptor.

Analysis of a human cDNA clone encoding zcytor19 (SEQ ID NO: 26) showed an open reading frame encoding 520 amino acids (SEQ ID NO: 27) comprising a secretion signal sequence (SEQ ID NO: 27, residues 1 (Met) to 20 (Gly)) and a mature zcytor19 cytokine receptor polypeptide (SEQ ID NO: 27, residues 21 (Arg) to 520 (Arg)), wherein the extracellular ligand binding domain has approximately 206 amino acid residues (SEQ ID NO: 27, residues 21 (Arg) to 226 (Asn)), the transmembrane domain has approximately 23 amino acid residues (SEQ ID NO: 27, residues 227 (Trp) to 249 (Trp)), and the intracellular domain has approximately 271 amino acid residues (SEQ ID NO: 27, residues 250 (Lys) to 520 (Arg)). Within the extracellular ligand binding domain, there are two fibronectin type III domains and a linker region. The first fibronectin type III domain comprises seq id no: 27 (Arg) to 119 (Tyr), and a linker comprising SEQ ID NO: 27 (Leu) 120 to Glu 124, and a second fibronectin type III domain comprising SEQ ID NO: 27 from residue 125 (Pro) to residue 223 (Pro). Thus, a polypeptide comprising seq id NO: the polypeptides of amino acids 21 (Arg) to 223 (Pro) of 27 are considered ligand-binding fragments. Furthermore, as is generally conserved among class II receptors, there are conserved tryptophan residues (including residue 43 (Trp) and residue 68 (Trp) as shown in SEQ ID NO: 27) and the amino acid residues located in SEQ ID NO: 27 at positions 74, 82, 195, 217.

In addition, a human cDNA clone encoding a zcytor19 variant in which 30 amino acid residues were deleted was also identified. The variant zcytor19 (shown in SEQ ID NO: 23) comprises an open reading frame encoding 491 amino acids (SEQ ID NO: 24) comprising a secretion signal sequence (SEQ ID NO: 24, residues 1 (Met) to 20 (Gly)) and a mature zcytor19 cytokine receptor polypeptide (SEQ ID NO: 24, residues 21 (Arg) to 491 (Arg)), wherein the extracellular ligand binding domain comprises approximately 206 amino acid residues (residues 21 (Arg) to 226 (Asn) of SEQ ID NO: 24), the transmembrane domain comprises approximately 23 amino acid residues (residues 227 (Trp) to 249 (Trp) of SEQ ID NO: 24), and the intracellular domain comprises approximately 242 amino acid residues (residues 250 (Lys) to 491 (Arg) of SEQ ID NO: 24). Within the extracellular ligand binding domain, there are two fibronectin type III domains and a linker region. The first fibronectin type III domain comprises SEQ id no: 24 (Arg) 21 to 119 (Tyr), and a linker comprising SEQ ID NO: 24 (Leu) 120 to Glu 124, and a second fibronectin type III domain, shorter, comprising SEQ ID NO: residues 125 (Pro) to 223 (Pro) of 24. Thus, a polypeptide comprising SEQ ID NO: the polypeptide of amino acids 21 (Arg) to 223 (Pro) of 24 is considered to be a ligand-binding fragment. Furthermore, as is generally conserved in class II receptors, there are conserved tryptophan residues (including residue 43 (Trp) and residue 68 (Trp) as shown in SEQ ID NO: 24) and the amino acid residues located in SEQ ID NO: 24, conserved cysteine residues at positions 74, 82, 195, 217.

It appears that mRNA which produces a truncated soluble form of the Zcytor19 receptor may be naturally expressed. Analysis of the human cDNA clone (SEQ ID NO: 28) encoding the truncated soluble zcytor19 revealed an open reading frame encoding 211 amino acids (SEQ ID NO: 29) comprising a secretory signal sequence (SEQ ID NO: 29, residues 1 (Met) to 20 (Gly)) and a mature truncated soluble zcytor19 receptor polypeptide (SEQ ID NO: 29, residues 21 (Arg) to 211 (Ser)), wherein the truncated extracellular ligand-binding domain is approximately 143 amino acid residues (residues 21 (Arg) to 163 (Trp) of SEQ ID NO: 29), has NO transmembrane domain, but has an extra domain of approximately 48 amino acid residues (residues 164 (Lys) to 211 (Ser) of SEQ ID NO: 29). Within the truncated extracellular ligand binding domain, there are two fibronectin type III domains and a linker region. The first fibronectin type III domain comprises SEQ ID NO: 29 (Arg) 21 to 119 (Tyr), and a linker comprising SEQ ID NO: 29 (Leu) 120 to Glu 124, and a second fibronectin type III domain comprising SEQ ID NO: residues 125 (Pro) to 163 (Trp) of 29. Thus, a polypeptide comprising SEQ ID NO: the polypeptide of amino acids 21 (Arg) to 163 (Trp) 29 is considered to be a ligand-binding fragment. Furthermore, as is generally conserved in class II receptors, there are conserved tryptophan residues (including residue 43 (Trp) and residue 68 (Trp) as shown in SEQ ID NO: 29), and the conserved cysteine residue in the truncated soluble form of the zcytor19 receptor is located in the amino acid sequence shown in SEQ ID NO: 29, bits 74 and 82.

The Zcytor19 receptor is a member of the same receptor subfamily as the class II cytokine receptor, which can bind to form homodimers of the transduction signal. Several members of the subfamily (e.g., receptors that bind interferon, IL-10, IL-19, and IL-TIF) combine with a second subunit (referred to as the β -subunit) to bind ligand and transduce signals. However, in many cases, a particular β -subunit binds to many particular cytokine receptor subunits. For example, class II cytokine receptors such as zcytor11 (U.S. Pat. No. 3, 5,965,704) and CRF2-4 receptors heterodimerize to bind the cytokine IL-TIF (see, WIPO publication WO 00/24758; Dumontier et al, J.Immunol.164: 1814. sup. 1819, 2000; Spencer, SD et al, J.exp.Med.187: 571. 578, 1998; Gibbs, VC and Pennica, Gene 186: 97-101, 1997(CRF2-4 cDNA); Xie, MH et al, J.biol. chem.275: 35. sup. 31339, 2000). The IL-10 β receptor is considered synonymous with CRF2-4 (Dumoutier, L. et al, Proc. Natl. Acad. Sci.97: 10144-18249, 2000; Liu Y et al, J. Immunol.152: 1821-1829, 1994(IL-10R cDNA). it is therefore expected that zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 will bind to the monomeric, homodimeric, heterodimeric and multimeric zcytor19 receptor. experimental evidence has identified CRF2-4(SEQ ID NOS: 40 and 41) as a putative binding partner for zcyto 19, again supporting the important role of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 in immune regulatory systems, affecting physiological functions such as the natural immune system and inflammatory response system.

The localization of receptor/ligand pair receptor expression may be important for the identification of the target cell or tissue for ligand action. This is particularly useful when the receptor/ligand complex involves a heterodimeric receptor and one subunit of the receptor is widely expressed while the other subunit is expressed in a localized manner (spatially or temporally limited). Expression of zcytor19 has been identified in skin cancer samples using in situ hybridization, where cancerous granular epithelium is strongly positive, while no positive signal is observed in normal skin. Other tissues expressing zcytor19 were identified including fetal liver, where signals were observed in a mixed population of sinusoid mononuclear cells; type II alveolar intraepithelial expression was observed in the lungs; and expression was observed in macrophage-like mononuclear cells of mesenchymal tissues. Northen analysis of Zcytor19 identified the expression of an approximately 4.5kb transcript, which is expressed in maximal amounts in cardiac, skeletal muscle, pancreatic and prostate tissues, as well as in burkitt's lymphoma (RAJI) cell line and SW-480 colorectal cancer cell line.

The present invention provides polynucleotide molecules, including DNA and RNA molecules, encoding the zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 polypeptides disclosed herein. It will be readily apparent to those skilled in the art that a considerable number of sequence variations are possible in these polynucleotide molecules in view of the degeneracy of the genetic code. SEQ ID NO: 3 is a degenerate DNA sequence comprising a nucleotide sequence encoding SEQ ID NO: 2, zcyto20 polypeptide. Those skilled in the art will appreciate that by substituting U for T, SEQ ID NO: 3 and encoding SEQ ID NO: 2. Thus, a polypeptide comprising SEQ ID NO: 3 from nucleotide 1 or 64 to nucleotide 615 encoding zcyto20 polypeptides and RNA equivalents thereof are contemplated by the present invention. Table 3 lists SEQ ID NOs: 3 to indicate degenerate nucleotide positions. "interpretations" are nucleotides represented by code letters. "complement" refers to the code of complementary nucleotides. For example, the code Y represents C or T, and its complement R represents A or G, where A is complementary to T and G is complementary to C.

SEQ ID NO: 46 is a degenerate DNA sequence comprising a nucleotide sequence encoding SEQ ID NO: 7, zcyto22 polypeptide. Those skilled in the art will appreciate that by substituting U for T, SEQ ID NO: 46 further provides a nucleic acid sequence encoding SEQ ID NO: 7. Thus, a polypeptide comprising SEQ ID NO: 46 and RNA equivalents thereof are contemplated by the present invention. Table 3 lists SEQ ID NOs: 46 to indicate degenerate nucleotide positions. "interpretation" is the nucleotides indicated by the code letters. "complement" refers to the code of complementary nucleotides. For example, the code Y represents C or T, and its complement R represents A or G, where A is complementary to T and G is complementary to C.

TABLE 3

SEQ ID NO: 3, all possible codons for a given amino acid are included.

TABLE 4

One skilled in the art will appreciate that some uncertainty is introduced in determining degenerate codons that represent all possible codons encoding each amino acid. For example, a degenerate codon for serine (WSN) may in some cases encode Arginine (AGR), while a degenerate codon for arginine (MGN) may in some cases encode serine (AGY). A similar relationship exists between codons encoding phenylalanine and leucine. Thus, certain polynucleotides encompassed by degenerate sequences may encode variant amino acid sequences, but one of skill in the art would recognize by reference to SEQ ID NO: 2 and SEQ ID NO: 7 can be readily identified. The functionality of variant sequences can be readily tested as described herein.

One skilled in the art will also appreciate that different species may exhibit "preferential codon usage". See generally 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 filiers, Gene 18: 199-209, 1982; holm, nuc. acids res.14: 3075-87, 1986; ikemura, j.mol.biol.158: 573-97, 1982. As used herein, the term "preferential codon usage" or "preferential codon" is a term of art that refers to the most frequently used codon for translation of a protein in a cell of a species, and thus biases one or more of the possible codons encoding various amino acids toward one or more of the representative codons (see Table 3). For example, threonine (Thr) can be encoded by ACA, ACC, ACG or ACT, but ACC is the most commonly used codon in mammalian cells; in other species, e.g., insect cells, yeast, viruses, or bacteria, a different Thr codon may be preferred. Preferential codons for a particular species can be introduced into the polynucleotides of the invention by a variety of methods known in the art. Introduction of a preferential codon sequence into recombinant DNA can enhance protein production, for example, by making translation of the protein more efficient in a particular cell type or species. Thus, SEQ ID NO: 3 and 46 can be used as templates to optimize the expression of the polynucleotides in different cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in different species and tested for functionality as disclosed herein.

As mentioned previously, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. Typically, RNA is isolated from tissues or cells that produce large amounts of zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 RNA. These tissues and cells can be identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77: 5201, 1980) or by screening conditioned media from different cell types for activity against the target cell or tissue. Once cells or tissues producing the activity or RNA are identified, total RNA can be prepared using guanidinium isothiocyanate extraction followed by centrifugation in CsCl gradients (Chirgwin et al, Biochemistry 18: 52-94, 1979). Poly (A) + RNA was prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69: 1408-12, 1972). Complementary DNA can be prepared from poly (A) + RNA using known methods. In another alternative, genomic DNA may be isolated. Polynucleotides encoding zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 polypeptides are then identified and isolated, for example, by hybridization or PCR.

Full-length clones encoding zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 can be obtained by conventional cloning methods. Complementary dna (cDNA) clones are preferred, but for some applications (e.g. expression in transgenic animals) it may be preferred to use genomic clones, or to modify cDNA clones to include at least one genomic intron. Methods for preparing cDNA and genomic clones are well known and are within the level of ordinary skill in the art and include the use of the sequences disclosed herein or portions thereof to screen libraries as probes or to amplify libraries as primers. Expression libraries can be probed using fragments of the zcytor19 receptor or other specific binding partners.

The invention also provides corresponding polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to, mammals, birds, amphibians, reptiles, fish, insects, and other vertebrate and invertebrate species. Of particular interest are zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine and other primate polypeptides. The information and compositions provided by the present invention, as well as conventional cloning techniques, can be used to clone the orthologs of human zcyto20, zcyto21, and zcyto 22. For example, cDNA may be cloned using mRNA obtained from tissues or cell types expressing zcyto20, zcyto21, and zcyto22 disclosed herein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed according to the sequences disclosed herein. Libraries are then prepared from the mRNA of positive tissues or cell lines. The cDNAs encoding zcyto20, zcyto21 and zcyto22 can then be isolated by a variety of methods, e.g., probing with whole or partial human cDNAs or with one or more sets of degenerate probes based on the disclosed sequences. The cDNA may also be cloned using polymerase chain reaction or PCR (Mullis, U.S. Pat. No. 4,683,202), using primers designed according to the representative human zcyto20 sequence disclosed herein. In another approach, a cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can then be detected using antibodies, binding studies, or activity assays for zcyto20 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.

Those skilled in the art will appreciate that SEQ ID NOS: 1. the sequences disclosed in 4 and 6 represent one allele of the human zcyto20, zcyto21, and zcyto22 bands, respectively, and allelic variation and variable splicing are expected. Allelic variants of the sequence can be cloned by probing cDNA or genomic libraries from different individuals using standard methods. SEQ ID NO: 1. 4 and 6, including those containing silent mutations and those in which the mutation results in an amino acid sequence change, are within the scope of the present invention as are the allelic variants of the DNA sequences shown in SEQ id nos: 2. proteins of allelic variants of 5 and 7 are also within the scope of the invention. cDNAs prepared from mRNAs produced by variable splicing that retain the properties of zcyto20, zcyto21, and zcyto22 polypeptides are included within the scope of the invention, as are the polypeptides encoded by these cDNAs and mRNAs. Allelic and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues using standard methods known in the art.

The invention also provides reagents useful in diagnostic applications. For example, probes comprising the zcyto20, zcyto21, and zcyto22 genes, including zcyto20, zcyto21, and zcyto22DNA or RNA or subsequences thereof can be used to determine whether the zcyto20, zcyto21, and zcyto22 genes are present on a human chromosome, such as chromosome 19, or whether a gene mutation has occurred. Zcyto20, Zcyto21 and Zcyto21 are located in the q13.13 region of chromosome 19. Detectable chromosomal aberrations at the zcyto20, zcyto21, and zcyto22 gene loci include, but are not limited to, aneuploidy, gene copy number changes, loss of heterogeneity (LOH), translocations, insertions, deletions, restriction site changes, and rearrangements. These aberrations can be detected using the polynucleotides of the present invention by using molecular genetic techniques such as Restriction Fragment Length Polymorphism (RFLP) analysis, Short Tandem Repeat (STR) analysis using PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al, supra; Ausubel et al, supra; Marian, Chest 108: 255-65, 1995).

Accurate information of gene location may be useful for many purposes, including: 1) determining whether the sequence is part of an existing contig and obtaining additional surrounding genetic sequences in a variety of formats such as YAC, BAC or cDNA clones; 2) providing a potential candidate gene for a genetic disease that exhibits linkage to the same chromosomal region; and 3) cross-reference to a model organism (e.g., a mouse), which can help determine the function a particular gene may have.

For example, Delague et al (am.J.Hum.Genet.67: 236-.

Diagnosis may assist physicians in determining the type of disease and appropriate associated treatments, or in genetic counseling. Likewise, using methods known in the art and described herein, the anti-zcyto 20 antibodies, polynucleotides, and polypeptides of the invention can be used to detect zcyto20 polypeptides, mRNA, or anti-zcyto 20 antibodies, as described herein, thereby acting as markers, and can be used directly to detect genetic disease or cancer. Furthermore, zcyto20, zcyto21, and zcyto22 polynucleotide probes can be used to detect abnormalities or genotypes associated with: human disease-related deletions and translocations in chromosome 19q13.13, or other translocations involved in malignant development of tumors, or other 19q13.13 mutations expected to be involved in chromosomal rearrangements in malignant tumors or other cancers. Similarly, zcyto20 polynucleotide probes can be used to detect abnormalities or genotypes associated with: trisomy 19q13.13 and chromosomal loss associated with human disease or spontaneous abortion. Thus, the zcyto20, zcyto21, and zcyto22 polynucleotide probes may be used to detect abnormalities or genotypes associated with these defects.

Generally, diagnostic methods for genetic linkage analysis to detect genetic aberrations or abnormalities in a patient are known in the art. Analytical probes are generally at least 20nt in length, although shorter probes (e.g., 14-17nt) may sometimes be used. The PCR primers are at least 5nt long, preferably 15nt or more, more preferably 20 to 30nt long. For global analysis of gene or chromosomal DNA, the zcyto20 polynucleotide probe may contain complete exons or more. One skilled in the art can readily determine exons by comparing the genomic DNA of zcyto20, zcyto21, and zcyto22 sequences (SEQ ID NOS: 1, 4, and 6, respectively) to zcyto20, zcyto21, and zcyto 22. Generally, diagnostic methods for genetic linkage analysis to detect genetic aberrations or abnormalities in a patient are known in the art. Most diagnostic methods include the steps of: (a) obtaining a genetic sample from a patient who may be diseased, a diseased patient, or a potentially non-diseased carrier of a recessive disease allele; (b) generating a first reaction product by incubating the genetic sample with a zcyto20 polynucleotide probe (wherein the polynucleotide hybridizes to a complementary polynucleotide sequence) in, for example, an RFLP assay or by incubating the genetic sample with sense and antisense primers in a PCR reaction under appropriate PCR reaction conditions; (c) visualizing the first reaction product by gel electrophoresis and/or other known methods, e.g., using a zcyto20 polynucleotide probe (wherein the polynucleotide will hybridize to a complementary polynucleotide sequence in the first reactant); and (d) comparing the visualized first reaction product with a second control reaction product from a genetic sample from a wild-type patient. A difference between the first reaction product and the control reaction product will indicate a genetic abnormality in the diseased or potentially diseased patient, or the presence of a heterozygous recessive carrier phenotype in a non-diseased patient, or the presence of a genetic defect in a tumor of a patient, or the presence of a genetic abnormality in a fetus or pre-implantation embryo. For example, a difference in restriction fragment pattern, PCR product length, repeat length of the zcyto20 genetic locus would indicate a genetic abnormality, genetic aberration, or allelic difference compared to a normal wild-type control. Controls may be from unaffected family members or unrelated individuals depending on the test being performed and the availability of the sample. Genetic samples for use in the present invention include genomic DNA, mRNA and cDNA isolated from any tissue or other biological sample of a patient (e.g., without limitation, blood, saliva, semen, embryonic cells, amniotic fluid, etc.). The polynucleotide probe or primer may be RNA or DNA and will comprise the sequence of seq id no: 1, SEQ ID NO: 1, or an RNA equivalent thereof. These methods for performing genetic linkage analysis of human disease phenotypes are well known in the art. For PCR-based diagnostic methods, see generally Mathew (eds.), Protocols in Human molecular genetics (Humana Press, Inc.1991), White (eds.), PCR Protocols: current Methods and Applications (Humana Press, Inc.1993), Cotter (eds.), Molecular diagnostics of Cancer (Humana Press, Inc.1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols (Humana Press Inc.1998), Lo (eds.), Clinical Applications of PCR (Humana Press, Inc.1998) and Meltzer (eds.), PCR in Bioanalysis (Humana Press, Inc. 1998)).

The nucleic acid molecules of the invention can be used for detection of single-stranded conformational polymorphisms (PCR-based short tandem repeat analysis), amplification refractory mutation system analysis (amplification-mutation system analysis), single-stranded conformational polymorphism detection, RNase cleavage method, denaturing gradient gel electrophoresis, mismatch analysis by fluorescence and other Genetic analysis techniques known in the art (see, e.g., Mathew (eds.), Protocols in Human Molecular Genetics (Human Press, Inc.1991), Marian, chess 108: 255 (Ts1995); Coleman and on crystals, Molecular diagnostics (Humana Press, Inc. 1996); Elles (ES) (Molecular diagnostics of Genetic Diseases (Humana Press, Inc. 1996); latex (Laboratory) Laboratory coding of Molecular diagnostics of Genetic Diseases (Human research, Inc. 1996); Molecular analysis of Molecular analysis (Bio of Molecular analysis, Bio of Molecular analysis (Human Press, Inc. 1996); nucleic acids (Bio-detection, Bio-Rad et al) (Molecular analysis & S.; Molecular analysis, Bio-detection, etc. (Bio-detection, detection, "Molecular diagnostic tests", Principles of Molecular Medicine, pp.83-88 (Humana Press, Inc.1998)), to detect mutations associated with the zcyto20, zcyto21 and zcyto22 loci. Direct analysis of mutations in the Zcyto20 gene can be performed using genomic DNA of the subject. Methods for amplifying genomic DNA obtained from, for example, peripheral blood lymphocytes, are well known to those skilled in the art (see, e.g., Dracopoli et al (ed.), Current Protocols in human Genetics, pp. 7.1.6-7.1.7 (John Wiley & Sons 1998)).

In embodiments of the invention, an isolated zcyto20 encoding nucleic acid molecule can hybridize under stringent conditions to a polynucleotide having the sequence of SEQ ID NO: 1, to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 nucleotides 64 to 618 or to a nucleic acid molecule having a nucleotide sequence which hybridizes with SEQ ID NO: 1 complementary nucleotide sequence. Generally, stringent conditions are selected to be about 5 ℃ lower than the thermal melting temperature (Tm) for a particular 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. In embodiments of the invention, an isolated zcyto22 encoding nucleic acid molecule can hybridize under stringent conditions to a polynucleotide having the sequence of SEQ ID NO: 6, and hybridizing to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 6 nucleotides 64 to 618 or to a nucleic acid molecule having a nucleotide sequence which is identical to the nucleotide sequence of SEQ ID NO: 6 complementary nucleotide sequence.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA, can hybridize if the nucleotide sequences of the pair have a degree of complementarity. Hybrids can tolerate mismatched base pairs in the duplex, but the stability of the hybrid is affected by the degree of mismatch. The Tm of mismatched hybrids decreases by 1 ℃ for every 1-1.5% base pair mismatch. Varying the stringency of the hybridization conditions allows control over the degree of mismatch present in the hybrids. The stringency increases as the hybridization temperature increases and the ionic strength of the hybridization buffer decreases.

It is within the ability of those skilled in the art to adapt these conditions to a particular polynucleotide hybrid. The Tm for a particular target sequence is the temperature (under defined conditions) at which 50% of the target sequence hybridizes to a perfectly matched probe sequence. Those conditions that affect Tm include: the size and base pair number of the polynucleotide probe, the ionic strength of the hybridization solution, and the presence of destabilizing agents in the hybridization solution. Many 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 lengths (see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 nd edition (Cold Spring Harbor Press 1989); Ausubel et al, eds., Current protocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to Molecular Cloning technologies, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev. Biom. mol. 26: 227 (1990)). Sequence analysis software such as OLIGO6.0 (LSR; Long Lake, MN) and Primer Premier 4.0(Premier Biosoft International; Palo alto, CA) and Internet sites are publicly available tools that can be used to analyze a given sequence and calculate Tm based on user-defined criteria. These programs can also analyze a given sequence under defined conditions and determine the appropriate probe sequence. Typically, hybridization of longer polynucleotide sequences (>50 base pairs) is performed at a temperature of about 20-25 ℃ below the calculated Tm. For smaller probes (<50 base pairs), hybridization is typically performed at a temperature at or below the calculated Tm of 5-10 ℃. This allows DNA-DNA and DNA-RNA hybrids to hybridize at maximum rates.

After hybridization, the nucleic acid molecules can be washed under stringent conditions or under highly stringent conditions to remove unhybridized nucleic acid molecules. Typical stringent wash conditions include a 55-65 ℃ wash in a solution of 0.5X-2 XSSC plus 0.1% Sodium Dodecyl Sulfate (SDS). That is, nucleic acid molecules encoding variants of zcyto20, zcyto21, and zcyto22 polypeptides can hybridize under stringent washing conditions to a nucleic acid molecule having the sequence of SEQ ID NOS: 1. 4 and 6 (or a complement thereof), wherein said wash stringency corresponds to 0.5 x-2 xssc plus 0.1% SDS at 55-65 ℃, comprises 0.5 xssc plus 0.1% SDS at 55 ℃ or 2 xssc plus 0.1% SDS at 65 ℃. Equivalent conditions can be easily designed by the skilled person by e.g. replacing the SSC in the wash solution by SSPE.

Typical high stringency washing conditions include 50-65 ℃ washes in a solution of 0.1 × -0.2 × SSC plus 0.1% Sodium Dodecyl Sulfate (SDS). That is, a nucleic acid molecule encoding a variant zcyto20 polypeptide can hybridize under high stringency wash conditions to a polypeptide having the sequence of SEQ ID NO: 1 (or a complement thereof), wherein said wash stringency corresponds to 0.1 x-0.2 x SSC plus 0.1% SDS at 50-65 ℃, including 0.1 x SSC plus 0.1% SDS at 50 ℃ or 0.2 x SSC plus 0.1% SDS at 65 ℃.

The invention also provides a polypeptide or polypeptide combination thereof which is respectively similar to SEQ ID NOS: 2. 5, 7, 9, 11 or orthologs thereof having substantially similar sequence identity to the isolated zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptides. The term "substantially similar sequence identity" is used herein to refer to the identity of SEQ ID NOS: 2. 5, 7, 9, 11 or orthologs thereof, having a sequence identity of at least 70%, at least 80%, at least 90%, at least 95%, or greater than 95%. The invention also includes a polypeptide comprising a sequence corresponding to SEQ ID NO: 2 or SEQ ID NO: 7 amino acid residues 1 to 205 or 21 to 205, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95%, 96%, 97%, 98%, 99% sequence identity. The invention also includes nucleic acid molecules encoding these polypeptides. The method for determining percent identity is described below.

The present invention also contemplates that two standard identified variants of zcyto20, zcyto21, and zcyto22 nucleic acid molecules may be used: and respectively comparing the encoded polypeptide with the polypeptide shown in SEQ ID NOS: 2. 5, 7, 9, 11, and/or a hybridization assay as described above. These zcyto20 variants include: (1) respectively with the sequences of SEQ ID NOS: 1. 4, 6, 8, 10 (or a complement thereof) under stringent wash conditions, wherein the wash stringency corresponds to 0.5 x-2 xssc plus 0.1% SDS at 55-65 ℃; or (2) encodes a polypeptide substantially similar to SEQ ID NO: 2, or a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or greater than 95% sequence identity to the amino acid sequence of seq id No. 2. Alternatively, the zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 variants may be characterized as: (1) respectively with the sequences of SEQ ID NOS: 1. 4, 6, 8, 10 (or a complement thereof) under high stringency washing conditions, wherein the washing stringency corresponds to 0.1 x-0.2 x SSC plus 0.1% SDS at 50-65 ℃; and (2) encoding sequences corresponding to SEQ ID NOS: 2. 5, 7, 9, 11, or a polypeptide having at least 70%, at least 80%, at least 90%, at least 95%, or greater than 95% sequence identity in its amino acid sequence.

The percent sequence identity can be determined by conventional methods. See, e.g., Altschul et al, bull.math.bio.48: 603(1986), and Henikoff, proc.natl.acad.sci.usa 89; 10915(1992). Briefly, two amino acid sequences were aligned to optimize alignment scores using a gap (gap) opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM 62" scoring matrix of Henikoff and Henikoff (supra) (see Table 4, amino acids are represented by standard one letter codes).

TABLE 5

A R N D C Q E G H I L K M F P S T W Y V

A 4

R -1 5

N -2 0 6

D -2 -2 1 6

C 0 -3 -3 -3 9

Q -1 1 0 0 -3 5

E -1 0 0 2 -4 2 5

G 0 -2 0 -1 -3 -2 -2 6

H -2 0 1 -1 -3 0 0 -2 8

I -1 -3 -3 -3 -1 -3 -3 -4 -3 4

L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4

K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5

M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5

F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6

P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7

S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4

T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5

W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11

Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7

V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4

It will be appreciated by those skilled in the art that there are many well-established algorithms for aligning two amino acid sequences. The "FASTA" similarity search algorithm by Pearson and Lipman is a suitable protein alignment method that can be used to detect the level of identity between the amino acid sequences disclosed herein and the amino acid sequence of the putative zcyto20 variant. The FASTA algorithm is described in Pearson and Lipman, proc.natl.acad.sci.usa85: 2444(1988) and Pearson, meth. enzymol.183: 63(1990).

In short, FASTA first characterizes sequence similarity by determining the regions that are common to the query sequence (e.g., SEQ ID NO: 2) and the test sequence, i.e., the regions that have the greatest density of identity (if ktup variable is 1) or pair-wise identity (if ktup ═ 2) without regard for conservative amino acid substitutions, insertions, or deletions. The amino acid substitution matrix is then used to compare the similarity of all paired amino acids, re-score the 10 regions with the greatest consistency density, and "trim" the ends of these regions to include only those residues contributing to this highest score. If there are several regions with scores greater than the "cutoff value" (calculated by a predetermined formula based on the sequence length and ktup value), then these clipped original regions are examined to determine if these regions can incorporate gaps to form approximate alignments. Finally, the maximum scoring regions of the two amino acid sequences are aligned using a modified Needleman-Wunsch-Senlers algorithm (Needleman and Wunsch, J.mol.biol.48: 444 (1970); Senlers, SIAM J.Appl.Math.26: 787 (1974)). Preferred parameters for the FASTA assay are: ktup ═ 1, gap opening penalty ═ 10, gap extension penalty ═ 1, and substitution matrix ═ BLOSUM 62. These parameters can be incorporated into the FASTA program by modifying the scoring matrix file ("SMATRIX"), for an explanation of which see Pearson, meth.enzymol.183; appendix 2 of 63 (1990).

Using the ratios disclosed above, FASTA can also be used to determine the sequence identity of nucleic acid molecules. For nucleotide sequence comparisons, ktup values may range from 1 to 6, preferably from 3 to 6, most preferably 3, while other parameters are set as default values.

The variant zcyto20, zcyto21, and zcyto22 polypeptides or polypeptides having substantially similar sequence identity are characterized as having one or more amino acid substitutions, deletions or insertions. These changes are preferably minor in nature, i.e., conservative amino acid substitutions (see table 5) 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; amino-or carboxy-terminal extensions, such as an amino-terminal methionine residue, small linker peptides or affinity tags of no more than about 20-25 residues. The present invention therefore provides a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 2, preferably at least 90%, more preferably 95%, 96%, 97%, 98%, 99% or more identical to the corresponding region of the polypeptide having about 154-235 amino acid residues. Polypeptides comprising an affinity tag may also contain a proteolytic cleavage site between the zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 polypeptides and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.

TABLE 6

Conservative amino acid substitutions

Alkalinity:	arginine
			Lysine
	Histidine
		Acidity:	glutamic acid
	Aspartic acid
		Polarity:	glutamine
	Asparagine
		And (3) hydrophobic:	leucine
	Isoleucine
			Valine
And (3) fragrance:	phenylalanine
			Tryptophan
	Tyrosine
		Small:	glycine
	Alanine
			Serine
	Threonine
			Methionine

Amino acid residues comprising regions or domains critical for maintaining structural integrity can be identified. Specific residues can be identified in these regions that are more or less resistant to alteration while maintaining the overall tertiary structure of the molecule. Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity, secondary structure propensity (proportionality), binary patterns, complementary stacking, and cryptic polar interactions (Barton, Current opin. Structure. biol. 5: 372. 376, 1995; and Cordes et al, Current opin. Structure. biol. 6: 3-10, 1996). Generally, when designing a molecular modification or identifying a particular fragment, the activity of the modified molecule will be evaluated while determining the structure.

Amino acid sequence changes can be made in the zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 polypeptides to minimize disruption of higher order structures necessary for biological activity. For example, when the zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 polypeptides comprise one or more helices, the amino acid residues will be altered so as not to disrupt the geometry of the helix and other molecular components in which conformational changes will attenuate some critical functions (e.g., binding of the molecule to its binding partner). The effect of amino acid sequence alterations can be predicted, for example, by computer modeling as disclosed above, or determined by crystal structure analysis (see, e.g., Lapthorn et al, nat. struct. biol. 2: 266-268, 1995). Other techniques well known in the art compare the folding of variant proteins to standard molecules (e.g., native proteins). For example, the cysteine distribution in the variant and standard molecules can be compared. Mass spectrometry and chemical modification using reduction and alkylation provide methods for determining cysteine residues associated with disulfide bonds or not forming such bonds (Bean et al, anal. biochem. 201: 216-266, 1992; Gray, Protein Sci.2: 1732-1748, 1993; and Patterson et al, anal. chem. 66: 3727-3732, 1994). It is generally believed that folding is affected if the modified molecule has a different cysteine distribution than the standard molecule. Another accepted well-known method for measuring folding is Circular Dichroism (CD). The measurement and comparison of the CD profiles generated by the modified and standard molecules is conventional (Johnson, Proteins 7; 205-. Crystallography is another well-known method of analyzing folding and structure. Nuclear Magnetic Resonance (NMR), mapping of digested peptides and epitope mapping are also known methods for analyzing folding and structural similarity between proteins and polypeptides (Schaanan et al, Science 257: 961-.

One can prepare SEQ ID NO: 2 (Hopp et al, Proc. Natl. Acad. Sci.78: 3824-3828, 1981; Hopp, J.Immun. meth.88: 1-18, 1986 and Triquier et al, protein engineering 11: 153-169, 1998). The profile is based on a sliding six-residue window. The cryptic G, S and T residues and the exposed H, Y and W residues were omitted. For example, in zcyto20, the hydrophilic region includes SEQ ID NO: 2, amino acid residues 169(Glu) to 174(Glu) of SEQ ID NO: 2 from amino acid residues 54(Lys) to 59(Ala), seq id NO: 2 from amino acid residue 53(Phe) to amino acid residue 58(Asp), SEQ ID NO: 2 from amino acid residue 168(Gln) to 173(Lys), and SEQ ID NO: 2 from 154(Pro) to 159 (Arg).

One can prepare SEQ ID NO: the Hopp/Woods hydrophilicity profile of the Zcyto22 protein sequence shown in FIG. 7 (Hopp et al, Proc. Natl. Acad. Sci.78: 3824-3828, 1981; Hopp, J. Immun. meth.88: 1-18, 1986 and Triquier et al, protein engineering 11: 153-169, 1998). The profile is based on a sliding six-residue window. The cryptic G, S and T residues and the exposed H, Y and W residues were omitted. For example, in zcyto22, the hydrophilic region includes SEQ ID NO: 7 from amino acid residues 169(Glu) to 174(Glu), SEQ ID NO: 7 from amino acid residues 54(Lys) to 59(Ala), seq id NO: 7 from amino acid residue 53(Phe) to amino acid residue 58(Asp), SEQ ID NO: 7 from amino acid residues 168(Gln) to 173(Lys), and SEQ ID NO: 7 amino acid residues 154(Pro) to 159 (Arg).

It will be appreciated by those skilled in the art that hydrophilic or hydrophobic properties should be considered when designing modifications in the amino acid sequences of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptides so as not to disrupt overall structure and biological status. Of particular interest for substitution are hydrophobic residues selected from the group consisting of Val, Leu and Ile or from the group consisting of Met, Gly, Ser, Ala, Tyr and Trp.

From the sequence similarity analysis between IFN- α and members of the zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 families (shown in tables 1 and 2), it is also possible to deduce which are essential amino acids. Using methods such as the "FASTA" analysis described above, regions of high similarity can be identified in protein families, which can be used to analyze the amino acid sequences of conserved regions. An alternative method for identifying variant polynucleotides based on structure is to determine whether nucleic acid molecules encoding potential zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 gene variants can be compared to a polynucleotide having the sequence of SEQ ID NOS: 1. 4, 6, 8 or 10.

Other methods by which essential amino acids in the polypeptides of the invention can be identified are those known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081(1989), Bass et al, Proc. Natl. Acad. Sci. USA 88: 4498(1991), Coombs and Corey, "site-directed mutagenesis and protein engineering", "Proteins: Analysis and Design," Angeletti (eds.), pages 259-311 (academic Press, Inc.1998)). In the latter technique, single alanine mutations are introduced at each residue position of the molecule and the resulting mutant molecules are tested for biological or biochemical activity according to the methods disclosed below to determine the amino acid residues that are critical to the activity of the molecule. See also Hilton et al, j.biol.chem.271: 4699(1996).

The invention also includes functional fragments of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptides and nucleic acid molecules encoding these functional fragments. As used herein, "functional" zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 or fragments thereof are characterized by their proliferative or differentiative activity, ability to induce or inhibit specialized cellular functions, or ability to specifically bind to anti-zcyto 20, zcyto21, zcyto22, zcyto24 and zcyto25 antibodies or zcytor19 receptors (soluble or immobilized). As previously described herein, zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 polypeptides are characterized by a 6-helix bundle. Accordingly, the present invention also provides a fusion protein comprising: (a) polypeptide molecules comprising one or more helices as described above; and (b) a functional fragment comprising one or more of these helices. The other polypeptide portion of the fusion protein may be provided by another helical bundle cytokine or interferon (e.g., IFN- α) or by a non-native and/or unrelated secretion signal peptide that promotes secretion of the fusion protein.

The zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptides of the present invention, including full-length polypeptides, biologically active fragments and fusion polypeptides, can be prepared according to conventional techniques using cells into which an expression vector encoding the polypeptide is introduced. As used herein, "a cell into which an expression vector is introduced" includes a cell directly subjected to a manipulation to introduce an exogenous DNA molecule and its progeny containing the introduced DNA. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and can be grown in culture, including bacterial, fungal cells and higher eukaryotic cells in culture. Techniques for manipulating cloned DNA molecules and introducing foreign DNA into a variety of host cells are disclosed in Sambrook et al, Molecular Cloning: a Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel et al, eds., Current protocols Molecular Biology, John Wiley and Sons, Inc. NY, 1987.

Generally, the DNA sequences encoding the zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 polypeptides are operably linked in an expression vector with other genetic elements necessary for their expression, typically including a transcription promoter and terminator. The vector will typically also contain one or more selectable markers and one or more origins of replication, but it will be appreciated by those skilled in the art that in some systems the selectable marker may be provided on a separate vector and that replication of the exogenous DNA may be effected by integration into the host cell genome. The choice of promoter, terminator, selectable marker, vector and other elements is a matter of routine design within the level of ordinary skill in the art. Many of these elements have been described in the literature and are available from commercial suppliers.

To direct zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 polypeptides into the secretory pathway of a host cell, a secretion signal sequence (also referred to as a leader sequence, prepro sequence, or pre sequence) may be provided in the expression vector. The secretion signal sequence may be that of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25, or may be derived from another secreted protein (e.g. t-PA; see U.S. Pat. No. 3, 5,641,655) or synthesized de novo. The secretion signal sequence is operably linked to the DNA sequences of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25, i.e., the two sequences are linked in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretion signal sequences are typically located 5' of the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be located elsewhere in the DNA sequence of interest (see, e.g., Welch et al, U.S. Pat. No. 5,037,743; Holland et al, U.S. Pat. No. 5,143,830).

Cultured mammalian cells can be used as hosts in the present invention. Methods for introducing foreign DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al, Cell 14: 725, 1978; Corsaro and Pearson, genomic Cell Genetics 7: 603, 1981; Graham and Vander Eb, Virology 52: 456, 1973), electroporation (Neumann et al, EMBO J.1: 841-845, 1982), DEAE-dextran-mediated transfection (Ausubel et al, supra), and liposome-mediated transfection (Hawley-Nelson et al, Focus 15: 73, 1993; Ciccarone et al, Focus 15: 80, 1993). Production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, in Levinson et al, U.S. patent 4,713,339; hagen et al, U.S. Pat. Nos. 4,784,950; palmiter et al, U.S. patent 4,579,821; and Ringold, us patent 4,656,134. Suitable mammalian cells for culture include COS-1(ATCC No. CRL1650), COS-7(ATCC No. CRL1651), BHK (ATCC No. CRL1632), BHK570(ATCC No. CRL10314), 293(ATCCNO. CRL 1573; Graham et al, J.Gen.Virol.36: 59-72, 1977) and Chinese hamster ovary cell lines (e.g., CHO-K1, ATCC No. CCL61; or CHO DG44, Chasin et al, Som.cell Molec.Genet.12: 555, 1986). Other suitable cell lines are known in the art and are available from public collections, such as the American type culture Collection, Manassas, Va. In general, strong transcription promoters are preferred, such as those from SV40 or cytomegalovirus. See, for example, U.S. patent 4,956,288. Other suitable promoters include promoters from the metallothionein gene (U.S. Pat. Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter. Expression vectors for mammalian cells include pZP-1 and pZP-9 (which are deposited at the American type culture Collection (Manassas, VA, USA) under accession numbers 98669 and 98668, respectively) and derivatives thereof.

Drug screening is commonly used to select cultured mammalian cells into which exogenous DNA is inserted. These cells are commonly referred to as "transfectants". Cells cultured in the presence of a selection agent and capable of transmitting a gene of interest to their progeny are referred to as "stable transfectants". Preferred selectable markers are genes encoding the antibiotic neomycin resistance. Selection is performed in the presence of neomycin, such as G-418, etc. Selection systems may also be used to increase the expression level of a gene of interest, a process known as "amplification". The embodiment of amplification is: transfectants are cultured in the presence of low levels of a selection agent and then the amount of selection agent is increased to select for cells that produce high levels of gene-introduced product. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g., hygromycin resistance, multidrug resistance, puromycin acetyltransferase) may also be used.

The adenovirus system may also be used for in vitro protein production. By culturing adenovirus-infected non-293 cells under conditions where the cells do not divide rapidly, the cells can produce proteins for extended periods of time. For example, BHK cells are cultured to confluence in a cell factory and then exposed to an adenoviral vector encoding the secreted protein of interest. The cells are then cultured under serum-free conditions, under which the infected cells are allowed to survive for several weeks without significant cell division. In an alternative method, adenovirus-infected 293 cells can be cultured to relatively high cell densities, either as adherent cells or as suspension cultures, to produce significant amounts of protein (see Garnier et al, Cytotechnol.15: 145-55, 1994). Using either approach, the isolation of the expressed secreted heterologous protein from cell culture supernatant, lysate or membrane debris can be repeated according to the distribution of the expressed protein in the cell. In production protocols using infected 293 cells, non-secreted proteins can also be efficiently obtained.

Insect cells can be infected with recombinant baculovirus, typically derived from Autographa californica nuclear polyhedrosis virus (AcNPV), according to methods known in the art. In a preferred method, recombinant baculoviruses are generated by using a transposon-based system (described in Luckow et al, J.Virol.67: 4566-4579, 1993). The system utilizes a transfer carrier and is commercially available in the form of a kit (Bac-to-Bac)^TMA kit; life Technologies, Rockville, Md.). The transfer vector (e.g., pFastBacl)^TM(ii) a Life Technologies) contains the Tn7 transposon so that the DNA encoding the protein of interest can be transferred into the baculovirus genome maintained as a large plasmid called a "bacmid" in E.coli. See, Hill-Perkins and Possee, j.gen.virol.71: 971-; bonning et al, j.gen.virol.75: 1551 1556, 1994; and Chazenbalk Rapoport, j.biol.chem.270: 1543-1549, 1995. In addition, the transfer vector may comprise an in-frame fusion to DNA encoding a polypeptide extension or affinity tag as disclosed above. Transfer vectors containing the zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 coding sequences can be transformed into E.coli host cells and cells with bacmid containing an interrupted lacZ gene (indicative of recombinant baculovirus) screened using techniques known in the art. Bacmid DNA containing the recombinant baculovirus genome is isolated using conventional techniques and used to transfect spodoptera frugiperda (spodoptera rugerida) cells, such as Sf9 cells. Recombinant viruses were subsequently generated that expressed zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 proteins. The recombinant virus stock is prepared by methods commonly used in the art.

For the production of proteins, host cells can be infected with the recombinant virus, typically from fall armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., High Five ni)^TMA cell; invitrogen, Carlsbad, CA). See, for example, U.S. Pat. No. 5,300,435. Cells were cultured and maintained using serum-free media. Suitable media formulations are known in the art and are available from commercial suppliers. From about 2-5X 10⁵Seeding Density of Individual cells were cultured to 1-2X 10⁶The density of individual cells, at which time a recombinant virus stock with a multiplicity of infection (MOI) of 0.1-10, more typically close to 3, is added. The methods used are generally known in the art.

Other higher eukaryotic cells may also be used as hosts, including plant cells and avian cells. For a review of Agrobacterium rhizogenes (Agrobacterium rhizogenes) as a vector for expressing genes in plant cells see Sinkar et al, j.biosci. (Bangalore) 11: 47-58, 1987.

Fungal cells, including yeast cells, may also be used in the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris and Pichia methanolica. Methods for transforming s.cerevisiae cells with exogenous DNA and preparing recombinant polypeptides therefrom are disclosed in, for example, Kawassaki, U.S. patent 4,599,311; kawasaki et al, U.S. Pat. Nos. 4,931,373; brake, U.S. patent 4,870,008; welch et al, U.S. Pat. No. 5,037,743; and Murray et al, U.S. Pat. No. 4,845,075. Transformed cells are selected for a phenotype determined by a selectable marker, typically drug resistance or ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for s.cerevisiae is the POTI vector system disclosed by Kawasaki et al (U.S. Pat. No. 4,931,373), which allows selection of transformed cells by growth in a medium containing glucose. Suitable promoters and terminators for yeast include those derived from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al, U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) and alcohol dehydrogenase. See also U.S. patent 4,990,446; 5,063,154, respectively; 5,139,936, and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha (Hansenula polymorpha), Schizosaccharomyces pombe, Kluyveromyces lactis (Kluyveromyces lactis), Kluyveromyces fragilis (Kluyveromyces fragilis), Ustilago zeae (Ustilago maydis), Pichia pastoris, Pichia methanolica, Pichia quarternia (Pichia guillensis) and Candida maltosa (Candida maltosa) are also known in the art. See, e.g., Gleeson et al, J.Gen.Microbiol.132: 3459 3465, 1986; cregg, U.S. patent 4,882,279; and Raymond et al, Yeast 14, 11-23, 1998. Aspergillus cells can be used according to the method of McKnight et al, U.S. Pat. No. 4,935,349. A method for transformation of Acremonium chrysogenum is disclosed in Suminio et al, U.S. Pat. No. 5,126,228. A method for transformation of Neurospora is disclosed in Lambowitz, U.S. Pat. No. 4,486,533. The preparation of recombinant proteins in Pichia methanolica is disclosed in U.S. Pat. Nos. 5,716,808, 5,736,383, 5,854,039 and 5,888,768.

Prokaryotic host cells, including strains of the bacteria Escherichia coli (Escherichia coli), Bacillus (Bacillus), and other genera, are also useful host cells in the present invention. Techniques for transforming such hosts and expressing the foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al, supra). When expressing zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 polypeptides in bacteria such as e.coli, the polypeptides may be retained in the cytoplasm, typically in the form of insoluble particles, or may be directed to the periplasmic space by bacterial secretion sequences. In the former case, the cells are lysed and the particles are recovered and denatured using, for example, guanidinium isothiocyanate or urea. The denatured polypeptide is then refolded and dimerized by diluting the denaturant, for example, by dialysis against a combination of urea solution and reduced and oxidized glutathione, followed by dialysis against buffered saline solution. In the latter case, the polypeptide may be recovered from the periplasmic space in a soluble functional form by disrupting the cells (e.g., by sonication or osmotic shock) to release the periplasmic space contents and recovering the protein, thereby eliminating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according to conventional methods in a medium containing nutrients and other components necessary for the growth of the selected host cell. A variety of suitable media, including media of known composition and complex media, are known in the art and generally include carbon sources, nitrogen sources, essential amino acids, vitamins and minerals. The medium may also contain components such as growth factors or serum, if desired. The growth medium typically selects for cells containing exogenously added DNA, for example, by drug screening or the absence of essential nutrients that can be supplemented by selectable markers carried by the expression vector or co-transfected into the host cell. The liquid culture is provided with sufficient air by conventional means, such as shaking a flask or fermenter and sparging.

The polypeptides and proteins of the invention are preferably purified to a purity of 80% or more, more preferably 90% or more, even more preferably 95% or more, particularly preferably in a pharmaceutically pure state, i.e.a purity of more than 99.9% relative to contaminating macromolecules, in particular other proteins and nucleic acids, and are free of infectious and pyrogenic agents. Preferably, the purified polypeptide or protein is substantially free of other polypeptides or proteins, especially those of animal origin.

The expressed recombinant zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins (including chimeric polypeptides and multimeric proteins) can be purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See generally affinity chromatography: principles and Methods (Affinity Chromatography: Principles & Methods), Pharmacia LKBBIOTechnique, Uppsala, Sweden, 1988; and Scopes, protein purification: principles and practices (Protein Purification: Principles and Practice), Springer-Verlag, New York, 1994. Proteins comprising a polyhistidine affinity tag (typically about 6 histidine residues) can be purified by affinity chromatography on nickel chelating resins. See, e.g., Houchuli et al, Bio/Technol.6; 1321-1325, 1988. The protein comprising the glu-glu tag can be purified by immunoaffinity chromatography according to conventional methods. See, e.g., Grussenmeyer et al, supra. Maltose binding protein fusions are purified on amylose columns according to methods known in the art.

The Zcyto20, Zcyto2l, Zcyto22, Zcyto24, and Zcyto25 polypeptides may also be prepared by chemical synthesis according to methods known in the art, including complete solid phase synthesis, partial solid phase synthesis methods, fragment condensation, or classical solution synthesis. See, e.g., Merrifield, j.am.chem.soc.85: 2149, 1963; stewart et al, Solid Phase peptide Synthesis (2 nd edition), Pierce Chemical Co., Rockford, IL, 1984; bayer and Rapp, chem.pept.prot.3: 3,1986, respectively; and Atherton et al, Solidphase Peptide Synthesis: a Practical Approach, IRL Press, Oxford, 1989. In vitro synthesis is particularly advantageous for the preparation of smaller polypeptides.

Monomers or multimers may be prepared using methods known in the art; (ii) glycosylated or unglycosylated; the pegylated (pegylated) or non-pegylated (non-pegylated) forms of the zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 proteins, which may or may not include an initiating methionine residue.

Target cells for the activity assays of zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 include, but are not limited to, vascular cells (especially endothelial cells and smooth muscle cells), hematopoietic (myeloid and lymphoid) cells, liver cells (including hepatocytes, windowed endothelial cells, kupffer cells, and Ito cells), fibroblasts (including human skin fibroblasts and lung fibroblasts), fetal lung cells, joint synoviocytes, pericytes, chondrocytes, osteoblasts, and prostate epithelial cells. Endothelial cells and hematopoietic cells are derived from a common ancestor, the hemangioblast (Choi et al, Development 125: 725-732, 1998).

The zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins of the present invention may be characterized by their activity, i.e., modulation of proliferation, differentiation, migration, adhesion or metabolism of responsive cell types. The biological activity of the zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 proteins can be tested using in vitro or in vivo assays designed to detect cell proliferation, differentiation, migration or adhesion, or alterations in cell 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, e.g., for determining the effect of amino acid substitutions, deletions or insertions. However, in view of the complexity of developmental processes (e.g., angiogenesis, wound healing), biological activity is generally validated and further characterized using in vivo assays. Certain in vitro models, such as the three-dimensional collagen gel matrix model of Pepper et al (biochem. Biophys. Res. Comm.189: 824-831, 1992) are sufficiently complex to allow testing of histological effects. Assays may be performed using exogenously produced proteins, or may be performed in vitro or in vivo using cells expressing the polypeptide of interest. Assays may be performed using the zcyto20 protein alone or in combination with other growth factors (e.g., VEGF family members) or hematopoietic cytokines (e.g., EPO, TPO, G-CSF, stem cell factor). Representative tests are disclosed below.

The activity of the Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 proteins can be measured in vitro using cultured cells or in vivo by administering the molecules of the invention to an appropriate animal model. Assays for measuring cell proliferation or differentiation are well known in the art. For example, assays for measuring proliferation include, for example, chemosensitivity to neutral red dyes (Cavanaugh et al, Investigational New Drugs 8: 347- "354, 1990), incorporation of radiolabeled nucleotides into the DNA of proliferating cells (disclosed, for example, in Rains and Ross, methods enzymol. 109: 749-773, 1985; Wahl et al, mol. cell biol. 8: 5016-5025, 1988; and Cook. et al, Analytical biochem. 179: 1-7, 1989), incorporation of 5-bromo-2' -deoxyuridine (BrdU) into the DNA of proliferating cells (Porstmann et al, J.Immunol. methods 82: 179, 1985), and use of tetrazolium salts (Mosmann, J.Immunol. methods 65: 55-63, 1983; Alley. 82: 179-169; 1988; Marchen. 1988; Reinival et al, 1988: 487. 1988; Reinival et al, Sc. 1988; 1995: 487. 1988; 1995, 1995). Differentiation may be tested using suitable precursor cells that can be induced to differentiate into a more mature phenotype. Assays for measuring Differentiation include, for example, measuring cell surface markers, enzymatic activity, functional activity or morphological changes associated with stage-specific expression of tissues (Watt, FASEB, 5: 281-284, 1991; Francis, Differentiation 57: 63-75, 1994; Res, adv. anim. CellBiol. technol. bioprocesses, 161-171, 1989; all incorporated herein by reference).

The activity of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 can also be tested using assays designed to detect the production of one or more other growth factors or other macromolecules induced by zcyto20, zcyto21, zcyto22, zcyto24 and zcyto 25. Preferred such assays include those that determine the presence or absence of Hepatocyte Growth Factor (HGF), Epidermal Growth Factor (EGF), transforming growth factor alpha (TGF α), interleukin-6 (IL-6), VEGF, acidic fibroblast growth factor (aFGF), angiogenin, and other macromolecules produced by the liver. Suitable assays include mitotic assays using target cells that respond to the macromolecule of interest, receptor binding assays, competitive binding assays, immunological assays (e.g., ELISA), and other formats known in the art. Can be prepared from treated primary human skinFibroblasts, synoviocytes and chondrocytes measure metalloprotease secretion. The relative levels of collagenase, gelatinase and matrilysin (stromalysin) produced in response to incubation in the presence of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins can be measured using zymographic gel (Loita and Stetler-Stevenson, Cancer Biology 1: 96-106, 1990). Procollagen/collagen synthesis by skin fibroblasts and chondrocytes in response to test proteins can be used ³Incorporation of H-proline in nascent secreted collagen was measured.³H-labeled collagen can be visualized by autoradiography after SDS-PAGE (Unemori and Amento, J.biol.chem.265: 10681-10685, 1990). Glycosaminoglycans (GAG) secreted from skin fibroblasts and chondrocytes can be measured using the 1, 9-dimethylmethylene blue dye binding assay (Farndale et al, biochem. Biophys. acta 883: 173-177, 1986). Collagen and GAG assays can also be performed in the presence of IL-1 α or TGF- α to test the ability of the zcyto20 protein to modify the response established against these cytokines.

Certain members of the protein family comprising zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 have been shown to increase the number of monocytes circulating in vivo. Activated monocytes are important for both innate and acquired immunity. For example, activation of monocytes has been shown to stimulate antigen presentation by several mechanisms. Antigen presentation promotes the activation and proliferation of T cells (cytotoxic and helper T cells). Maturation and activation of dendritic cells also promotes T cell activation and natural and acquired immunity. Furthermore, it has been demonstrated that an increase in activated monocytes and macrophages can increase the cytolytic activity. Thus, zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 can be used as anti-infective agents to enhance natural, cell-mediated, and humoral immune responses. Enhanced ICAM staining was observed in CD14+ monocytes, suggesting that zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 play an important role in monocyte activation. Although the data show that members of this family can promote an antiviral response to viruses, they can also affect bacteria and parasites.

Monocyte activation assays can be performed to: (1) the ability of the zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins to further stimulate monocyte activation, and (2) the ability of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins to modulate monocyte activation induced by attachment or by exotoxin (Fuhlbridge et al, J.Immunol.138: 3799-3802, 1987). The levels of IL-1 α and TNF α produced in response to activation can be measured by ELISA (Biosource, inc. Monocytes/macrophages are extremely sensitive to exotoxins via CD14(LPS receptor), and proteins with appropriate levels of exotoxin-like activity can activate these cells.

The increase in monocyte levels suggests that zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 may have a direct effect on myeloid progenitor cells in the bone marrow. The increase in differentiation of myeloid progenitor cells into monocytes is necessary to restore immunocompetence, for example, following chemotherapy. Thus, administration of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 to patients receiving chemotherapy can promote recovery and improve their ability to fight infections commonly associated with chemotherapy. Accordingly, the present invention provides a method of expanding the number of monocytes or monocyte progenitors by: the use of the molecules of the invention for culturing bone marrow or peripheral blood cells in order to achieve an increase in the number of monocytes or monocyte progenitors in vitro or ex vivo. The invention also provides methods of administering a molecule of the invention in vivo to a mammal in need of an increase in monocytes or monocyte progenitors. The increase in monocytes and monocyte progenitors can be measured using clinicians, physicians and methods well known to those skilled in the art. Monocytes belong to the myeloid hematopoietic lineage and therefore their effect on other cells in this lineage is not unusual. For example, when one factor favors the differentiation or proliferation of a class of cells in myeloid or lymphoid lineages, this may also affect the production of other cells that have a common progenitor or stem cell.

The hematopoietic activity of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 proteins can be tested on a variety of hematopoietic cells in culture. Preferred test methods include primary bone marrow colony analysis and late lineage restriction colony analysis, which are known in the art (e.g., Holly et al, WIPO publication WO 95/21920). Myeloid cells seeded on a suitable semi-solid medium (e.g., 50% methylcellulose containing a mixture of 15% fetal bovine serum, 10% bovine serum albumin, and 0.6% PSN antibiotics) are cultured in the presence of the test polypeptide and then examined microscopically for colony formation. Known hematopoietic factors were used as controls. The mitogenic activity of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 polypeptides on hematopoietic cell lines can be measured as disclosed above.

Cell migration was determined essentially as described by Kahler et al (Arteriosclerosis, Thrombosis, and Vascular Biology 17: 932-939, 1997). A protein is considered chemotactic if it induces migration of cells from a region of low protein concentration to a region of high protein concentration. A typical test was performed using a modified Boyden chamber (two chambers separated by a polystyrene membrane) (Transwell; Corning Costar Corp.). Test samples diluted in medium containing 1% BSA were added to the lower chamber of a Transwell-containing 24-well plate. Then, cells were placed on Transwell inserts previously treated with 0.2% gelatin. Cell migration was measured after 4 hours incubation at 37 ℃. Non-migrating cells on top of the Transwell membrane were removed and cells attached to the lower surface of the membrane were fixed and stained with 0.1% crystal violet. The stained cells were then extracted with 10% acetic acid and the absorbance was measured at 600 nm. Migration is then calculated from the standard calibration curve. Cell migration can also be measured using the matrigel method of Grant et al ("angiogenesis is a component of Epithelial-Mesenchymal Interaction", Goldberg and Rosen, Epithelial-Mesenchymal Interaction in Cancer, Birkhauser Verlag, 1995, 235-248; Baatout, Anticancer Research 17: 451-456, 1997).

Cell adhesion activity was determined essentially as described by LaFleur et al (J.biol.chem.272: 32798-32803, 1997). Briefly, microtiter plates were coated with test proteins, non-specific sites blocked with BSA and placed at approximately 10⁴-10⁵Cells (such as smooth muscle cells, leukocytes, or endothelial cells) are seeded at a density of individual cells/well. The wells are incubated at 37 ℃ (typically for about 60 minutes) and then the non-adherent cells are removed by gentle washing. The number of adherent cells is quantified by conventional methods (e.g., staining with crystal violet, lysing the cells and determining the optical density of the lysate). Control wells were coated with a known adhesion protein such as fibronectin or vitronectin.

The activity of the Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 proteins can be measured with a silicon-based biosensiometer (microphysiometer) which measures the rate of extracellular acidification or proton excretion associated with receptor binding and subsequent cellular physiological reactions. An example of such a device is the Cytosensor manufactured by Molecular Devices (Sunnyvale, Calif.)^TMA microphysiological meter. Various cellular responses, such as cell proliferation, ion transport, energy production, inflammatory responses, modulation, and receptor activation, can be measured by this method. See, for example, McConnell et al, Science 257: 1906-; pitchford et al, meth.enzymol.228: 84-108, 1997; arigili et al, j.immunol.meth.212: 49-59, 1998; and Van Liefde et al, Eur.J.Pharmacol.346: 87-95, 1998. The microphysiologic meter can be used to analyze adherent or non-adherent eukaryotic or prokaryotic cells. By measuring changes in extracellular acidification in cell culture media over time, the microphysiometer can directly measure the response of cells to a variety of stimuli, including Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 proteins, their agonists and antagonists. Preferably, the microscopic physiological meter is used to measure the response of the Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 responsive eukaryotic cells relative to control eukaryotic cells that do not respond to the Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 polypeptides. The Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 responsive eukaryotic cells include cells responding to Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 by transfecting receptors of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25, and cells naturally responding to Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto 25. Exposure to Zcyto20, z as measured by a change (e.g. increase or decrease) in extracellular acidification The difference in response between cells of cyto21, Zcyto22, Zcyto24, and Zcyto25 and controls (not exposed to Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25) is a direct measure of the cellular response modulated by Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto 25. Moreover, such responses modulated by Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 can also be measured under a variety of stimuli. Accordingly, the present invention provides methods for identifying agonists and antagonists of the Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 proteins, comprising providing cells responsive to Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 polypeptides, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of the test compound, and detecting a change, such as an increase or decrease, in cellular response in the second portion of cells relative to the first portion of cells. Changes in cellular response are manifested as measurable changes in extracellular acidification rates. The third portion of the cells described above was cultured in the presence of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 proteins but without the test compound, which provided a positive control for Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 responsive cells and a control for comparing the agonist activity of the test compound to the activity of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 polypeptides. Antagonists of Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 may be identified by exposing cells to Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 proteins in the presence and absence of a test compound, whereby a decrease in the stimulatory activity of Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 indicates that the test compound has antagonist activity.

The expression of the Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 polynucleotides in animals provides a model for further studies of the biological effects of overproduction or inhibition of proteins in vivo. The encoding and antisense polynucleotides for Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 can be introduced into a test animal, such as a mouse, using a viral vector or naked DNA, or a transgenic animal can be prepared.

One in vivo method for testing the proteins of the invention utilizes a viral delivery system. Example viruses for this purpose include adenovirus, herpes virus, retrovirus, vaccinia virus and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is the best currently studied gene transfer vector for delivery of heterologous nucleic acids. For a review see Becker et al, meth.cell biol.43: 161-89, 1994; and Douglas and Curiel, Science & Medicine 4: 44-53, 1997. Adenovirus systems have several advantages. Adenoviruses can (i) accommodate relatively large insert DNA; (ii) growing to a high titer; (iii) a broad spectrum of mammalian cell types; and (iv) use with many different promoters, including ubiquitous, tissue-specific, and regulatable promoters. Since adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.

By deleting part of the adenoviral genome, larger heterologous DNA inserts (up to 7kb) can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In one exemplary system, the essential E1 gene is deleted from the viral vector so that the virus will not replicate unless the E1 gene is provided by the host cell (e.g., human 293 cell line). When administered intravenously to intact animals, adenoviruses are primarily targeted to the liver. If the adenovirus delivery system has a deletion in the E1 gene, the virus cannot replicate in the host cell. However, host tissues (e.g., liver) will express and process (and, if a signal sequence is present, secrete) the heterologous protein. The secreted protein will enter the circulation in the highly vascularized liver and its effect on infected animals can be determined.

Another alternative method of gene delivery involves taking cells of the body and introducing the vector into the cells in the form of a naked DNA plasmid. The transformed cells are then reimplanted into the body. Methods for introducing naked DNA vectors into host cells are known in the art and include transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter. 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 the genes Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25, and mice exhibiting complete deletions of the genes Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25, the latter being referred to as "knockout mice" (Snouwaert et al, Science 257: 1083, 1992), can also be prepared (Lowell et al, Nature 366: 740-742, 1993). The Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 genes and proteins encoded thereby can be studied in an in vivo system using these mice. Transgenic mice are particularly useful for studying the role of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 proteins in early development, as they allow identification of dysplasias or disruptions due to over or under expression of specific factors. See also maison pierre et al, Science 277: 55-60, 1997 and Hanahan, Science 277: 48-50, 1997. Preferred promoters for transgene expression include promoters from the metallothionein and albumin genes.

Loss of normal inhibitory control of muscle contraction is associated with injury or disorder of selected γ aminobutyric acid secreting neurons. For example, stiff person syndrome exhibits significant muscular stiffness, which is thought to be mediated by interfering with the function of neurons that produce gamma aminobutyric acid (GABA). Other related neuromuscular diseases include myotonia, metabolic myopathy, Isaacs syndrome, dystonia and spasticity (Valldeoriola, J.Neurol.246: 423-431, 1999).

Similarly, Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 polypeptides, or loss of their expression in a tissue, can be measured directly in a tissue or cell undergoing tumor progression. The increase in cell invasiveness and motility, or the gain or loss of expression of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 in the case of precancerous or cancerous conditions, as compared to normal tissue, can be used to diagnose transformation, invasion and metastasis in the course of a tumor. As such, information about tumor progression or metastatic stage will help physicians select the most appropriate treatment or aggressiveness of treatment for a given unique cancer patient. Methods for measuring gain and loss of expression (of mRNA or protein) are well known in the art and described herein, and may be applied to the expression of Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto 25. For example, the presence or absence of polypeptides that modulate cell motility can be used to aid in the diagnosis and prognosis of prostate Cancer (Banyard, J. and Zetter, B.R., Cancer and Metast. Rev.17: 449-458, 1999). The gain or loss of expression of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25, as effectors of cell motility or as liver-specific markers, can be used to diagnose brain and other cancers. Moreover, similar to prostate-specific antigen (PSA), increased levels of Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 polypeptides or anti-Zcyto 20, anti-Zcyto 21, anti-Zcyto 22, anti-Zcyto 24, and anti-Zcyto 25 antibodies in patients relative to normal controls may be indicative of brain and other cancers (see, e.g., Mulders, TMT et al, Eur. J. Surgical Oncol.16: 37-41, 1990). The strong expression of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 in tissues normally found not expressing these polynucleotides may be used to diagnose abnormalities in cell or tissue types, invasion or metastasis of cancerous liver tissue to non-liver tissue, and may help physicians guide further testing or research or help physicians guide treatment.

In addition, Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 polynucleotide probes, anti-Zcyto 20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 antibodies, and detection of the presence of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 in tissues can also be used to assess the presence of brain or other tissues normally expressing Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25, for example, after surgery involving the removal of diseased or cancerous liver or nerve tissue. In this regard, the polynucleotides, polypeptides and antibodies of the invention may be used to help determine whether all tissue has been excised following surgery (e.g., post-surgery for brain and other cancers). In these cases, it is particularly important to remove all potentially diseased tissue in order to achieve maximum recovery and minimal recurrence following cancer. Preferred embodiments include fluorescent, radiolabeled, or calorimetrically labeled anti-Zcyto 20 antibodies and Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 polypeptide binding partners that can be used histologically or in situ.

Furthermore, the activity and effect on tumor progression and metastasis of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 can be measured in vivo. Several syngeneic mouse models have been developed to study the effect of polypeptides, compounds or other treatments on tumor progression. In these models, subcultured tumor cells were implanted into mice of the same strain as the tumor donor. Cells will develop into tumors of similar characteristics in recipient mice and metastasis will occur in some models. For our study, suitable tumor models include, inter alia, Lewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No. CRL-6323). Both of these are commonly used tumor lines, isogenic to C57BL6 mice, and easy to culture and manipulate in vitro. Tumors arising from implantation of either cell line were able to metastasize to the lung in C57BL6 mice. The Lewis lung cancer model has recently been used to identify angiogenesis inhibitors in mice (O' Reilly MS et al, Cell 79: 315-328, 1994). C57BL6/J mice were treated with the test agent by daily injections of recombinant protein, agonist or antagonist or by single injections of recombinant adenovirus. 3 days after this treatment, 10 ⁵To 10⁶The individual cells were transplanted under the skin of the back. Alternatively, the cells themselves may be infected with a recombinant adenovirus (e.g., a recombinant adenovirus expressing Zcyto20, Zcyto21, Zcyto22, Zcyto24, or Zcyto 25) prior to transplantation, so that the protein is synthesized at the tumor site or within the cells rather than systemically. The mice normally develop visible tumors within 5 days. The tumors were allowed to grow for up to 3 weeks, during which time they could reach 1500-³Size. Tumor size and body weight were carefully monitored throughout the experiment. At sacrifice, tumors were removed with lung and liver and weighed. Lung re-visualization showed a strong correlation with metastatic tumor burden. As another measurement, lung surface metastases were counted. Treated using methods known in the art and described hereinTumors, lungs and liver were excised for histopathological examination, immunohistochemical analysis and in situ hybridization. The effect of the expressed polypeptide of interest, e.g., Zcyto20, Zcyto21, Zcyto22, Zcyto24 or Zcyto25, on the ability of the tumor to recruit the vasculature and to metastasize can thus be assessed. Furthermore, in addition to using adenovirus, it is also possible to transiently transfect implanted cells with Zcyto20, Zcyto21, Zcyto22, Zcyto24 or Zcyto 25. The use of stable Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 transfectants and the use of inducible promoters to activate Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 expression in vivo is known in the art and can be used in this system to evaluate the induction of metastasis by Zcyto 20. Also, the purified conditioned medium of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 or Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 can be directly injected into the mouse model and thus used in the system. See, generally, O' Reilly MS et al, Cell 79: 315-; and Rusciano D et al, murine model of liver Metastasis, Invasion Metastasis 14: 349-361, 1995.

Antisense methodology can be used to inhibit the transcription of the zcyto20 gene to detect the in vivo effects of this inhibition. Polynucleotides complementary to segments of the encoding polynucleotides of Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 (e.g., the polynucleotides set forth in SEQ ID NO: 1) are designed so that they bind to and inhibit translation of the mRNA encoding Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto 25. These antisense oligonucleotides may also be used to inhibit the expression of genes encoding Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 polypeptides in cell culture.

Most cytokines and other proteins produced by activated lymphocytes play important biological roles in cell differentiation, activation, recruitment, and cell homeostasis throughout the body. Inhibitors of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 and their activity are expected to have a variety of therapeutic uses. Such therapeutic uses include the treatment of diseases requiring immune modulation, including autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and systemic lupus erythematosusAnd diabetes. Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 may be important for the regulation of inflammation and thus may be used in the treatment of rheumatoid arthritis, asthma and sepsis. Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 may have a role in mediating tumorigenesis, and therefore Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 antagonists may be useful in the treatment of cancer. Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 may be used to modulate the immune system whereby antagonists of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 and Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 may be used to reduce transplant rejection, prevent graft versus host disease, enhance immunity to infectious diseases, treat immunocompromised patients (e.g. HIV ⁺Patient) or improved vaccines.

Members of the protein family of the invention have been shown to have antiviral effects similar to interferon alpha. In the united states, interferons have been approved for the treatment of autoimmune diseases, condyloma acuminatum, chronic hepatitis c, bladder cancer, cervical cancer, laryngeal papillomatosis, mycosis fungoides, chronic hepatitis b, kaposi's sarcoma in patients infected with human immunodeficiency virus, malignant melanoma, hairy cell leukemia, and multiple sclerosis. In addition, Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 can be used to treat various forms of arteriosclerosis, such as atherosclerosis, by inhibiting cell proliferation. Thus, the present invention contemplates the use of proteins, polypeptides and peptides having zcyto20 activity in the treatment of these diseases as well as in the treatment of retinopathy. The invention also contemplates the use of proteins, polypeptides and peptides having Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 activity in the treatment of lymphoproliferative diseases including B-cell lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, non-hodgkin's lymphoma, multiple myeloma, acute myelogenous leukemia, chronic myelogenous leukemia.

Interferons have also been shown to induce antigen expression in cultured cells (see, e.g., Auth et al, Hepatology 18: 546(1993), Guadagni et al, int.J.biol.Markers9: 53(1994), Girolomoni et al, Eur.J.Immunol.25: 2163(1995), and Maciejewski et al, Blood 85: 3183 (1995)). This activity enhances the ability to identify new tumor associated antigens in vitro. Moreover, the ability of interferons to increase the expression levels of human tumor antigens suggests that interferons can be used as adjuvants in immunotherapy or to enhance immunoscintigraphy using anti-tumor antigen antibodies (Guadagni et al, Cancer Immunol. Immunother.26: 222 (1988); Guadagni et al, int.J.biol. Markers 9: 53 (1994)). Thus, the invention includes the use of proteins, polypeptides and peptides having Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 activity as adjuvants for immunotherapy or for improving immunoscintigraphy using anti-tumor antigen antibodies.

Methods for detecting and diagnosing viral infections are well known to those skilled in the art. The exact method used to measure viral reduction in response to administration of the molecules of the invention will depend on the patient, the type of viral infection, and the like. For example, methods include, but are not limited to, measuring changes in the number of CD4 cells, serological assays, measuring viral DNA and viral RNA by conventional real-time quantitative polymerase chain reaction assays, virus-induced antibody levels, immunofluorescence and enzyme-linked immunosorbent assays, cytotropic viral effects, and histology.

Furthermore, Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 can bind to CD4 or another leukocyte receptor and exhibit antiviral effects, such as anti-Human Immunodeficiency Virus (HIV) or Human T-cell lymphotropic virus (HTLV). Alternatively, the zcyto20 polypeptide may compete with the virus for viral receptors or co-receptors to block viral infection. Zcyto20 can be administered parenterally to prevent viral infection or to reduce ongoing viral replication and reinfection (Gayowski, T. et al, Transplantation 64: 422-. Thus, zcyto20 may be used as an antiviral therapeutic Agent, for example, in the treatment of viral leukemia (HTLV), AIDS (HIV), or gastrointestinal viral infections, hepatitis B and C caused by viruses such as rotavirus, calicivirus (e.g., Norwalk Agent) and certain pathogenic strains of the gonadal virus.

Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 are also useful in the treatment of myocarditis, a disease caused when the heart is involved in inflammatory processes. Lymphocyte infiltration and myocyte lysis are thought to be the result of infection by viruses, bacteria, fungi or parasites (see, e.g., Brodison et al, J. infection 37: 99 (1998)). Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 can be injected by intravenous or subcutaneous routes to treat infections associated with myocarditis. Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 can also be administered by intravenous route as immunomodulatory cytokines to treat autoimmune myocarditis. The dose of interferon can be inferred using the A/J mouse myocarditis autoimmune model (Donemeyer et al, J.Exp.Med.182: 1291 (1995)).

Exogenous administration of interferon tau to sheep increases pregnancy rates (Aggarwal, HumanCytokines III, (B1ackwell Science 1997)). As described herein, zcyto20mRNA is expressed in the placenta. Thus, the invention includes the use of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25, such as human Zcyto20, Zcyto21 and Zcyto22, as disclosed herein, for promoting and protecting fetal growth. By way of illustration, Zcyto20, Zcyto21, and Zcyto22 can be used to protect a developing fetus from infection by a virus (e.g., human immunodeficiency virus, human papilloma virus, etc.). In addition, Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 may be used to promote in vitro fertilization.

Recent reports suggest that type I interferons may play a role in preventing virus-induced diabetes early in viral infection by inducing a strong antiviral state in pancreatic beta cells (Flodstroem et al, Nature Immunology 3, 373-382 (2002)). This can prevent β cell loss due to virus-induced cell death and concomitant autoimmunity. Zcyto20, 21 and 22 can also induce antiviral states in cells expressing their receptor zcytor 19. Zcyto 19 is highly expressed in pancreatic tissue, therefore, zcyto20-22 can prevent virus-induced diabetes caused by beta cell death. In addition, the role of type I interferons in preventing virus-induced diabetes may be extended to other virus-induced autoimmune diseases, and thus zcyto20-22 may also serve to prevent other diseases such as muscle sclerosis (muscle sclerosis), lupus and virus-induced autoimmune diseases in tissues expressing zcyto20-22 receptor zcytor 19.

Chronic systemic expression of type I interferons is also associated with the pathology of type I diabetes. Due to the similarity in biological activity and gene induction between type I interferon and zcyto20-22, chronic systemic expression of zyto20, 21 or 22 may also play a role in the pathology of type I diabetes. Therefore, inhibitors of zcyto20-22 activity in the pancreas may be beneficial for the prevention of type I diabetes.

The Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 polypeptides may be administered alone or in combination with other angiogenic or angiogenic agents, including VEGF. When Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 are used in combination with other agents, the two compounds may be administered simultaneously or sequentially as appropriate for the particular disease being treated.

Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 are useful for treating tumorigenesis and thus may be used for treating cancer. Zcyto20 can inhibit normal B cells stimulated by anti-IgM, with similar effects observed in B cell tumor lines, suggesting that treatment of patients with Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 to induce B cell tumor cells into a less proliferative state may have therapeutic benefit. The ligand may be administered in combination with other agents already in use, including conventional chemotherapeutic agents and immunomodulators such as interferon alpha. Interferon-alpha/beta has been shown to be effective in the treatment of certain leukemia and animal disease models, and the growth inhibitory effects of interferon-alpha and zcyto20 may be additive for B cell tumor-derived cell lines.

The present invention provides a method of reducing proliferation of neoplastic B or T cells comprising administering to a mammal having a B or T cell neoplasm a zcyto20 composition in an amount sufficient to reduce proliferation of neoplastic B or T cells. zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 stimulate lytic NK cells from bone marrow progenitor cells and stimulate T cell proliferation upon antigen receptor activation, which will enhance treatment of patients receiving allogeneic bone marrow transplantation, and thus, zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 can all enhance the generation of anti-tumor responses with or without infusion of donor lymphocytes.

In another aspect, the invention provides a method of reducing proliferation of a neoplastic B or T cell comprising administering to a mammal having a B or T cell neoplasm an amount of a zcyto20 antagonist composition sufficient to reduce proliferation of the neoplastic B or T cell. Furthermore, the zcyto20 antagonist can be a ligand/toxin fusion protein.

The fusion toxins of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 with saporin can be used against a similar group of leukemias and lymphomas, thereby expanding the range of leukemias that can be treated with zcyto20, zcyto21, zcyto22, zcyto24 and zcyto 25. Fusion toxin-mediated activation of the zcyto20 receptor provides two independent means for inhibiting target cell growth, the first being the same effect as observed with ligand alone, and the second being due to toxin delivery via receptor internalization.

For pharmaceutical use, the zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins may be formulated for local or parenteral, especially intravenous or subcutaneous administration, according to conventional methods. In general, pharmaceutical formulations include zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 polypeptides and a pharmaceutically acceptable carrier, such as saline, buffered saline, 5% aqueous dextran, and the like. The formulation may also include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, and the like. Formulation methods are well known in the art and are disclosed, for example, in Remington: the Science and Practice of pharmacy, Gennaro eds, Mack Publishing Co., Easton, Pa, 19 th edition, 1995. The zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 are preferably used at a concentration of about 10 to 100. mu.g/ml in total volume, but concentrations of 1ng/ml to 1000. mu.g/ml may also be used. For topical use, e.g. to promote wound healing, mayAccording to the ratio of 0.1-10 mu g/cm²The wound area is treated with the protein and the exact dosage is determined by the clinician according to accepted criteria, taking into account the nature and severity of the disease to be treated, the nature of the patient, etc. Determination of dosage is well within the level of ordinary skill in the art. Administration may be daily or intermittent during the treatment period. Intravenous administration can be by bolus injection or infusion over a typical time period of one to several hours. Sustained release formulations may also be used. Generally, a therapeutically effective amount of zcyto20 refers to an amount sufficient to cause a clinically significant change in the treated disease (e.g., a clinically significant change in hematopoiesis or immune function, a significant decrease in morbidity, or a significant increase in histological score).

The zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 proteins, agonists, and antagonists may be used to modulate the expansion, proliferation, activation, differentiation, migration, or metabolism of responsive cell types, including primary cells and cultured cell lines. Of particular interest in this regard are hematopoietic cells, mesenchymal cells (including stem cells and mature myeloid and lymphoid cells), endothelial cells, epithelial cells, smooth muscle cells, fibroblasts, hepatocytes, neural cells, and embryonic stem cells. The zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 polypeptides were added to the tissue culture medium of these cell types at concentrations of about 10pg/ml to about 100 ng/ml. It will be clear to the skilled person that the zcyto20 protein may advantageously be used in combination with other growth factors in the culture medium.

In the field of laboratory research, the zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins may also be used as molecular weight standards or as reagents in assays to determine the circulating levels of the protein, such as for diagnosing diseases characterized by excessive or low production of zcyto20 protein or for analyzing cellular phenotypes.

Inhibitors of the activity of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 proteins may also be identified. Test compounds were added to the above assay to identify compounds that inhibit the activity of zcyto20 protein. In addition to the above tests, various settings are possible The inhibition of zcyto20 activity by the test sample in an assay designed to measure receptor binding or stimulation/inhibition of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 dependent cellular responses. For example, zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 responsive cell lines may be transfected with reporter gene constructs that respond to the cellular pathways stimulated by zcyto20, zcyto21, zcyto22, zcyto24 and zcyto 25. Such reporter gene constructs are known in the art and typically include a Serum Response Element (SRE) activated by zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 operably linked to a gene encoding a detectable protein, such as luciferase. Testing candidate compounds, solutions, mixtures or extracts on target cells for their ability to inhibit zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 activity can be demonstrated by the reduction in reporter expression stimulated by zcyto20, zcyto21, zcyto22, zcyto24 and zcyto 25. Such assays can detect compounds that directly block the binding of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 to cell surface receptors as well as compounds that block processes occurring in the cellular pathway following receptor-ligand binding. In alternative methods, attachment of a detectable label (e.g., attachment of a detectable label) may be used ¹²⁵I. Biotin, horseradish peroxidase, FITC, etc.) zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25, compounds or other samples that directly block the binding of zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 to the receptor were tested. In such assays, the ability of the test sample to inhibit the binding of the labeled zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 to the receptor is indicative of inhibitory activity, which can be verified by other assays. The receptor used in the binding assay may be a cellular receptor or an isolated immobilized receptor.

As used herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof, such as F (ab')₂And Fab fragments, single chain antibodies, and the like, including genetically engineered antibodies. Non-human antibodies can be of human origin by grafting non-human CDRs onto human framework and constant regions, or by integrating intact non-human variable regions (optionally "masking" them with a human-like surface by replacing exposed residues, with the result that "veneered" antibodies are obtained)And (4) transforming. In some cases, humanized antibodies can retain non-human residues in the human variable region framework to increase proper binding characteristics. By humanizing antibodies, biological half-life can be increased and the likelihood of an adverse immune response following administration to a human is reduced. One skilled in the art can prepare humanized antibodies with specific and different constant regions (i.e., different Ig subtypes) to promote or inhibit different immune functions associated with the specific antibody constant regions. An antibody is defined as specifically binding if it has at least 10-fold greater binding affinity for the antibody to Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 polypeptides or proteins than for control (non-Zcyto 20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25) polypeptides or proteins. The affinity of monoclonal antibodies can be readily determined by one of ordinary skill in the art (see, e.g., Scatchard, Ann. NY Acad. Sci.51: 660-627, 1949).

Methods for making Monoclonal and polyclonal Antibodies are well known in the art (see, e.g., Hurrell, J.G.R. eds., Monoclonal Hybridoma Antibodies: Techniques and applications, CRC Press, Inc., Boca Raton, FL, 1982, incorporated herein by reference). Of particular interest is the preparation of antibodies directed against hydrophilic antigenic sites including, for example, the amino acid sequences of SEQ ID NOs: 2 amino acid residues 169(Glu) to 174(Glu), SEQ ID NO: 2 amino acid residues 54(Lys) to 59(Ala), SEQ ID NO: 2 amino acid residues 53(Phe) to 58(Asp), SEQ ID NO: 2 from 168(Gln) to 173(Lys), and SEQ ID NO: 2 amino acid residues 154(Pro) to 159 (Arg). For example, in zcyto22, the hydrophilic region includes SEQ ID NO: 7 amino acid residues 169(Glu) to 174(Glu), SEQ ID NO: 7 amino acid residues 54(Lys) to 59(Ala), SEQ ID NO: 7 (Phe) to 58(Asp), SEQ id no: 7 amino acid residues 168(Gln) to 173(Lys), and SEQ ID NO: amino acid residues 154(Pro) to 159(Arg) of 7, which are useful. It will be apparent to those of ordinary skill in the art that polyclonal antibodies can be prepared from a variety of warm-blooded animals such as horses, cattle, goats, sheep, dogs, chickens, rabbits, mice and rats. The immunogenicity of Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 polypeptides may be enhanced by the use of adjuvants such as alum (aluminum hydroxide) or freund's complete or incomplete adjuvant. Polypeptides for immunization also include fusion polypeptides, such as the fusions of Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 polypeptides or portions thereof with immunoglobulin polypeptides or with maltose binding proteins. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide moiety is "hapten-like", the moiety may advantageously be conjugated or linked to a macromolecular carrier such as Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA) or tetanus toxoid for immunization.

Alternative techniques for making or screening antibodies include in vitro exposure of lymphocytes to Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 polypeptides, and screening of antibody display libraries in phage or similar vectors (e.g., by using immobilized or labeled Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 polypeptides). Human antibodies can be made in transgenic non-human animals that have been engineered to contain human immunoglobulin genes, as disclosed in WIPO publication WO 98/24893. Endogenous immunoglobulin genes in these animals are preferably inactivated or eliminated, for example by means of homologous recombination.

A variety of assays known to those of skill in the art can be used to detect antibodies that specifically bind to the Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 polypeptides. Exemplary assays are described in detail in Antibodies: a Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: parallel immunoelectrophoresis (convurrentimmunoelectrophoresis), radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot analysis, Western blot analysis, inhibition or competition assays and sandwich assays.

Antibodies to Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 can be used for affinity purification of these proteins, for determining circulating levels of these proteins in diagnostic assays; for detecting or quantifying soluble Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 polypeptides as markers of underlying pathology or disease; for immunolocalization in whole animals or tissue sections, including immunodiagnostic applications; for immunohistochemistry; and as antagonists to block protein activity in vivo and in vitro. Antibodies to Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 may also be used to label cells expressing Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto 25; for affinity purification of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 polypeptides and proteins; for use in an assay method using FACS; for screening expression libraries; and for the preparation of anti-idiotype antibodies. Antibodies and other compounds, including therapeutic and diagnostic agents, can be linked using known methods such that these compounds can target cells expressing receptors for Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto 25. For certain applications, including in vitro and in vivo diagnostic applications, it is advantageous to use labeled antibodies. Suitable direct labels or tags include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles, and the like; indirect labels or tags may similarly use biotin-avidin or other complement/anti-complement pairs as intermediates. The antibodies of the invention may also be conjugated directly or indirectly to drugs, toxins, radionuclides, and the like, and such conjugates may be used for in vivo diagnostic or therapeutic applications (e.g., inhibition of cell proliferation). See generally Ramakrishan et al, Cancer Res.56: 1324-1330, 1996.

The polypeptides and proteins of the invention can be used to identify and isolate receptors. Receptors for Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 may be involved in growth regulation, angiogenesis and other developmental processes in the liver. For example, the Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 proteins and polypeptides can be immobilized on a column and the membrane preparation passed through the column (see generally Immolified Affinity Ligand and technologies, Hermanson et al, Academic Press, San Diego, CA, 1992, p. 195-202). Proteins and polypeptides may also be radiolabeled (Methods enzymol. Vol. 182, "guide to protein purification", M.Deutscher eds., Academic Press, SanDiego, 1990, 721-. Similarly, cells transfected with a cDNA expression library can be used in binding assays to clone cognate receptors using radiolabeled zcyto20 protein and polypeptide.

Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 polypeptides may also be used to teach analytical techniques such as mass spectrometry, circular dichroism to determine the conformation of, inter alia, a four alpha helix, x-ray crystallography to determine three-dimensional structure in atomic detail, nuclear magnetic resonance spectroscopy to reveal the structure of proteins in solution. For example, students can be given a kit containing Zcyto20, Zcyto21, Zcyto22, Zcyto24, and Zcyto25 for analysis. Since the amino acid sequence is known to the teacher, the student can be given the protein as a test to determine or develop the student's skills, and the teacher will then know whether the student correctly analyzed the polypeptide. Since each polypeptide is unique, teaching the use of zcyto20 will be unique in itself.

Antibodies that specifically bind to Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 can be used as teaching aids to teach students how to prepare affinity chromatography columns to purify Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25, as well as to clone and sequence polynucleotides encoding the antibodies and thus serve as practical lessons to teach students how to design humanized antibodies. The Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 genes, polypeptides or antibodies may be packaged by reagent companies and sold to teaching institutions for students to master the skills in the field of molecular biology. Since each gene and protein is unique, each gene and protein constitutes a unique challenge and learning experience for students in experimental classes. Such teaching kits containing ZCYTO20 gene, polypeptide or antibody are considered to be within the scope of the present invention.

In summary, the present invention provides an isolated polypeptide having at least 80% or 95% or 100% identity to a polypeptide selected from the group consisting of: (a) comprises the amino acid sequence of SEQ ID NO: 2 from amino acid residue 22 to amino acid residue 205; (b) comprises the amino acid sequence of SEQ ID NO: 7 from amino acid residue 22 to amino acid residue 205; (c) comprises the amino acid sequence of SEQ ID NO: 9 from amino acid residue 29 to amino acid residue 202; and (d) comprises SEQ ID NO: 11 from amino acid residue 29 to amino acid residue 202.

In another embodiment, the isolated polypeptide binds to SEQ ID NOS: 24. 27 or 29 in the form of a monomeric or homodimeric receptor or a receptor of SEQ ID NOS: 24. 27 or 29 and SEQ ID NO: 41 in combination.

In another aspect, the invention includes a polypeptide comprising SEQ ID NO: 2 from amino acid residue 22 to amino acid residue 205 or SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 205.

In another aspect, the invention includes a polypeptide comprising SEQ ID NO: 7 from amino acid residue 22 to amino acid residue 205 or SEQ ID NO: 7 from amino acid residue 1 to amino acid residue 205.

In another aspect, the invention includes a polypeptide comprising SEQ ID NO: 9 from amino acid residue 29 to amino acid residue 202 or SEQ ID NO: 9 from amino acid residue 1 to amino acid residue 202.

In another aspect, the invention includes a polypeptide comprising SEQ ID NO: 11 from amino acid residue 29 to amino acid residue 202 or SEQ ID NO: 11 from amino acid residue 1 to amino acid residue 202.

In another aspect, the invention includes a polypeptide having the sequence of SEQ ID NO: 2 from amino acid residue 22 to amino acid residue 205 or SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 205, wherein the polypeptide stimulates an antigenic response (antigenic response) in a mammal.

In another aspect, the invention includes a method having the SFQ ID NO: 7 from amino acid residue 22 to amino acid residue 205 or SEQ ID NO: 7 from amino acid residue 1 to amino acid residue 205, wherein the polypeptide stimulates an antigenic response in a mammal.

In another aspect, the invention includes a polypeptide having the sequence of SEQ ID NO: 9 from amino acid residue 29 to amino acid residue 202 or SEQ ID NO: 9 from amino acid residue 1 to amino acid residue 202, wherein the polypeptide stimulates an antigenic response in a mammal.

In another aspect, the invention includes a polypeptide having the sequence of SEQ ID NO: 11 from amino acid residue 29 to amino acid residue 202 or SEQ ID NO: 11 from amino acid residue 1 to amino acid residue 202, wherein the polypeptide stimulates an antigenic response in a mammal.

The invention includes pharmaceutical compositions comprising a polypeptide as described herein in a pharmaceutically acceptable carrier.

The invention also includes fusion proteins comprising the polypeptides described herein.

In other aspects, the invention includes an isolated polynucleotide encoding a polypeptide, wherein the nucleic acid molecule is selected from the group consisting of: (a) comprises the amino acid sequence shown in SEQ ID NO: 3; (b) after stringent washing conditions still compared to the sequence defined by SEQ ID NO: 1 from nucleotide 64 to 618, or SEQ ID NO: 1 from nucleotide 64 to 618.

In another embodiment, the invention includes an isolated polynucleotide encoding a polypeptide, wherein the nucleic acid molecule is selected from the group consisting of: (a) comprises the amino acid sequence shown in SEQ ID NO: 36; (b) after stringent washing conditions still compared to the sequence defined by SEQ ID NO: 6 from nucleotide 64 to 618, or SEQ ID NO: 6 from nucleotide 64 to 618.

In another aspect, the invention includes a polypeptide comprising SEQ ID NO: 1 from nucleotide 64 to nucleotide 618 or SEQ ID NO: 1 from nucleotide 1 to nucleotide 618.

In another embodiment, the invention includes a polypeptide comprising SEQ ID NO: 6 from nucleotide 64 to nucleotide 618 or as shown in SEQ ID NO: 6 from nucleotide 1 to nucleotide 618.

In another embodiment, the invention includes a polypeptide comprising SEQ ID NO: 8 from nucleotide 67 to nucleotide 606 or SEQ ID NO: 1 from nucleotide 1 to nucleotide 606.

In another embodiment, the invention includes a polypeptide comprising SEQ ID NO: 10 from nucleotide 67 to nucleotide 606 or SEQ ID NO: 10 from nucleotide 1 to nucleotide 606.

The present invention provides expression vectors comprising an isolated nucleic acid molecule as described herein and a transcription promoter and a transcription terminator, wherein the promoter is operably linked to the nucleic acid molecule and the nucleic acid molecule is operably linked to the transcription terminator.

The present invention provides a recombinant host cell comprising an expression vector as described herein, wherein the host cell is selected from the group consisting of a bacterium, a yeast cell, a fungal cell, an insect cell, a mammalian cell, and a plant cell.

In another aspect, the present invention provides a method of preparing a polypeptide comprising the steps of: culturing a recombinant host cell comprising an expression vector as described herein and producing the polypeptide.

The invention provides antibodies or antibody fragments that specifically bind to the polypeptides described herein.

In another aspect, the invention provides a method of expanding a monocyte, or monocyte progenitor cell, comprising culturing a bone marrow or peripheral blood cell with a composition comprising a polypeptide described herein, wherein the amount of the polypeptide described herein is sufficient to cause an increase in the number of monocytes or monocyte progenitors in the bone marrow or peripheral blood cell relative to bone marrow or peripheral blood cells cultured in the absence of the administered polypeptide.

The present invention also provides a method of stimulating an immune response in a mammal exposed to an antigen or pathogen comprising: (1) determining the level of antigen or pathogen specific antibody; (2) administering a composition comprising a polypeptide described herein in a pharmaceutically acceptable carrier; (3) determining the level of antigen or pathogen-specific antibody after administration; (4) comparing the level of antibody in step (1) with the level of antibody in step (3), wherein an increase in the level of antibody indicates that an immune response is stimulated.

In other aspects, the invention provides methods of generating an antiviral response in a mammal comprising administering to a mammal infected with a virus a composition of polypeptides described herein in an amount sufficient to cause reduction of the virus.

The invention will thus be more generally described and will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

Examples

Example 1

Mammalian expression plasmids

Expression plasmids containing polynucleotides encoding Zcyto20, Zcyto21, Zcyto22, Zcyto24, or Zcyto25 can be constructed by homologous recombination. The use of the 5 'and 3' terminal flanking regions corresponding to the vector sequence surrounding the zcyto20 insertion site SEQ ID NO: 1, and isolating a cDNA fragment, such as zcyto20 cDNA, by PCR. Primers ZC40923 and ZC40927 are shown in SEQ ID NO: 12 and 13.

The PCR reaction mixture was electrophoresed on a 1% agarose gel and QIAquick was used^TMThe gel extraction kit (Qiagen, Valencia, Calif.) gel extracts a band corresponding to the size of the insert. Plasmid pZMP21 is a mammalian expression vector comprising an expression cassette having an MPSV promoter, a plurality of restriction sites for insertion of coding sequences, a stop codon, an e.coli (e.coli) origin of replication; a mammalian selectable marker expression unit comprising the SV40 promoter, enhancer, and origin of replication, the DHFR gene, and the SV40 terminator; and URA3 and CEN-ARS sequences necessary for selection and replication in s.cerevisiae (s.cerevisiae). This plasmid was constructed from pZP9 (deposited at the American type culture Collection, 10801University boulevard, Manassas, VA20110-2209, accession number 98668) using the yeast genetic element taken from pRS316 (deposited at the American type culture Collection, 10801University boulevard, Manassas, VA20110-2209, accession number 77145), the Internal Ribosome Entry Site (IRES) element from poliovirus, and the extracellular domain of CD8 truncated at the C-terminus of the transmembrane domain. Plasmid pZMP21 was digested with BglII and used for recombination with the PCR insert.

Mu.l of yeast (Saccharomyces cerevisiae) competent cells were mixed with 10. mu.l or more of each DNA mixture, respectively, and transferred to a 0.2cm electroporation cuvette. The yeast/DNA mixture was electrically shocked using a power supply (BioRad laboratories, Hercules, Calif.) set at 0.75kV (5kV/cm), ∞ ohms, 25. mu.F. 600. mu.l of 1.2M sorbitol was added to each cup, and yeast was inoculated in two 300. mu.l aliquots onto two URA-D plates and incubated at 30 ℃. After about 48 hours, Ura from individual plates⁺Yeast transformants were resuspended in 1ml H₂O, briefly spun to pellet the yeast cells. The cell pellet was resuspended in 1ml lysis buffer (2% Triton X-100, 1% SDS, 100mM NaCl, 10mM Tris, pH8.0, 1mM EDTA). Add 500. mu.l of lysis mixture to Eppendorf tubes containing 300. mu.l of acid-washed glass beads and 200. mu.l of phenol-chloroform, vortex for 1 minute, 2 or 3 times, and then centrifuge at the Eppendorf maximum rateAnd centrifuged for 5 minutes. Mu.l of the aqueous phase was transferred to a new tube and the DNA was precipitated with 600. mu.l of ethanol (EtOH) and centrifuged at 4 ℃ for 10 minutes. Resuspend DNA pellet in 10. mu. l H₂And (4) in O.

Electrocompetent E.coli host cells (Electromax DH 10B) were transformed with 0.5-2ml yeast DNA preparation and 40. mu.l cells ^TMA cell; obtained from Life Technologies, inc., Gaithersburg, MD). Cells were electrically pulsed at 1.7kV, 25. mu.F and 400 ohms. After electroporation, 1ml SOC (2% Bato)^TMTryptone (Difco, Detroit, MI), 0.5% yeast extract (Difco), 10mM NaCl, 2.5mM KCl, 10mM MgCl₂、10mM MgSO₄20mM glucose) was added to 250. mu.l aliquots and then plated on 4 LB AMP plates (LB liquid medium (Lennox), 1.8% Bact^TMAgar (Difco), 100mg/L ampicillin).

Individual clones containing the correct zcyto20 expression construct were identified by restriction digestion to verify the presence of the zcyto20 insert and to confirm that the individual DNA sequences were correctly ligated to each other. The inserts of the positive clones were subjected to sequence analysis. Plasmid DNA was isolated on a larger scale using a commercial kit (QIAGEN Plasmid Maxi kit, Qiagen, Valencia, CA) according to the manufacturer's instructions. The correct construct was named zcyto20-CEE/pZMP 21.

Plasmids containing zcyto21, zcyto22, zcyto24, or zcyto25 were prepared in a similar manner using nucleotide specific primers.

Example 2

Expression in Chinese hamster ovary cells

CHO DG44 cells (Chasin et al, Som. cell mol. Genet. 12: 555-666, 1986) were seeded in 10cm tissue culture dishes at 37 ℃ and 5% CO ₂In Ham's F12/FBS medium (Ham's F12 medium (Life Technology), 5% fetal bovine serum (Hyclone, Logan, UT), 1% L-glutamine (JRH Bioscience, Lenexa, KS), 1% sodium pyruvate (Life Technology)logies)) to approximately 50% to 70% confluence. Then by liposome-mediated transfection, 3:1(w/w) polycationic lipid 2, 3-dioleyloxy-N- [2 (spermine carboxamido) ethyl]Liposome formulation of (E) -N, N-dimethyl-1-propaninium trifluoroacetate and neutral lipid dioleoylphosphatidylethanolamine in Membrane filtered Water^TMReagent, Life Technologies), cells were transfected with a plasmid containing Zcyto20, Zcyto21, Zcyto22, Zcyto24 or Zcyto25, such as Zcyto20/pZMP6, in a serum-free (SF) medium formulation (Ham's F12, 10mg/ml transferrin, 5mg/ml insulin, 2mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate). Zcyto20/pZMP6 was diluted with SF media in 15ml tubes to a total final volume of 640. mu.l. Make 35. mu.l Lipofectamine^TMAnd 605. mu.l of SF medium. The resulting mixture was added to the DNA mixture and allowed to incubate at room temperature for about 30 minutes. To DNA: lipofectamine^TM5ml SF medium was added to the mixture. Cells were washed once with 5ml SF medium, aspirated, after which DNA: lipofectamine ^TMAnd (3) mixing. Cells were incubated at 37 ℃ for 5 hours, then 6.4ml Ham's F12/10% FBS, 1% PSN medium was added to each plate. The plates were incubated overnight at 37 ℃ and the next day the DNA was replaced with fresh 5% FBS/Ham's F12: lipofectamine^TMAnd (3) mixing. On day three post-transfection, cells were split and placed in growth medium in T-175 shake flasks. On day 7 post-transfection, cells were stained with FITC-anti-CD 8 monoclonal antibody (Pharmingen Biotec, Auburn, CA), followed by anti-FITC conjugated magnetic beads (Miltenyi Biotec, Auburn, CA). CD8 positive cells were isolated using a commercial column (mini-MACS column; Miltenyi Biotec) according to the manufacturer's instructions and placed in DMEM/Ham's F12/5% FBS (selection medium) without nucleotides but with 50nM methotrexate.

Cells were seeded in selective medium in 96-well plates at densities of 0.5, 1 and 5 cells per well for subcloning, and allowed to grow for approximately 2 weeks. The evaporation of the medium in the wells was checked and each well was allowed to recover to 200. mu.l if necessary during this process. When most colonies on the plate were close to confluency, 100. mu.l of medium was collected from each well for dot blot analysis,and fresh selection medium was added to the cells. The supernatant was loaded on a nitrocellulose filter in a dot blot apparatus, and the filter was then treated in a vacuum oven at 100 ℃ to denature the protein. The filters were incubated for 10 minutes at 65 ℃ in 625mM Tris-glycine (pH9.1), 5mM β -mercaptoethanol and then overnight at 4 ℃ on a rotary shaker in Western A buffer (0.25% gelatin, 50mM Tris-HCl pH7.4, 150mM NaCl, 5mM EDTA, 0.05% Igepal CA-630) in 2.5% dry skimmed milk. The filters were incubated with antibody-HRP conjugate in Western A buffer of 2.5% dry skimmed milk powder for 1 hour at room temperature on a rotary shaker. The filters were then washed three times each for 15 minutes at room temperature in PBS supplemented with 0.01% Tween 20. Using chemiluminescent reagents (ECL) ^TMDirectly labeling the kit; amersham Corp., arlington heights, IL) filters were developed according to the manufacturer's instructions and then exposed to film (HyperfilmECL, Amersham Corp.) for about 5 minutes. Positive clones in 96-well dishes were trypsinized, transferred to selection medium in 6-well dishes for amplification and Western blot analysis.

Example 3

Expression in baby hamster kidney cells

Full-length zcyto24 and zcyto25 proteins were produced in BHK cells. For example, BHK cells were transfected with zcyto24-CEE/pZMP21 or zcyto25-CEE/pZMP21 (example 1). BHK570 cells (ATCC CRL-10314) were seeded into T75 tissue culture flasks at 37 ℃ and 5% CO₂Grown overnight to approximately 50% to 70% confluence in growth medium (SL7V4, 5% fetal bovine serum (Hyclone, Logan, UT), 1% penicillin/streptomycin). Followed by liposome-mediated transfection (using Lipofectamine)^TM(ii) a Life technologies) transfected cells in serum-free (SF) medium with either zcyto24-CEE/pZMP21 or zcyto25-CEE/pZMP 21. The plasmid was diluted in 1.5ml tubes with SF medium to a total final volume of 640. mu.l. Mu.l of the lipid mixture was mixed with 605. mu. lSF of the medium, and the resulting mixture was incubated at room temperature for about 30 minutes. 6ml of SF medium was then added to the DNA/lipid mixture. Washing the cells with 5ml SF Medium The cells were pipetted once and then the DNA/lipid mixture was added. Cells were incubated at 37 ℃ for 5 hours, and then 15ml growth medium was added to each plate. The plates were incubated overnight at 37 ℃ and the next day the DNA/lipid mixture was replaced with selection medium (SL7V4, 5% fetal bovine serum (Hyclone, Logan, UT), 1% penicillin/streptomycin, 1. mu.M MTX). Approximately 7-10 days after transfection, the anti-methotrexate colonies were trypsinized, and the cells were re-seeded in T-162 shake flasks and then transferred to large-scale culture.

Example 4

Construction of adenovirus vectors

To construct the adenoviral vector, the protein coding region of Zcyto20, Zcyto21, Zcyto22, Zcyto24, or Zcyto25 was amplified by PCR using primers that added PmeI and AscI restriction sites at the 5 'and 3' ends, respectively. Amplification was performed in a PCR reaction using the full-length cDNA template as follows: one cycle was performed at 95 ℃ for 5 minutes; then 15 cycles of 95 ℃ for 1 minute, 61 ℃ for 1 minute, and 72 ℃ for 1.5 minutes; then 7 minutes at 72 ℃; then, the temperature is kept at 4 ℃. The PCR reaction products were loaded onto a 1.2% low melting agarose gel in TAE buffer (0.04M Tris-acetate, 0.001M EDTA). The PCR product was excised from the gel and applied to a commercial kit comprising a silica gel membrane spin column PCR purification kit and gel purification kit; qiagen, Inc.) were purified according to kit instructions. The PCR product was then digested with PmeI and AscI, phenol/chloroform extracted, EtOH precipitated, and then rehydrated in 20mM TE (Tris/EDTA pH8.0). The zcyto20 fragment was then ligated into the PmeI-AscI site of the transgenic vector pTG12-8 and transformed into E.coli DH10B by electroporation^TMA competent cell. Vector pTG12-8 was derived from p2999B4(Palmiter et al, mol. cell biol. 13: 5266-5275, 1993) by inserting the rat insulin II intron (approximately 200bp) and a polylinker (Fse I/Pme I/AscI) at the Nru I site. The vector contains the mouse metallothionein (MT-1) promoter (approximately 750bp) surrounded by 10kb MT-15 'flanking sequence and 7kb MT-13' flanking sequence and human growth hormoneThe untranslated region of the hormone (hGH) and the polyadenylation signal (approximately 650 bp). The cDNA was inserted between the insulin II and hGH sequences. Clones containing the Zcyto20, Zcyto21, Zcyto22, Zcyto24, or Zcyto25cDNA were identified by plasmid DNA miniprep followed by digestion with PmeI and AscI. Positive clones were sequenced to ensure that there were no deletions or other abnormalities in the construct.

DNA was prepared using a commercial kit (Maxi kit, Qiagen, Inc.) and cDNA released from pTG12-8 vector using PmeI and AscI enzymes. The cDNA was separated on a 1% low melting agarose gel and excised from the gel. The excised gel strips were thawed at 70 deg.C, DNA was extracted twice with equal volumes of Tris-buffered phenol, precipitated with EtOH and resuspended at 10. mu. l H ₂And (4) in O.

The cDNA was cloned into the EcoRV-AscI site of modified pAdTrack-CMV (He, T-C. et al, Proc. Natl. Acad. Sci. USA 95: 2509-one 2514, 1998). The construct contains a Green Fluorescent Protein (GFP) marker gene. The CMV promoter driving GFP expression was replaced with the SV40 promoter and the polyadenylation signal of SV40 was replaced with the human growth hormone polyadenylation signal. In addition, the original polylinker was replaced with FseI, EcoRV and AscI sites. This modified form of pAdTrack-CMV was designated pZyTrack. Using commercial DNA ligation and screening kit (Fast-Link)A kit; epicentre Technologies, Madison, WI). Clones containing zcyto20 were identified by digesting mini-prep DNA with FseI and AscI. To linearize the plasmid, approximately 5. mu.g of the resulting pZyTrack zcyto20, zcyto21, zcyto22, zcyto24 or zcyto25 plasmid was digested with PmeI. Approximately 1. mu.g of linearized plasmid was cotransformed into E.coli BJ 5183 cells (He et al, supra) with 200ng of supercoiled pAdEasy (He et al, supra). Co-transformation was performed using a Bio-Rad Gene pulser at 2.5kV, 200 ohm and 25. mu.Fa. All co-transformation mixtures were plated on 4 LB plates containing 25. mu.g/ml kanamycin. The smallest colonies were picked and amplified in LB/kanamycin and recombinant adenovirus DNA was identified by standard DNA miniprep methods. Recombinant adenovirus DNA transfer using miniprep Coli DH10BTM competent cells were transformed and DNA was prepared using Maxi kit (Qiagen, Inc.) according to the kit instructions.

Approximately 5. mu.g of recombinant adenovirus DNA3 were digested with PacI enzyme (New England Biolabs) at 37 ℃ in a reaction volume of 100. mu.l containing 20-30U PacI. Digested DNA was extracted twice with equal volumes of phenol/chloroform and precipitated with ethanol. The DNA pellet was resuspended in 10. mu.l of distilled water. QBI-293A cells (Quantum Biotechnologies, Inc., Montreal, Qc. Canada) in T25 shake flasks that were seeded the previous day and grown to 60% -70% confluence with PacI digested DNA were transfected. PacI digested DNA was diluted to a total volume of 50. mu.l with sterile HBS (150mM NaCl, 20mM HEPES). IN a separate tube, 20. mu.l of 1mg/ml N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium salt (DOTAP) (Boehringer Mannheim, Indianapolis, IN) was diluted with HBS to a total volume of 100. mu.l. The DNA was added to DOTAP, gently mixed by pipetting up and down, and then left at room temperature for 15 minutes. The medium was removed from 293A cells and washed with 5ml of serum-free Minimal Essential Medium (MEM) alpha containing 1mM sodium pyruvate, 0.1mM MEM nonessential amino acids and 25mM HEPES buffer (reagents obtained from Life technologies, Gaithersburg, Md.). 5ml of serum-free MEM was added to 293A cells and the mixture was placed at 37 ℃. The DNA/lipid mixture was added dropwise to 293A cells in T25 shake flasks, mixed gently, and incubated for 4 hours at 37 ℃. After 4 hours, the medium containing the DNA/lipid mixture was aspirated and replaced with 5ml of complete MEM containing 5% fetal bovine serum. Transfected cells were monitored for GFP expression and formation of foci (viral plaques).

7 days after transfection of 293A cells with recombinant adenoviral DNA, the cells expressed GFP protein and began to form foci (viral "plaques"). The crude virus lysate was collected using a cell scraper to collect all 293A cells. The lysate was transferred to a 50ml conical tube. To release most of the virus particles from the cells, 3 freeze/thaw cycles were performed in a dry ice/ethanol bath and a water bath at 37 ℃.

The crude lysate was amplified (primary (1 °) amplification) to obtain a working "stock" of zcyto20 rAdV lysate. Pre-incubation for 20 hours to obtain 10cm plates where 293A cells were near confluency (80% -90%), 200ml of crude rAdV lysate was added to each 10cm plate, then the cells were monitored for CPE (cytopathic effect) under a white light microscope for 48 to 72 hours and GFP expression under a fluorescent microscope. When all 293A cells exhibited CPE, the stock lysates were collected and subjected to freeze-thaw cycles as described above.

The zcyto20 rAdV was then amplified twice (2 ℃). 20 293A cells of 15cm tissue culture dish were prepared so that the cells reached 80-90% confluence. Only 20ml of 5% MEM medium was left and each plate was inoculated with 300-500ml of the primary amplified rAdV lysate. After 48 hours, 293A cells were lysed for virus production and the lysate was collected in 250ml polypropylene centrifuge bottles and rAdV was purified.

NP-40 detergent was added to the vial of crude lysate to a final concentration of 0.5% in order to lyse all cells. The bottles were placed on a rotating platform for 10 minutes, with shaking performed as quickly as possible without turning the bottles over. Centrifuge at 20,000 XG for 15 minutes to pellet debris. The supernatant was transferred to a 250ml polycarbonate centrifuge bottle, and then 0.5 volume of 20% PEG8000/2.5M NaCl solution was added. Shake the bottle on ice overnight. The bottles were centrifuged at 20,000 XG for 15 minutes and the supernatant was discarded into the bleaching solution. Using a sterile cell scraper, 2 bottles of white virus/PFG pellets were resuspended in 2.5ml PBS. The resulting virus solution was placed in a 2ml microcentrifuge tube and centrifuged in a microfuge at 14,000 XG for 10 minutes to remove all other cellular debris. The supernatant from a 2ml microcentrifuge tube was transferred to a 15ml polypropylene capped tube and adjusted to a density of 1.34g/ml with CsCl. The solution was transferred to a 3.2ml polycarbonate thin-walled centrifuge tube and centrifuged at 348,000 XG for 3-4 hours at 25 ℃. The virus formed a white band. Viral bands were collected using a wide-bore tip.

Using commercial ion exchange columns (e.g. prepacked with G-25M PD-10 column; pharmacia Biotech, Piscataway, NJ) on viral preparationsAnd (4) carrying out desalination treatment. The column was equilibrated with 20ml PBS. The virus was loaded and then passed through the column. 5ml PBS was added to the column and fractions were collected as 8-10 drops. The optical density at 260nm of a 1:50 dilution of each fraction was determined on a spectrophotometer. The peak fractions were pooled and the Optical Density (OD) of the 1:25 dilution was determined. Using the formula: (260nm OD) (25) (1.1X 10)¹²) OD was converted to viral concentration as virions/ml.

To store the virus, glycerol was added to the purified virus to a final concentration of 15%, gently but efficiently mixed, and stored in aliquots at-80 μ C.

Recombinant virus infectivity was measured according to the protocol developed by Quantum Biotechnologies, Inc. Briefly, for each recombinant virus tested, 1X 10 per well was used⁴Individual 293A cells were seeded in two 96-well tissue culture plates in MEM (containing 2% fetal bovine serum). After 24 hours, 10-fold dilutions of each virus (from 1X 10) were made in MEM containing 2% fetal bovine serum^-2To 1X 10^-14). One dilution (100. mu.l) was added to each of the 20 wells. After 5 days of incubation at 37 ℃, the wells were observed for positive or negative CPE and the value of "plaque forming units/ml" (PFU) was calculated.

Example 5

Cloning of zcyto20, zcyto22, zcyto24 and zcyto25

A: zcyto20 and zcyto22

PCR primers common to zcyto20 and zcyto22 were designed in the predicted coding sequence. They were named ZC39339(SEQ ID NO: 47) and ZC39393(SEQ ID NO: 48). PCR was performed on a panel of human cDNA libraries from different tissues. Products were observed in brain, pancreatic islets (pancreas), prostate, placenta, testis, HPVS (prostate epithelium) and CD3+ libraries. PCR primers were then designed based on the 5 'genomic sequence of the start methionine and the 3' genomic sequence of the stop codon of zcyto20 (highly similar to zcyto 22). They were named ZC39340(SEQ ID NO: 49) and ZC39341(SEQ ID NO: 50). PCR was performed on the above library. 4 libraries contained full length clones. The PCR products from these clones were sequenced, resulting in 3 libraries containing zcyto22 (prostate, testis and CD3+) and one library containing zcyto20(HPVS (prostate epithelium)). PCR was also performed using primers specific for the predicted coding sequence of zcyto 22. These primers are designated ZC39295(SEQ ID NO: 51) and ZC39298(SEQ ID NO: 52). The positive library of this PCR was brain, prostate, CD3+ and testis. Sequencing confirmed the B zcyto20 sequence.

B: zcyto24 and zcyto25

PCR primers common to zcyto24 and zcyto25 were designed in the predicted coding sequence. These were named ZC39687(SEQ ID NO: 53) and ZC 397641 (SEQ ID NO: 54). PCR was performed on a set of mouse cDNA libraries from different tissues. Products were observed in the following library: heart, lung, placenta, Torres prostate, skin, small intestine, testis, and thymus. PCR primers were then designed for the positions of the initiation methionine 5' and the stop codon. The 5' primer, designated ZC 3972 (SEQ ID NO: 55), matched both the zcyto24 and zcyto25 sequences. The Zcyto 243' primer was named ZC 3977 (SEQ ID NO: 56). The ZCyto 253' primer was named ZC39688(SEQ ID NO: 57). PCR was performed on the positive library described above. For zcyto24, all libraries except heart and Torres prostate were positive. Sequencing confirmed the sequence of zcyto24 from placenta, testis, and small intestine libraries. For zcyto25, all libraries except heart and Torres prostate were positive. Sequencing confirmed the zcyto25 sequence from the lung library.

Example 6

Expression in baculovirus

A: constructs for expression of z cyto20

Preparation of expression vector pzBV 37L: zCyto20 to express zCyto20 polypeptide in insect cells. Primers ZC41932(SEQ ID NO: 14) and ZC41933(SEQ ID NO: 15) and Expand High Fidelity PCR System (Boer) hinger Mannheim) A536 bp fragment containing the sequence of zcyto20 and encoding Bspel and XbaI restriction sites at the 5 ' and 3 ' ends, respectively, was PCR amplified from plasmid zcyto20 according to the manufacturer's instructions. The PCR conditions were as follows: 1 cycle at 94 ℃ for 4 minutes; 30 cycles at 94 ℃ for 30 seconds, 50 ℃ for 30 seconds and 72 ℃ for 1 minute; 1 cycle at 74 ℃ for 4 minutes; then, the temperature is kept at 4 ℃. The small amount of PCR product was observed by gel electrophoresis (1% NuSieve agarose), confirming that the fragment was approximately 550bp in length. The remaining reaction mixture was purified by Qiagen PCR purification kit according to the manufacturer's instructions and diluted in 30. mu.l of water. The cDNA was digested with BspEI and XbaI (New England Biolabs, Beverly, Mass.) in a volume of 36. mu.l at 37 ℃ under appropriate buffer conditions. The digested PCR product bands were separated by 1% agarose TAE gel electrophoresis, excised and subjected to QIAquick^TMGel extraction kit (Qiagen, Cat. No.28704) was extracted and then diluted in 30. mu.l EB buffer. The digested zcyto20PCR product was ligated into the vector pZBV37L at BspEI and XbaI multiple cloning sites. The pZBV37L vector is a modified pFastBacl^TM(Life Technologies) expression vector in which the polyhedrin promoter has been removed and replaced upstream of the MCS with a late activating alkaline protein promoter and an EGT leader sequence. Mu.l of the restriction enzyme digested zcyto20PCR fragment and 4. mu.l of the corresponding pZBV37L vector were ligated in a volume of 20. mu.l of the appropriate buffer at 15 ℃ for 72 hours. Transformation of 33. mu.l ElectroMAX with 5. mu.l ligation mixture by electroporation in 2 mm-spaced electroporation cuvettes (BTX, Model No.620) at 400 ohm, 2.00kV and 25. mu.F ^TM DH12s^TMCells (Life Technologies, Cat. No. 18312-017). The transformed cells were diluted in 500. mu.l of LB medium, grown at 37 ℃ for 1 hour, and 10. mu.l and 20. mu.l of the dilutions were plated on LB plates containing 100. mu.g/ml ampicillin. Clones were analyzed by PCR and positive clones were selected, inoculated and sequenced. The sequence is then verified.

B. Construction and expression of tagged zcyto20

Preparation of expression vector pZBV 32L: zCyto20cee to express zCyto20cee polypeptides in insect cells. Design pZBV 32L: zCyto20cee to express zCyto20 polypeptide with a C-terminal GLU-GLU tag (SEQ ID NO: 16). This construct can be used to determine the N-terminal amino acid sequence of zcyto20 after cleavage of the signal peptide.

pZBV 32L: construction of zCyto20cee

PCR amplification from a plasmid containing the zcyto20cDNA using primers zc40240 and zc40241 (SEQ ID NOS: 17 and 18, respectively) yielded a 625bp zcyto20 fragment containing BamHI and XbaI restriction sites at the 5 'and 3' ends, respectively. The PCR conditions were as follows: 1 cycle at 94 ℃ for 4 minutes in a 100. mu.l volume of reaction containing 5% DMSO using the Expand High Fidelity PCR system (Boehringer Mannheim); 30 cycles at 94 ℃ for 30 seconds, 50 ℃ for 30 seconds and 72 ℃ for 60 seconds; 1 cycle at 72 ℃ for 4 minutes; then, the temperature is kept at 4 ℃. Gel electrophoresis (1% NuSieve agarose) observed 5. mu.l of the PCR fragment. The remaining reaction mixture was purified by Qiagen PCR purification kit according to the manufacturer's instructions and diluted in 30. mu.l of water. The cDNA was digested with BamHI and XbaI (New England Biolabs, Beverly, Mass.) in a volume of 35. mu.l under appropriate buffer conditions at 37 ℃ for 2 hours. The digested PCR product bands were separated by 1% agarose TAE gel electrophoresis, excised and subjected to QIAquick ^TMGel extraction kit (Qiagen) extraction, then diluted in 30. mu.l water. The purified digested zCyto20cee PCR product was ligated into the multicloning site of the vector pZBV32L previously prepared and digested with restriction enzymes BamHI and XbaI. The vector pZBV32L is a modified pFastBacl^TM(Life Technologies) expression vector in which the polyhedrin promoter has been removed and replaced with a late activated basic protein promoter, and a Glu-Glu tag (SEQ ID NO: 10) and a coding sequence for a termination signal are inserted at the 3' end of the polyclonal region. Mu.l of the restriction digested ZCyto20 insert and 40ng of the corresponding pZBV32L vector were ligated in a 20. mu.l volume at 16 ℃ overnight. 50 μ l of ElectroMAX was transformed with 5 μ l of ligation mixture by electroporation in 2mm spaced electroporation cuvettes at 400 ohm, 2.00kV and 25 μ F^TM DH12s^TMCells (Life Technologies). The transformed cells were diluted in 500. mu.l SOC medium (2% Bacto tryptone, 0.5% Bacto yeast extract, 10ml 1M NaCl, 1.5mM KCl, 10mM MgCl)₂、10mM MgSO₄And 20mM glucose), 50. mu.l of the dilution was plated on LB plates containing 100. mu.g/ml ampicillin. Clones were analyzed by PCR and restriction digestion. Positive clones were selected and inoculated for sequencing. Once the correct sequence was confirmed, 66. mu.l of DH10Bac was transformed with 25ng of positive clone DNA by heat shock in a 42 ℃ heat block for 45 seconds ^TMMAX EfficiencyCompetent cells (GIBCO-BRL). The transformed DB10Bac^TMCells were diluted in 600. mu.l SOC medium (2% Bacto tryptone, 0.5% Bacto yeast extract, 10ml 1M NaCl, 1.5mM KCl, 10mM MgCl)₂、10mM MgSO₄And 20mM glucose) and grown at 37 ℃ for 1 hour, and then 100. mu.l was plated on Luria agar plates containing 50. mu.g/ml kanamycin, 7. mu.g/ml gentamicin, 10. mu.g/ml tetracycline, 40. mu.g/ml IPTG and 200. mu.g/ml Bluo Gal. The plates were incubated at 37 ℃ for 48 hours. Color selection was used to identify those cells with transposed viral DNA (called "bacmid"). White colonies were picked for analysis. Colonies were analyzed by PCR and positive colonies (containing the desired bacmid) were selected for culture, followed by bacmid DNA purification. Primers for the hub element in the bacmid were used: ZC447(SEQ ID NO: 19) and ZC976(SEQ ID NO: 20), DNA was amplified by PCR to screen for clones with inserts of the correct molecular weight. The PCR reaction conditions were as follows: 1 cycle at 94 ℃ for 4 minutes; 25 cycles at 94 ℃ for 30 seconds, 50 ℃ for 30 seconds, and 72 ℃ for 2.5 minutes; 1 cycle at 72 ℃ for 4 minutes; then, the temperature is kept at 4 ℃. The PCR products were electrophoresed on a 1% agarose gel to verify the size of the insert. Those with the correct size insert were used to transfect Spodoptera Frugiperda (Sf9) cells.

2. Transfection

At a rate of 1X 10 per hole⁶Sf9 cells were seeded into 6-well plates and allowed to adhere for 1 hour at 27 ℃. Approximately 5. mu.g of bacmid DNA was diluted to 100. mu.l with Sf-900II SFM (Life Technologies). Mu.l Lipofectamine using Sf-900II SFM^TMReagents (Life Technologies) were diluted to 100. mu.l. Gentle mixing rodThe plasmid DNA and lipid solutions were incubated for 45 minutes at room temperature. Mu.l of Sf-900II SFM was added to the lipid-DNA mixture. The medium in the wells was aspirated and 1ml of DNA-lipid mixture was added to the cells. Cells were incubated overnight at 27 ℃. The DNA-lipid mixture in each well was aspirated and replaced with 2ml of Sf-900II medium. The plates were incubated at 27 ℃ and 90% humidity for approximately 7 days before harvesting the virus.

3. Amplification of

At a rate of 1X 10 per hole⁶Individual cells Sf9 cells were seeded onto 6-well plates. Mu.l of virus from the transfection plate was added to the wells, and the plate was incubated at 27 ℃ and 90% humidity for 96 hours before harvesting the virus (primary amplification).

Transfer 100. mu.l of virus from the primary amplification plate to a plate containing 1X 10⁶Individual cells/well, for a second round of expansion. Plates were incubated for 96 hours prior to harvest.

A further round of amplification (third amplification) was performed. Sf9 cells were grown to approximately 1X 10 in 50ml Sf-900IISFM in 250ml shake flasks ⁶Density of individual cells/ml. Then infected with 1ml of virus stock from the above plate, followed by incubation at 27 ℃ for 6 days, after which the virus was harvested.

The virus stock was titrated by a growth inhibition curve, allowing a titration culture indicating a multiplicity of infection (MOI) of 1 to proceed for a total of 48 hours. Supernatants were analyzed by reduction Western using a monoclonal primary antibody specific for the Glu-Glu tag followed by HRP-conjugated goat anti-mouse secondary antibody. The result showed a band of approximately 20 kDa. The supernatant was also used for activity analysis.

A large quantity of virus stock was then prepared by the following method: sf9 cells were cultured to approximately 1X 10 in 2800ml shake flasks of 1LSf-900II SFM⁶Density of individual cells/ml. The flasks were then infected with 5ml of virus stock from the flask, incubated at 27 ℃ for 4 days, after which the virus was harvested.

Larger scale infection was done to provide material for downstream purification.

Similarly, zcyto21 and zcyto22 were expressed in baculoviruses.

Example 7

Protein purification

For any of the proteins described herein, a recombinant protein can be prepared.

Purification of zcyto20

Recombinant zcyto20 tagged with Glu-G1u at its carboxy terminus was prepared from recombinant baculovirus infected insect cells, stable or transient BHK cell lines. The culture was harvested and the medium was sterile filtered using a 0.2 μm filter.

Proteins were purified from conditioned medium by a combination of anti-Glu (anti-EE) peptide antibody affinity chromatography and Superdex75 gel exclusion chromatography. The medium from BV (pH6.0, conductivity 7mS) was adjusted to pH 6.7. NaCl was then added to 300mM to both BV and BHK media before loading it on a 10X 70mM (5-ml column volume) Poros protein A anti-EE antibody affinity column at a flow rate of 2-5m 1/min. The column was then washed with 5 Column Volumes (CV) of 5 XPBS (pH 7.2). The bound protein was eluted with 0.5M acetic acid, 0.5M NaCl (pH 3.0). Fractions were collected at 2ml and 2M Tris was added to neutralize the eluate. Samples from the anti-FE antibody affinity column were analyzed by SDS-PAGE for the presence of zcyto20 protein by silver staining and western blotting. Fractions containing zcyto20 protein were pooled and concentrated to approximately 2ml using a Biomax-5 concentrator (Millipore) and loaded onto a 16X 600mm Superdex X75 gel filtration column (Amersham Pharmacia Biotech). Fractions containing the purified zcyto20 protein were pooled, filtered through a 0.2 μm filter, aliquoted into 100 μ l aliquots, and frozen at-80 ℃. The concentration of the final purified protein was determined by BCA assay (Pierce, Rockford, IL) and HPLC-amino acid analysis.

SDS-PAGE and Western blot analysis of zcyto20 protein

By SDS-PAGE (Nupage 4-12% Bis-Tris, Invitrogenn, Calsbad, CA) the recombinant zcyto20 protein was analyzed by Silver staining (Fast Silver, geno technology, inc., st. louis, MO) and Western blotting with anti-EE antibody. Using Xcell II^TMMINI-CELL (Invitrogen, Calsbad, CA) electrophoresis conditioned Medium or purified protein, using Xce1l II with stirring at room temperature^TMThe blot assembly (Invitrogen) was transferred to nitrocellulose (0.2_ m; Bio-Rad Laboratories, Hercules, Calif.) according to the instructions provided in the Instrument manual. The transfer was performed at 500mA in a buffer containing 25mM Tris base, 200mM glycine and 20% methanol for 45 minutes. The filters were then blocked with 10% skim dry milk powder in PBS for 10 minutes at room temperature. The nitrocellulose membrane was washed quickly and then primary antibody in PBS containing 2.5% dry skim milk powder was added. The blot was incubated at room temperature for 2 hours or at 4 ℃ overnight with gentle shaking. After incubation, the blots were washed three times in PBS for 10 minutes each. A secondary antibody (rabbit anti-mouse IgG conjugated to horseradish peroxidase; obtained from Pierce chemical Co., Rockford, Ill.) diluted 1:2000 in PBS containing 2.5% dry skim milk was added, and the blot was incubated at room temperature for two hours with gentle shaking. The blot was then washed three times in PBS for 10 minutes each, then in H ₂Fast wash in O. Use of commercial chemiluminescent substrate reagent (SuperSignal)A 1:1 mixture of ULTRA reagents 1 and 2; reagents were obtained from Pierce Chemical Co.) developed blots, and signals were captured over 10 seconds to 5 minutes or as needed using Lumi-Imager's Lumi analysis 3.0 software (Boehringer Mannheim GmbH, Germany).

C. Summary of protein purification and analysis

Purified zcyto20-CEE protein from BV medium migrated as a 21kDa monomer on 4-12% Bis-Tris gels, however, a faint 36kDa doublet band was also observed. Dimeric proteins become monomeric upon reduction with a reducing agent, suggesting that zcyto20-CEE dimerizes via disulfide bonds, and this is consistent with an odd number of cysteine residues (7 in total), resulting in interchain disulfide bonds.

Zcyto21, zcyto22, zcyto24 and zcyto25 polypeptides were purified in a similar manner.

Example 8

Interferon-like transcriptional regulatory region of zcyto20

Local sequence alignments of the characterized IFN-alpha, IFN-beta, IFN-gamma promoters and promoters of many other cytokines with the upstream regions of zcyto20, zcyto21 and zcyto24 were performed to identify whether these genes were co-regulated. The hypothesis is that: the common nature of gene regulation should be reflected in the sequence similarity of the upstream regions of the genes.

Due to the low binding specificity of TF, the prediction of each binding site has a high proportion of false positives. Therefore, isolated site prediction is not useful in order to identify binding sites that have a functional role in vivo. However, predicted binding sites that may have sequence-specific functions may be selected by conservative-based filtering: regulatory regions are often much more conserved between species than other non-coding regions, and this biological observation can be quantified to reveal a conserved pattern called phylogenetic footprints (Fickett et al, curr. Opin. Biotechnol. 11: 19-24, 2000). In particular, human-rodent comparisons have proven to be a valuable resource for identifying functional regulatory elements (Wasserman et al, nat. Genet.26: 225-.

Alignment revealed a significant match between the promoters of the different IFN-alpha genes, while there was little evidence of similarity between the promoters of zcyto20-22 and other cytokines based on this analysis.

Pairwise sequence alignments were performed with DBA (Jareborg et al, Genome Res.9: 815-824, 1999). Each transcription factor binding site was retrieved using a standard position weight matrix (Fickett, mol. cell biol. 16; 437 + 441, 1996) from the TRANSFAC database (version 3.0, Winger et al, Nucleic Acids Res.28: 316 + 319, 2000)) and the alignment of regions 1-4 was calculated using SSEARCH (version 3.1t12, Pearson, Genomics 11; 635 + 650, 1991). Based on published studies on other TF groups, it is expected that most natural binding sites that are well conserved between mouse and human could be detected within the score range used (Fickett, mol. cell biol. 16; 437-441, 1996; Wasserman et al, J. mol. biol. 278: 167-181, 1998).

The results of the calculations suggest that the characteristics shared by the zcyto20 family and other cytokines in regulation, reflected by sequence similarity, would be expected to occur at the level of a single binding site rather than a broad match.

Based on a search of the binding sites of a set (32) of TFs, putative sites were identified for a limited number of factors known to be involved in the transcriptional regulation of interferon. The results of the comparison demonstrate that putative binding sites for TF that play an important role in the transcriptional regulation of IFN- β [ NF-. kappa.B, ISRE (IRF-binding element) ] and IFN-. gamma.AP-1, CREB, GATA, NF-. kappa.B, NF-. kappa.AT) are also present in conserved non-coding regions. For example, a pair of adjacent AP-1/NF-AT sites in region 1 is an example of well-defined synergistic binding that occurs in one of many cytokine promoters (Holloway et al, mol. Immunol.38; 567. cndot. 580, 2000).

The binding site for TF has been identified as critical for cytokine regulation, suggesting that a cluster of transcription factor binding sites in the promoter region of zcyto20 is a candidate functional region.

The result of the alignment of region 2 to the panel of known cytokines is: in the AK155 promoter, there was a match at position-415 with respect to the transcription initiation site of AK155 (given by Knapper et al, J.Virol.74: 3881-3887, 2000). The fact that the Zcyto20 promoter had a putative NF-KB binding site at this position suggests that an NF-KB site may be present in the AK155 promoter.

Example 9

Transgenic animals

Transgenic animals expressing zcyto21 gene were prepared using adult fertile males (gerbils) (B6C3f1, 2-8 months of age (Taconic Farms, Germantown, NY)), vasectomized males (castrated mice) (CD1, 2-8 months of age (Taconic Farms)), fertile females (donors) (B6C3f1, 4-5 weeks of age (Taconic Farms)) and adult fertile females (recipients) (CD1, 2-4 months of age (TaconicFarms)).

Donors were acclimated for 1 week, then approximately 8IU of pregnant mare serum gonadotropin (Sigma, st. louis, MO) was injected intraperitoneally (I.P.) per mouse, and approximately 8IU of human chorionic gonadotropin (hcg (Sigma)) was injected intraperitoneally (I.P.) per mouse after 46-47 hours to induce superovulation. Donor and breeding rats were mated, followed by injection of hormones. Ovulation typically occurs within 13 hours after hCG injection. Mating occurrence was confirmed by the presence of vaginal plugs in the morning after mating.

Fertilized eggs were collected under a surgical microscope (Leica MZ12 Stereo Microspcoppe, Leica, Wetzlar, DE). The oviducts were collected and the eggs released onto a urinalysis (urinalysis) slide containing hyaluronidase (Sigma). Eggs were washed once in hyaluronidase in Whitten's W640 medium (Table 7) (already at 5% CO) ₂、5％O₂And 90% N₂Incubated at 37 ℃) were washed twice. Eggs were then stored at 37 ℃/5% CO prior to microinjection₂An incubator.

10-20. mu.g of plasmid DNA containing cDNA of the zcyto21 gene was linearized, gel purified and resuspended in 10mM Tris pH7.4, 0.25mM EDTA pH8.0 to a final concentration of 5-10ng per microliter for microinjection.

Microinjection of plasmid DNA into harvested eggs contained in a drop of W640 medium, overlaid with CO₂Balanced warm paraffin oil. DNA was aspirated into injection needles (drawn from 0.75mmID, 1mm OD borosilicate glass capillaries) and then injected into individual eggs. For all eggs, the injection needle penetrated into one or two haploid pronuclei.

Picoliter DNA was injected into the pronuclei, the needle was withdrawn and not contacted with the nucleolus. This process was repeated until all eggs had completed injections. Will be successfully micro-injectedThe ejected eggs were transferred to organ tissue culture dishes containing pre-deflated (compressed) W640 medium at 37 deg.C/5% CO₂The incubators were stored overnight.

The following day, 12-17 healthy 2 cell embryos from the previous day injection were implanted into the recipient. The ampulla is positioned, the fallopian tube between the ampulla and the bursa (burst) is secured, and a 28G needle is used to make an incision in the fallopian tube at a position close to the bursa while ensuring that the ampulla or the bursa is not punctured. The embryo is implanted through this incision and then the reproductive organs are guided back into the abdominal cavity by fixation to the peritoneal wall.

The receptors were returned in pairs to cages and allowed to gestation for 19-21 days. The animals injected with zcyto21cDNA died before birth.

TABLE 7

WHITTEN 640 culture medium

	mgs/200m	mgs/500/ml
			NaCl	1280	3200
KCl	72	180
			KH2PO4	32	80
MgSO4·7H2O	60	150
			Glucose	200	500
Calcium lactate	106	265
			K Penn (penicillin potassium)	15	37.5
Streptomycin sulfate	10	25
			NaHCO3	380	950
Pyruvic acid sodium salt	5	12.5
			H2O	200	500
EDTA	100μl	250μl
			5% phenol Red	200μl	500μl
BSA	600	1500

All reagents were obtained from Sigma.

Example 10

Antibody preparation

A: zcytor19 polyclonal antibody

2 female New Zealand white rabbits were immunized with the purified recombinant protein huzcytor19/MBP-6H to prepare polyclonal antibodies. Each rabbit was initially injected intraperitoneally (ip) with 200. mu.g of purified protein in complete Freund's adjuvant, followed by an intraperitoneal booster injection of 100. mu.g of peptide in incomplete Freund's adjuvant every 3 weeks. 7-10 days after the second booster injection (3 injections total), animal blood was collected and serum was collected. Animals were then boosted and bled every 3 weeks.

Huzcytor 19/MBP-6H-specific rabbit serum was pre-adsorbed with anti-MBP antibodies using a CNBr-SEPHAROSE4B protein column (Pharmacia LKB, Peapack, N.J.) prepared using 10mg purified recombinant MBP per gram of CNBr-SEPHAROSE. Huzcytor 19-specific polyclonal antibody was affinity purified from rabbit serum using a CNBr-SEPHAROSE4B protein column (prepared using 10mg of the purified recombinant protein-specific antigen huzcytor19/MBP-6H, followed by 20 x dialysis overnight in PBS). Huzcytor19 specific antibodies were characterized in an ELISA using 500ng/ml of purified recombinant protein huzcytor19/MBP-6H or huzcytor19-Fc4 as antibody targets. The Lower Limit of Detection (LLD) of the rabbit anti-huzcytor 19/MBP-6H affinity purified antibody to the specifically purified recombinant antigen huzcytor19/MBP-6H and to the purified recombinant huzcytor19-Fc4 was determined.

B: polyclonal antibodies Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25

Polyclonal antibodies were prepared by immunizing female New Zealand white rabbits with the purified recombinant proteins zcyto20/MBP-6H, zcyto21/MBP-6H and zcyto22/MBP-6H and mouse zcyto24/MBP-6H or zcyto 25/MBP-6H. Each rabbit was initially injected intraperitoneally (ip) with 200. mu.g of purified protein in complete Freund's adjuvant, followed by an intraperitoneal booster injection of 100. mu.g of peptide in incomplete Freund's adjuvant every 3 weeks. 7-10 days after the second booster injection (3 injections total), animal blood was collected and serum was collected. Animals were then boosted and bled every 3 weeks.

Zcyto20/MBP-6H, zcyto21/MBP-6H, zcyto22/MBP-6H, zcyto24/MBP-6H or zcyto25/MBP-6H specific rabbit serum can be pre-adsorbed with anti-MBP antibodies using a CNBr-SEPHAROSE4B protein column (Pharmacia LKB, Peapack, N.J.) prepared using 10mg purified recombinant MBP per gram of CNBr-SEPHAROSE. Polyclonal antibodies specific for Zcyto20, Zcyto21, Zcyto22, Zcyto24 or Zcyto25 were affinity purified from rabbit sera using a CNBr-SEPHAROSE4B protein column (prepared using 10mg of purified recombinant protein-specific antigen followed by 20 x dialysis in PBS overnight). Antibodies were characterized in an ELISA using 500ng/ml purified recombinant protein as antibody target. The Lower Limit of Detection (LLD) of the purified antibody against its purified specific recombinant antigen was determined.

A similar method was used to prepare a polyclonal antibody to zcyto21 by immunizing rabbits with CEE-tagged zcyto21 protein and purifying the antibody.

Example 11

Polyinosinic acid-polycytidylic acid induced Zcyto20, Zcyto21, Zcyto22, Zcyto24 And zcyto25 Gene expression

A: various cell types

Cultures of primary cells (normal human bronchial epithelial cells, normal human dermal fibroblasts, human umbilical vein endothelial cells, human microvascular endothelial cells, and human smooth muscle cells; CLONTICS, Inc.; Walkersville, MD) and human choriocarcinoma cell lines (Jar, BeWo, and 3A-SubE cells; ATCC, Manassas, VA) were grown in the presence of polyinosinic-polycytidylic acid (poly I: C; 100. mu.g/ml) (SIGMA; St. Louis, MO) or in simple culture medium. In some cases, 10ng/ml hTNFa or 10ng/ml hIL1b was also tested. After 4 hours of incubation, total RNA from the cells was isolated and treated with RNase-free DNase. First strand cDNA was synthesized using 1. mu.g total RNA using the Advantage RT-for-PCR kit as recommended by the manufacturer (Clontech, Palo Alto, Calif.). The following primer pairs were used: zcyto20, ZC40134(SEQ ID NO: 30) and ZC40214(SEQ ID NO: 31); zcyto21, ZC40209(SEQ ID NO: 32) and ZC40213(SEQ ID NO: 33); zcyto22, ZC39295(SEQ ID NO: 34) and ZC39298(SEQ ID NO: 35) were subjected to polymerase chain reaction using 5% cDNA reaction according to the manufacturer's recommendations (Clontech). Primers to G3PDH were used as a control.

Low levels of zcyto20mRNA were detected in all cell types tested. Increased zcyto20mRNA levels were observed in NHBE, HUVEC, JAR, 3-A Sub E and BeWo cells stimulated with poly I: C. Low levels of zcyto22mRNA were detected in all cell types tested. Increased zcyto22mRNA levels were observed in NHBE, JAR, 3-A Sub E and BeWo cells stimulated with poly I: C. These results indicate that C, the interferon inducer, is known to stimulate the synthesis of zcyto20 and zcyto22 mRNA.

Low levels of zcyto21mRNA were detected in all cell types tested. Increased zcyto21mRNA levels were observed in poly I: C-stimulated NHBE, HUVEC, NHDF, SMC, HMVEC, JAR, 3-A Sub E, and BeWo cells. Increased levels of zcyto21mRNA were also observed in IL1b treated 3-A Sub E placental cells. These results suggest that the interferon inducer poly I: C is known to stimulate zcyto21mRNA synthesis and that in certain cell types cytokine IL1b may also stimulate its synthesis.

B: peripheral blood mononuclear cells

Total peripheral blood mononuclear cells were isolated from human blood using Ficoll Hypaque. T cells were purified from peripheral blood mononuclear cells using a VarioMacs positive screening column according to the manufacturer's instructions (Miltenyi Biotec inc., Auburn, CA). Samples from each population were stained and analyzed by Fluorescent Antibody Cell Sorting (FACS) (Bectin Dickinson, san jose, CA) analysis to determine the percentage enrichment. CD3+ T cells were approximately 95% pure. Whole peripheral blood leukocytes or CD3+ T cells were cultured in poly I: C (100. mu.g/ml) or in pure medium. After 4 hours of incubation, total RNA from the cells was isolated and treated with RNase-free DNase. RT-PCR was performed using Superscript one-step RT-PCR and Platinum Taq kit (Invitrogen, Frederick, Md.) with 100ng of total RNA as template to synthesize cDNA. The primer pairs used were as follows: zcyto 20: ZC40134(SEQ ID NO: 30) and ZC40214(SEQ ID NO: 31); zcyto 21: ZC40209(SEQ ID NO: 32) and ZC40213(SEQ ID NO: 33); zcyto 22: ZC39295(SEQ ID NO: 34) and ZC39298(SEQ ID NO: 35). Aliquots of each RNA were also tested against a primer pair specific for MHC class I (Clontech).

Zcyto20 mRNA was detected in poly I: C stimulated whole peripheral blood mononuclear cells. This result indicates that the known interferon stimulator, poly I: C, can stimulate the synthesis of zcyto20 mRNA by peripheral blood mononuclear cells.

Zcyto21mRNA was detected in poly I C-stimulated whole peripheral blood mononuclear cells and CD3+ T cells. This result indicates that the known interferon stimulator, poly I: C, can stimulate the synthesis of zcyto21mRNA by peripheral blood mononuclear cells, including CD3+ T cells.

Zcyto22 mRNA was detected in poly I C-stimulated whole peripheral blood mononuclear cells and CD3+ T cells. This result indicates that the known interferon stimulator, poly I: C, can stimulate the synthesis of zcyto22 mRNA by peripheral blood mononuclear cells, including CD3+ T cells.

These results indicate that poly I: C will have an effect on both zcyto24 and zcyto25, as well as other family members.

Example 12

Expression analysis of human primary immune cells and immune cell lines using RT-PCR

A panel of primary human immune cell populations and RNAs into immune cell lines were screened for expression of zcyto21, zcyto21, and zcyto22 using RT-PCR. This set of RNAs was homemade and comprised RNAs from 16 different resting and activated cells described below. All primary immune cell populations were isolated from the blood of several anonymous donors. The fractions were then fractionated using labelled Microbeads and a magnetic cell separation System (Miltenyi Biotec) Various subtypes of immune cells (CD3+, CD14+, CD19+, and CD56 +). Using RNease Midiprep^TMThe kit (Qiagen, Valencia, CA) prepared RNA according to the manufacturer's instructions. CD56+ NK cell RNA was isolated from these cells in the resting state. A CD3+ population was activated using a combination of 500ng/ml ionomycin and 5.0ng/ml PMA (phorbol 12-myristate 13-acetate). Another CD3+ population was stimulated using supernatant from Conconavalin a-stimulated rat splenocytes (a medium known to be rich in cytokines and growth factors). CD3+ cells were harvested at activation times of 0, 4 and 16 hours for RNA isolation. A CD19+ sample was isolated from human tonsils and activated with 0.5. mu.g/ml ionomycin and 10ng/ml PMA. Cells were then harvested at 0, 4 and 24 hours and RNA was isolated. Human CD14+ monocytes were activated with 0.1ng/ml Lipopolysaccharide (LPS) or 1.0ng/ml LPS for 20 hours. The resting and activated cells are then collected and RNA isolated. In addition, RNA was isolated from resting and activated human monocytic cell lines HL-60, THP-1 and U937. HL-60 cells were activated with 10ng/ml PMA overnight. THP-1 cells were activated with 1.0ng/ml LPS +10ng/ml IFN γ overnight. Finally U937 cells were activated with 10ng/ml PMA overnight. The cDNA was synthesized by RT-PCR using Superscript one-step RT-PCR and Platinum Taq kit (Invitrogen) and using 100ng of total RNA as a template. The PCR primer pairs used were as follows: zcyto 20: ZC40632(SEQ ID NO: 36) and ZC40633(SEQ ID NO: 37); zcyto 21: ZC40209(SEQ ID NO: 32) and ZC40213(SEQ ID NO: 33); zcyto 22: ZC40638(SEQ ID NO: 38) and ZC40639(SEQ ID NO: 39). Aliquots of each RNA were also tested against a primer pair specific for MHC class I (Clontech).

Zcyto20 mRNA was detected in LPS and interferon gamma treated THP-1 cells. Zcyto20 mRNA was also detected in CD3+ cells treated with PMA for 4 hours. Low levels of zcyto21 mRNA were detected in resting U937 cells and resting THP-1 cells. The level of zcyto21 mRNA increased when THP-1 cells were treated with LPS and interferon gamma. Zcyto21 mRNA was also detected in CD3+ cells treated with PMA for 4 hours. Zcyto22 mRNA was detected in THP-1 cells treated with LPS and interferon gamma. Zcyto22 mRNA was also detected in CD3+ cells treated with PMA for 4 and 16 hours and in CD3+ cells treated with conavalin a for 4 and 16 hours. These results indicate that activated monocyte cell lines and primary CD3+ immune cells can stimulate the synthesis of zcyto20, zcyto21, and zcyto22 mRNA.

These results indicate that poly I: C will have an effect on zcyto24 and zcyto25, as well as other family members.

Example 13

Antiviral activity: cytopathic effects in Hela and L929 cells

Initial functional assays for antiviral activity were performed using conditioned media from transiently transfected Human Embryonic Kidney (HEK) cells. The conditioned medium was prepared as follows. The full-length cDNA of zcyto20, zcyto21, zcyto22, zcyto24, or zcyto25 was cloned into pzp7Z vector using standard methods. 293HFK cells were transfected with this Zcyto20, Zcyto21, Zcyto22, Zcyto24 or Zcyto25 construct. Briefly, 700,000 cells/well (6 well plate) were seeded in 2ml dmem + 10% fetal calf serum approximately 18 hours prior to transfection for each construct. For each well, a total of 100. mu.l DMEM in 6. mu.l Fugene6 reagent (Roche Biochemicals) was added with 1.5. mu.g Zcyto20, Zcyto21, Zcyto22, Zcyto24 or Zcyto25 DNA and 0.5. mu.g pIRES2-EGFP DNA (Clontech). Mu.g pIRES2-EGFP DNA alone was used as a negative control. These transfection mixtures were added to the pre-seeded 293 cells after 30 minutes. After 24 hours, the cell culture medium was removed and DMEM + 0.1% bovine serum albumin was added. Conditioned media was collected after 48 hours, filtered through a 0.45 micron filter, and then used for antiviral and reporter assays.

Antiviral assays were performed using human cervical cancer cells (HeLa) and mouse fibroblasts (L929). On the first day, conditioned medium containing zcyto20, zcyto21, zcyto22, zcyto24, or zcyto25 was diluted and seeded with 50,000 cells in 96-well flat-bottom microtiter plates. After incubation at 37 ℃ for 24 hours, the medium was removed and replaced with a medium containing encephalomyocarditis virus with a multiplicity of infection of 0.1. The cells were incubated again at 37 ℃ for 24 hours. The presence of cytopathic effects in the cultured cells was then scored by observation on a 4-point scale and then converted to% CPE, as shown in table 8. Conditioned media from cells transfected with GFP and purified human interferon a-2a alone or murine interferon alpha alone were included as controls.

Table 8: determination of cytopathic effects

Sign	Observation of cytopathic Effect (CPE)
			Without CPE
+/-	There may be CPE (about 1% single layer surface)
		+	CEP confined to one plaque (approximately 5% surface)
+1	CEP localized in three plaques affecting less than 25% of the monolayer
		1	25％CPE
1-2	37％CPE
		2	50％CPE
2-3	62％CPE
		33	75％CPE
3-4	87％CPE
		4	100％CPE

Table 9 shows that conditioned media containing Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 inhibited infection of HeLa cells (% CPE) by the virus in a dose-dependent manner, while control GFP conditioned media did not significantly block the appearance of cytopathic effects. As shown in Table 10, the conditioned media containing Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 did not inhibit infection of L929 cells by the virus. Purified interferon showed positive antiviral activity in both experiments.

Table 9: percentage cytopathic effect of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 in HeLa cells using Conditioned Medium (CM)

Table 10: percentage cytopathic effect of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 in L929 cells using Conditioned Medium (CM)

As a further study, conditioned media from Sf9 cells infected with baculoviruses expressing Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 were used for antiviral assays. Conditioned medium from wild-type baculovirus-infected Sf9 cells was used as a control.

The results of antiviral assays using conditioned medium from baculovirus were similar to those using conditioned medium of transiently transfected 293. Table 11 shows that baculovirus-derived conditioned medium containing zcyto21 inhibited infection of HeLa cells by the virus in a dose-dependent manner, whereas control baculovirus-conditioned medium did not block the appearance of cytopathic effects.

Table 11: percentage cytopathic Effect in HeLa cells Using baculovirus-derived zcyto21 Conditioned Medium (CM)

Baculovirus constructs and conditioned media were prepared as described above.

Example 14

Anti-human interferon alpha receptor 2 beta chain antibodies fail to block antiviral activity

Additional antiviral assays were performed using human cervical cancer cells (HeLa). On the first day, anti-human IFN receptor mabs (Research Diagnostics Inc.) and isotype-matched negative control mabs were diluted in 96-well flat-bottom microtiter plates. Conditioned medium from baculovirus-infected Sf9 cells expressing zcyto20, zcyto21, or zcyto22 was added to give a final concentration of 0.0625 xcm, and then 50,000HeLa cells were seeded per well. After incubation at 37 ℃ for 24 hours, the medium was removed and replaced with a medium containing encephalomyocarditis virus with a multiplicity of infection of 0.1. The cells were incubated for a further 24 hours at 37 ℃. The cultured cells were then scored by observation for the presence of cytopathic effect (CPE) as shown in table 8. Purified human interferon-a-2 a at a concentration of 0.01ng/ml was included as a positive control.

Table 12 shows that conditioned media containing zcyto20, zcyto21, and zcyto22 all have antiviral activity in HeLa cells (as demonstrated by 0% CPE) in the presence or absence of neutralizing antibodies against the 2 β chain of human interferon alpha receptor. In contrast, in the presence of neutralizing antibodies against the 2 β chain of the human interferon alpha receptor, the antiviral activity of interferon-a-2 a is specifically inhibited in a dose-dependent manner. These data indicate that zcyto20, zcyto21 and zcyto22 interact with receptors other than human interferon alpha receptor, or that it interacts with human interferon alpha receptor in a mechanism different from the interaction of human interferon-a-2 a with human interferon alpha receptor.

Table 12: percentage of cytopathic effect in HeLa cells using zcyto20, zcyto21, or zcyto22 Conditioned Medium (CM) and neutralizing antibodies against the 2 beta chain of human interferon alpha receptor

Example 15

Antiproliferative assays using the BAF3 cell line

BaF3 was used to determine whether zcyto20 has anti-proliferative properties. Baby Hamster Kidney (BHK) cells were stably transfected with BRL lipofectamine using an expression vector containing the CMV promoter and intron a upstream of the zcyto20cDNA or an unrelated cDNA called Z α 30. Stably transfected cells were seeded in serum-free medium in cell factories, conditioned medium was harvested after 3 days of culture, and then concentrated 10-fold in 5K filters. Concentrated conditioned medium samples were stored at 4 ℃.

Antiproliferative activity was tested on BaF3 using the following assay. On a 96-well plate, 8 1:2 serial dilutions in a final volume of 100. mu.l were made in growth medium alone (RPMI 1640, 10% fetal bovine serum, 1mM sodium pyruvate, 2mM L-glutamine) or murine IL-3 (initial 50pg/ml in growth medium). 50 microliters of the following substances were added to the lane of simple growth medium or mIL-3 dilution: human interferon alpha (100ng/ml, 10ng/ml or 1ng/ml diluted in growth medium), human interferon beta (100ng/ml, 10ng/ml or 1ng/ml diluted in growth medium), murine interferon alpha (100ng/ml, 10ng/ml or 1ng/ml diluted in growth medium), murine interferon beta (100ng/ml, 10ng/ml or 1ng/ml diluted in growth medium), zcyto20(2.5 x, 0.5 x or 0.1 x) and murine Z alpha 20(2.5 x, 0.5 x or 0.1 x).

The BaF3 cell line was washed three times in growth medium, the pellet was resuspended in growth medium, the cells were counted and diluted in growth medium to 5,000 cells/50 μ l. Then 50. mu.l of the diluted cells were added to each sample dilution. The test plates were incubated in an incubator at 37 ℃ for 3 to 4 days. Then 20. mu.l of Alomar blue was added to each well and the plates were incubated overnight at 37 ℃. The plates were read on a fluorescence plate reader at 544 excitation and 590 emission wavelengths.

Example 16

Signaling through interferon response pathways

The interaction of type 1 interferons with their specific receptors will induce a number of genes responsible for antiviral/antiproliferative activity. These genes include 2 '-5' oligoadenylate synthetase (2-5A synthetase), double-stranded RNA-dependent Pkr kinase (Pkr), phospholipid scramblase, and intercellular adhesion molecule-1 (ICAM-1). Genes of unknown function at present, such as the interferon-stimulated 56kDa gene product (ISG-56k), were also induced. To determine whether treatment of cells with zcyto20 induced some or all of the genes, human Daudi B lymphoid cells were treated for 72 hours with conditioned media from baculovirus-infected Sf9 cells expressing zcyto 20. Conditioned medium from wild-type baculovirus-infected Sf9 cells was used as a negative control. After treatment, cells were collected and lysed to isolate total RNA. Mu.g of total RNA was converted to cDNA using reverse transcriptase, and this was used as template and polymerase chain reaction was performed using oligonucleotide primers specific for the human interferon-stimulated gene described above. Oligonucleotide primers directed against human glycerol-3-phosphate dehydrogenase (G3PDH) were used as non-interferon stimulated gene control. The results show that treatment of cells with zcyto20 induced significantly ISG-56k, Pkr, 2-5A synthetase and phospholipid scramblase. No induction of ICAM-1 or non-interferon stimulated gene control G3PDH was observed.

Example 17: identification of zcytor19 as the zcyto21 receptor

A: COS cell transfection and secretion trapping

Biotinylated zcyto21 was tested for binding to known or orphan cytokine receptors. COS cells were transfected with pZP7 expression vectors containing cdnas for cytokine receptors (including human IFN α R1, IFN β R2, IFN α R1, IFN β R2, IL-10R, CRF2-4, zcytoR7, DIRS1, zcytoR19, and tissue factor) and tested for binding of biotinylated zcyto20 to transfected COS cells using the secretion trapping assay described below. Positive binding in this assay indicates a receptor-ligand pair.

Transfection of COS cells

COS cells were transfected as follows: COS cells were seeded (1X 10)⁵Individual cells/well) were incubated overnight at 37 ℃ on fibronectin-coated 12-well tissue culture plates (Becton Dickinson, Bedford, MA). Cytokine receptor DNA (0.75. mu.g) was mixed with 50. mu.l serum-free DMEM medium (55 mg sodium pyruvate, 146mg L-glutamine, 5mg transferrin, 2.5mg insulin, 1. mu.g selenium and 5mg fetuin in 500ml DMEM) and then with 5. mu.l Lipofectamine in 45. mu.l serum-free DMEM medium^TM(Invitrogen, Carlsbad, Calif.) and incubated for 30 minutes at room temperature. Then 400. mu.l of serum-free DMEM medium was added. Cells were washed with serum-free DMEM and 500. mu.l of DNA mixture was added. Cells were incubated at 37 ℃ for 5 hours, during which time 500. mu.l of 20% FBS DMEM medium (100 ml FBS in 500ml DMEM, 55mg sodium pyruvate and 146mg L-glutamine) was added, and the cells were incubated overnight.

Secretion trapping test

Secretory capture was performed as follows: the medium was aspirated and the cells were washed with 1% BSA in PBS. Cells were blocked with TNB in water (0.1M Tris-HCl, 0.15M NaCl and 0.5% blocking agent (NENRenaissance TSA-Direct kit, NEN Life Science Products, Boston, Mass.). for 1 hour. Cells were incubated with 3. mu.g/ml of biotinylated zcyto21 protein in TNB (example 27) for 1 hour. Cells were then washed 3 times with 1% BSA in PBS and then incubated with streptavidin-HRP (NEN kit) diluted 1:300 in TNB for an additional 1 hour. Cells were washed again 3 times with 1% BSA in PBS and then fixed with 1.8% formaldehyde in PBS for 15 minutes. The cells were then washed 3 times with TNT (0.1M Tris-HCl, 0.15M NaCl and 0.05% Tween-20 in water).

Positive binding was detected with fluorescein tyramide reagent diluted 1: 50 in dilution buffer (NEN kit), incubated for 4.5 minutes and washed with TNT. Cells were preserved with Vectashield Mounting medium (Vector Labs Burlingame, CA) diluted 1:5 in TNT. Cells were observed under a fluorescent microscope using a FITC filter.

Positive binding was detected on cells transfected with human zcytor19cDNA and incubated with biotinylated zcyto 21. None of the other transfected receptors bound zcyto21, and zcytor19 did not bind to the control biotinylated protein. These data indicate that zcytor19 is the receptor for zcyto 21.

Other experiments have shown that both human and murine zcytor19 bind positively to biotinylated zcyto 21. Positive binding was also detected on cells transfected with human zcytor19cDNA and incubated with biotinylated zcyto20 and zcyto 24.

Example 18

Signal transduction reporter assay

The functional interaction of zcyto20, zcyto21, zcyto22, zcyto24 and zcyto25 with zcytor19 can be determined using signal transduction reporter assays. Human Embryonic Kidney (HEK) cells were transfected with a reporter plasmid containing an Interferon Stimulated Response Element (ISRE) that drives transcription of a luciferase reporter gene in the presence or absence of a pZP7 expression vector containing cDNA for class II cytokine receptors including human DIRS1, IFN α R1, IFN α R2, and zcytor19(SEQ id nos: 23 and 26). Luciferase activity following stimulation of transfected cells with class II ligands (including zcyto20(SEQ ID NO: 1), zcyto21(SEQ ID NO: 4), zcyto22(SEQ ID NO: 6), zcyto10, human IL10 and human IFNa-2a) reflects ligand interaction with transfected and native cytokine receptors on the cell surface. The results and methods are as follows.

Cell transfection

293HEK cells were transfected as follows: approximately 18 hours prior to transfection, 700,00 293 cells/well (6 well plate) were seeded in 2ml DMEM + 10% fetal calf serum. For each well, 1. mu.g pISRE-luciferase DNA (Stratagene), 1. mu.g cytokine receptor DNA, and 1. mu.g pIRES2-EGFP DNA (Clontech) were added to a total of 9. mu.l Fugene 6 reagent (Roche Biochemicals) in 100. mu.l DMEM. When cytokine receptor DNA was excluded, 2. mu.g of pIRES2-EGFP DNA was used. After 30 minutes the transfection mixture was added to pre-seeded 293 cells. After 24 hours, the transfected cells were detached from the plate using trypsin-EDTA and reseeded in 96 well microtiter plates at approximately 25,000 cells/well. Approximately 18 hours prior to ligand stimulation, the medium was changed to DMEM + 0.5% FBS.

Signal transduction reporter assay

The signal transduction reporter assay was performed as follows: after incubation in DMEM + 0.5% FBS at 37 ℃ for 18 hours, transfected cells were stimulated with dilutions of the following class II ligands (in DMEM + 0.5% FBS): zcyto20, zcyto21, zcyto22, zcyto10, human IL10(huIL10) and human IFN-a-2a (huIFNa-2 a). After 4 hours incubation at 37 ℃, cells were lysed and Relative Light Units (RLU) were measured on a luminometer after addition of luciferase substrate. The results obtained are shown as the fold induction of RLU in the experimental samples relative to RLU in the medium alone control (RLU in experimental samples/RLU in medium alone ═ fold induction). Table 14 shows that zcyto20, zcyto21, and zcyto22 induced ISRE signaling in ISRE-luciferase transfected 293 cells, resulting in 15-17 fold induction of luciferase activity compared to medium alone. The addition of zcytor19DNA to the transfection mixture resulted in an additional 6-8 fold increase in zcyto20, zcyto21, and zcyto22 induced ISRE signaling, resulting in a total of 104 to 125 fold induction. All other transfected class II cytokine receptor DNA did not result in increased ISRE signaling. These results suggest that zcyto20, zcyto21, and zcyto22 functionally interact with the zcytor19 cytokine receptor. Table 13 also shows that human IFNa-2a can induce ISRE signaling in ISRE-luciferase transfected 293 cells, resulting in 205-fold induction of luciferase activity compared to medium alone. However, addition of zcytor19DNA to the transfectants resulted in an 11-fold decrease in ISRE signaling (compared to ISRE-luciferase DNA alone), suggesting that overexpression of zcytor19 negatively affected interferon signaling, and conversely, overexpression of zcytor19 positively affected zcyto20, zcyto21, and zcyto22 signaling.

Table 13: interferon Stimulation Response Element (ISRE) signalling (fold induction) of transfected 293 cells following class II cytokine stimulation

Ligands	ISRE-luciferases	ISRE-luciferase/Zcytor 19
			Zcyto20(125ng/ml)	15	125
Zcyto21(125ng/ml)	17	108
			Zcyto22(125ng/ml)	17	104
HuIFNa-2a(100ng/ml)	205	18
			Zcyto10(125ng/ml)	1.3	1
HuIL10(100ng/ml)	1	0.5

Example 19: identification of IL10Rb (CRF2-4) as a receptor subunit of zcytor19

A: IL10Rb neutralizing antibodies inhibit ISRE signaling

The functional interactions of zcyto20, zcyto21 and zcyto22 with zcytor19 and IL10Rb (CRF2-4) were determined using signal transduction reporter experiments. Human Embryonic Kidney (HEK) cells or Human Embryonic Kidney (HEK) cells stably overexpressing human zcytor19 are transfected with a reporter plasmid containing an interferon-stimulated response element (ISRE) that will drive transcription of a luciferase reporter. Stimulation of luciferase activity following transfection of cells with class II ligands (including zcyto20, zcyto21, zcyto22 and human IFNa-2a) in the presence or absence of neutralizing antibodies to IL10Rb (CRF2-4) reflects ligand interaction with cytokine receptors on the cell surface. The results and methods are as follows.

Cell transfection:

to prepare 293HEK cells stably overexpressing human zcytor19, 293 cells were transfected as follows: 300,000 293 cells/well (6-well plate) were seeded in 2ml DMEM + 10% fetal bovine serum approximately 6 hours prior to transfection. For each well, 2. mu.g of pZP7 expression vector containing cDNA of human zcytor19 (SEQ ID NO: 23) was added to 6. mu.l Fugene 6 reagent (Roche Biochemicals) in a total of 100. mu.l DMEM. After 30 minutes the transfection mixture was added to pre-seeded 293 cells. After 48 hours, the transfected cells were placed under 2. mu.g/ml puromycin selection. Puromycin resistant cells are expressed as a population of cells.

293HEK cells (wild type or overexpressing human zcytor19) were transfected as follows: 700,000 293 cells/well (6-well plate) were seeded in 2ml DMEM + 10% fetal calf serum approximately 18 hours prior to transfection. For each well, 1. mu.g of pISRE-luciferase DNA (Stratagene) and 1. mu.g of pIRES2-EGFP DNA (Clontech) were added to 6. mu.l of Fugene 6 reagent (Roche Biochemicals) in a total of 100. mu.l DMEM. After 30 minutes the transfection mixture was added to pre-seeded 293 cells. After 24 hours the transfected cells were detached from the plate using trypsin-EDTA and reseeded in 96 well microtiter plates at approximately 25,000 cells/well. Approximately 18 hours prior to ligand stimulation, the medium was changed to DMEM + 0.5% FBS.

Signal transduction reporter assay

The signal transduction reporter assay was performed as follows: after incubation in DMEM + 0.5% FBS at 37 ℃ for 18 hours, the transfected cells were pretreated with neutralizing polyclonal goat antibody to IL10Rb (2.5. mu.g/ml for zcytor 21; 8. mu.g/ml for zcyto20 and zcyto22, R & D Systems) or PBS37 ℃ for 1 hour. For experiments involving zcyto20 and zcyto22, Human Embryonic Kidney (HEK) cells stably overexpressing human zcyto R19 were also pre-treated with non-neutralizing polyclonal goat antibodies to IFNAR1 (8 μ g/ml, R & D Systems) as antibody controls. Pre-treated cells were stimulated with dilutions of the following class II ligands (in DMEM + 0.5% FBS): zcyto20, zcyto21, or zcyto 22. Human IFNa-2a was used as a control in each experiment. After 4 hours incubation at 37 ℃, cells were lysed and Relative Light Units (RLU) were measured on a luminometer after addition of luciferase substrate. The results obtained are shown as fold induction of RLU in the experimental samples compared to the medium-only control (RLU in experimental sample/RLU in medium-only).

Tables 14 and 15 show that pre-treatment of wild type 293 cells or 293 cells overexpressing human zcytoR19 with neutralizing antibodies to IL10Rb can inhibit the induction of ISRE signaling by zcyto 20. Little or no inhibition of human IFNa-2 a-induced ISRE signaling was observed. These results indicate that, for maximum induction of ISRE signaling, zcyto20 needs to interact with IL10Rb (CRF2-4) and that the receptor for zcyto20 is a heterodimeric combination of zcytoR19 and IL10Rb (CFR 2-4).

Table 14: IL10Rb inhibition (fold induction) of ISRE signaling in transfected wild type 293 cells following stimulation with class II cytokines

Concentration of cytokine (ng/ml)	Zcyto20	Zcyto20+ IL10Rb neutralizing antibodies	HuIFNa-2a	Huifna-2a + IL10Rb neutralizing antibody
					100	8.4	0.8	152	102
10	4	0.9	160	117
					1	1	0.9	90	69
0.1	1	1	12	6
					0.01	1	0.8	1.2	1
0	1	1	1	1

Table 15: IL10Rb inhibition (fold induction) of ISRE signaling in transfected 293 cells overexpressing zcytoR19 following stimulation with class II cytokines

Concentration of cytokine (ng/ml)	Zcytoo20	Alizing in Zcyto20+ IL10Rb	HuIFNa-2a	Huifna-2a + IL10Rb neutralizing antibody
					100	91	60	16	16
10	97	23	14	15
					1	68	1.3	8	8.4
0.1	6	1.1	1.5	1.9
					0.01	1.1	1.2	1.2	1.3
0	1	1	1	1

Tables 16 and 17 show that pre-treatment of wild type 293 cells or 293 cells overexpressing human zcytoR19 with neutralizing antibodies to IL10Rb can inhibit zcyto 21-induced ISRE signaling. No inhibition was observed for human IFNa-2 a-induced ISRE signaling. These results suggest that, for maximum induction of ISRE signaling, zcyto21 needs to interact with IL10Rb (CRF2-4) and that the receptor for zcyto21 is a heterodimeric combination of zcytoR19 and IL10Rb (CRF 2-4).

Table 16: IL10Rb inhibition (fold induction) of ISRE signaling in transfected wild type 293 cells following stimulation with class II cytokines

Concentration of cytokine (ng/ml)	Zcyto21	Zcyto21+ IL10Rb neutralizing antibodies	HuIFNa-2a	Huifna-2a + IL10Rb neutralizing antibody
					100	4.1	1.8	31	30
10	3.2	1.4	32	31
					1	1.5	1.3	16.3	15
0.1	1.1	1.3	1.4	2
					0.01	1.2	1.3	1.1	1.2
0.001	1.2	1.3	0.9	2.1
					0	1	1	1	1

Table 17: IL10Rb inhibition (fold induction) of ISRE signaling in transfected 293 cells overexpressing zcytoR19 following stimulation with class II cytokines

Concentration of cytokine (ng/ml)	Zcyto21	Zcyto21+ IL10Rb neutralizing antibodies	HuIFNa-2a	Huifna-2a + IL10Rb neutralizing antibody
					100	45	31	9	7.7
10	48	28	9	8.5
					1	35	5.8	4.3	4.3
0.1	3.5	1	1.4	1.3
					0.01	1.5	1.1	0.9	1.2
0.001	1.1	1	1.2	1
					0	1	1	1	1

Tables 18 and 19 show that pre-treatment of wild type 293 cells or 293 cells overexpressing human zcytoR19 with neutralizing antibodies to IL10Rb can inhibit zcyto 22-induced ISRE signaling. For human IFNa-2 a-induced ISRE signaling, no or little inhibition was observed. These results suggest that, for maximum induction of ISRE signaling, zcyto22 needs to interact with IL10Rb (CRF2-4) and that the receptor for zcyto21 is a heterodimeric combination of zcytoR19 and IL10Rb (CRF 2-4).

Table 18: IL10Rb inhibition (fold induction) of ISRE signaling in transfected wild type 293 cells following stimulation with class II cytokines

Concentration of cytokine (ng/ml)	Zcyto22	Zcyto22+ IL10Rb neutralizing antibodies	HuIFNa-2a	Huifna-2a + IL10Rb neutralizing antibody
					100	11	1.2	152	102
10	8	1	160	117
					1	1.8	0.8	90	69
0.1	1.2	0.8	12	6
					0.01	0.9	0.9	1.2	1
0	1	1	1	1

Table 19: IL10Rb inhibition (fold induction) of ISRE signaling in transfected 293 cells overexpressing zcytoR19 following stimulation with class II cytokines

Concentration of cytokine (ng/ml)	Zcyto22	Zcyto22+ IL10Rb neutralizing antibodies	HuIFNa-2a	Huifna-2a + IL10Rb neutralizing antibody
					100	82	76	16	16
10	97	39	14	15
					1	69	2.3	8	84
0.1	8.4	1.1	1.5	1.9
					0.01	1	1.3	1.2	1.3
0	1	1	1	1

B: anti-IL 10Rb antibodies block antiviral activity

An antiviral assay was performed to determine the ability of anti-IL 10Rb antibodies to block the antiviral activity of zcyto 20. The assay was performed using 293HEK cells (wild type or overexpressing human zcytoR 19). On the first day, antibodies (anti-human IL10R β, anti-human Leptin receptor, R & DSystems) were diluted at 5 μ g/ml in cell culture medium and then seeded in 96-well plates with 50,000 cells/well. After 1 hour incubation at 37 ℃, zcyto20-CEE (from example 3) (200ng/ml for wild type 293 cells, 0.5ng/ml for 293 cells overexpressing human zcytoR19) or human interferon a-2a (1ng/ml for wild type 293 cells, 100ng/ml for 293 cells overexpressing human zcytoR19) was added to the wells and incubated overnight at 37 ℃. The following day, the medium was removed and replaced with a medium containing encephalomyocarditis virus (EMCV) with a multiplicity of infection of 0.1. Cells were then incubated overnight at 37 ℃. Subsequently, 25. mu.l of 5mg/ml Methylthiazoltetrazolium (MTT) (Sigma) was added to each well, incubated at 37 ℃ for 2 hours, and then each well was extracted with 100. mu.l of extraction buffer (12.5% SDS, 45% DMF). After overnight incubation at 37 ℃, the optical density of 570nM was measured on a Spectromax plate reader (Molecular Devices, CA). A decrease in optical density (570nm) indicates a decrease in cell survival (loss of antiviral activity). The optical density (570nm) for the different test conditions is shown in Table 20 below. The results show that blocking human IL10 receptor beta specifically inhibits the antiviral activity of zcyto20, but does not affect the activity of interferon-a-2 a. This suggests that human IL10 receptor beta is part of a receptor complex (including human zcytor19) involved in the antiviral activity of zcyto 20.

Table 20: optical Density (570nm) of ECMV infected cytokine-treated cells

Cytokine	Wild type 293 cells: anti-IL 10Rb	Wild type 293 cells: anti-leptin R	293 cells overexpressing human zcytor 19: anti-IL 10Rb	293 cells overexpressing human zcytor 19: anti-leptin R
					zcyto20-CEE	.94	.88	.95	.24
Human IFNa-2a	.58	.4	.18	.05

C: co-expression of zcytoR19 and IL10Rb increased zcyto20, zcyto21 and signal transduction of zcyto22

The functional interactions of zcyto20, zcyto21 and zcyto22 with zcytor19 and IL10Rb (CRF2-4) were determined using a signal transduction reporter assay. Hamster Kidney (BHK) cells were transfected with a reporter plasmid containing an Interferon Stimulus Response Element (ISRE) that would drive transcription of a luciferase reporter gene, with or without a pZP7 expression vector containing the cDNA of the class II cytokine receptors zcytor19 and IL10Rb (CRF 2-4). The luciferase activity following stimulation of transfected cells with class II ligands (including zcyto20, zcyto21 and zcyto22) reflects the interaction of the ligand with transfected and native cytokine receptors on the cell surface. The results and methods are as follows.

Cell transfection:

BHK-570 cells were transfected as follows: 200,000 BHK cells/well (6-well plate) were seeded approximately 5 hours prior to transfection in 2ml DMEM + 5% fetal calf serum. For each well, 1. mu.g of pISRE-luciferase DNA (Stratagene), 1. mu.g of cytokine receptor DNA and 1. mu.g of pIRES2-EGFP DNA (Clontech) were added to 9. mu.l of Fugene6 reagent (Roche Biochemicals) in a total of 100. mu.l DMEM. When cytokine receptor DNA was not included, 2. mu.g of pIRES2-EGFP DNA was used. The transfection mixture was added to the pre-seeded BHK cells after 30 minutes. After 24 hours the transfected cells were detached from the plate using trypsin-EDTA and reseeded in 96 well microtiter plates at approximately 25,000 cells/well. Approximately 18 hours prior to ligand stimulation, the medium was changed to DMEM + 0.5% FBS.

Signal transduction reporter assay

The signal transduction reporter assay was performed as follows: after incubation in DMEM + 0.5% FBS at 37 ℃ for 18 hours, transfected cells were stimulated with dilutions of zcyto20, zcyto21, zcyto22, zcyto24 or zcyto25 ligand (in DMEM + 0.5% FBS). After 4 hours incubation at 37 ℃, cells were lysed and Relative Light Units (RLU) were measured on a luminometer after addition of luciferase substrate. The results obtained are shown as fold induction of RLU in the experimental samples compared to the medium-only control (RLU in experimental sample/RLU in medium-only). Table 21 shows that zcyto20, zcyto21, and zcyto22 induced ISRE signaling in a dose-dependent manner in ISRE-luciferase and zcytor19 transfected BHK cells. Addition of IL10Rb (CRF2-4) DNA to the transfection mixture resulted in half the maximal induction of signal transduction at a 10-100 fold lower cytokine dose. No reaction was observed when only ISRE transfection was used. These results suggest that the ability of zcyto20, zcyto21, and zcyto22 to signal response elements through interferon stimulation may be potentiated by the co-expression of zcyto R19 and IL10Rb (CRF2-4), suggesting that the receptors for zcyto20, zcyto21, and zcyto22 are heterodimer combinations of zcyto R19 and IL10Rb (CRF 2-4).

Table 21: interferon Stimulated Response Element (ISRE) signalling (fold induction) of transfected BHK cells following class II cytokine stimulation

Class II ligand concentration (ng/ml)	zcyto 20/simple zcytor19 transfected cells	zcyto20/zcytor19 and IL10Rb (CRF2-4) transfected cells	zcyto 21/simple zcytor19 transfected cells	zcyto21/zcytor19 and IL10Rb (CRF2-4) transfected cells	zcyto 22/simple zcytor19 transfected cells	zcyto22/zcytor19 and IL10Rb (CRF2-4) transfected cells
							1000	2.25	2.1	3.3	2.2	1.8	2.2
100	2.2	2.6	2.6	2.5	2	2.2
							10	2.1	2.4	2.4	2.6	1.9	2.7
1	1.3	2.5	2	2.5	1.5	2.7
							0.1	1.25	2.1	1.4	2.2	1.1	2.4
0.01	1.2	1.6	1.4	1.6	1.2	1.7
							0.001	1.4	1.5	1.3	1.3	1.2	1.3
0	1	1	1	1	1	1

Example 20

Construction of soluble receptor expressing zcytor 19: mammalian expression vectors for zcytor19CEE, zcytor19CFLG, zcytor19CHIS and zcytor19-Fc4

For expression of the soluble extracellular domain of the zcytor19 polypeptide, an expression vector pC4zcytor19CEE was prepared, wherein the construct was designed to express a zcytor19 polypeptide containing a predicted initiation methionine and truncated near the predicted transmembrane domain with a C-terminal Glu-Glu tag (SEQ ID NO: 16).

A zcytor19DNA fragment comprising the extracellular domain of zcytor19 or the cytokine binding domain of zcytor19 described herein was constructed using PCR and purified using standard methods. The excised DNA was subcloned into a plasmid expression vector having a signal peptide (e.g., the native zcytor19 signal peptide) and a Glu-Glu tag (SEQ ID NO: 16) was added to the coding nucleotide sequence at the C-terminus of the zcytor19 polypeptide. The mammalian expression vector contains an expression cassette with a mammalian promoter, a multiple cloning site for insertion of a coding sequence, a stop codon, and a mammalian terminator. The plasmid may also have an origin of replication of E.coli, a mammalian selectable marker expression unit (with SV40 promoter, enhancer and origin of replication, DHRF gene and SV40 terminator).

The restriction digested zcytor19 insert was ligated to the pre-digested vector using standard molecular biology techniques, then electroporated into competent cells such as DH10B competent cells (GIBCO BRL, Gaithersburg, Md.) according to the manufacturer's instructions, followed by plating on LB plates containing 50mg/ml ampicillin and incubating overnight. Colonies were screened by restriction analysis of DNA prepared from each colony. The insert sequence of the positive clone was verified by sequence analysis. Use ofMass production kits (Qiagen) plasmids were prepared on a large scale according to the manufacturer's instructions.

The same procedure was used to prepare a zcytor19 soluble receptor with a C-terminal His tag (consisting of a row of 6 His residues); and with C-terminal FLAGZcytor19CFLAG of the tag (SEQ ID NO: 42). To construct these constructs, the glu-glu tag (SEQ ID NO: 16) in the aforementioned vector was replaced with C-HIS or FLAGAnd (4) a label.

The expression vector zcytor19/Fc4/pzmp20 was prepared to express the soluble zcytor19 version (human zcytor19-Fc4) with an Fc4 tag at the C-terminus in BHK cells. A zcytor19cDNA fragment comprising a polynucleotide sequence of the extracellular domain of the zcytotr 19 receptor is fused in-frame with an Fc4 polynucleotide sequence (SEQ ID NO: 43) to produce a zcytor19-Fc4 fusion. The pzmp20 vector is a mammalian expression vector containing the Fc4 polynucleotide sequence and a cloning site that allows for the rapid construction of a C-terminal Fc4 fusion using standard molecular biology techniques.

A630 base pair fragment containing the extracellular domain of human zctor19 and encoding BamHI and Bgl2 sites at the 5 'and 3' ends, respectively, was generated by PCR. The PCR fragment was prepared by amplifying a human brain cDNA library using primers ZC37967(SEQ ID NO: 44) and ZC37972(SEQ ID NO: 45). The PCR reaction conditions were as follows: 30 cycles at 94 ℃ for 20 seconds and 68 ℃ for 2 minutes; 1 cycle at 68 ℃ for 4 minutes; then the temperature is kept at 10 ℃. The fragment was digested with BamHI and Bgl2 restriction enzymes, followed by purification by 1% gel electrophoresis and band purification using QiaQuick gel extraction kit (Qiagen). The resulting purified DNA was ligated to pzmp20 vector (which contained Fc4 3' to Bgl2 site) previously digested with Bgl2 for 5 hours at room temperature.

Electroporation was carried out in 37. mu.l of DH10B electrocompetent E.coli (Gibco) with 1. mu.l of ligation mixture according to the manufacturer's instructions. The transformed cells were diluted in 400. mu.l LB medium and plated on LB plates containing l 00. mu.g/ml ampicillin. Clones were analyzed by restriction digestion, and positive clones were DNA sequenced to verify the sequence of the fusion construct.

Example 21

Mammals express the human zcytor19 soluble receptor: zcytor19/Fc4

BHK570 cells (ATCC NO: CRL-10314) were seeded in T-75 tissue culture flasks at 37 ℃ and 5% CO₂Growth was performed down to approximately 50% to 70% confluence in DMEM/FBS medium (DMEM, Giboco/BRL high glucose (Gibco BRL, Gaithersburg, Md.), 5% fetal bovine serum, 1mM L-glutamine (JRH Biosciences, Lenea, KS), 1mM sodium pyruvate (Gibco BRL)). The plasmids zcytor19/Fc4/pzmp20 (example 4B) and Lipofectamine were then used^TM(Gibco BRL), cells were transfected in serum-free (SF) medium preparation (DMEM, 10mg/ml transferrin, 5mg/ml insulin, 2mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate). Mu.g of plasmid DNAzcytor19/Fc4/pmzp20 (example 4B) were diluted in SF medium in 15ml tubes to a final volume of 500. mu.l. 50 μ l Lipofectamine was mixed with 450 μ l SF medium. The Lipofectamine mixture was added to the DNA mixture, followed by incubation at room temperature for about 30 minutes. 4ml of SF medium was added to the DNA: lipofectamine mixture. Cells were washed once with 5ml SF medium, aspirated and the DNA: lipofectamine mixture. Incubation at 37 deg.CCells were incubated for 5 hours, then 5ml DMEM/10% FBS medium was added. The shake flasks were incubated overnight at 37 ℃ before separating the cells and adding 1:2, 1:10 and 1:50 to 150mm plates of selection medium (DMEM/FBS medium as above plus 1. mu.M methotrexate (Sigma Chemical Co. St. Louis, Mo.). Approximately 10 days after transfection, 1 μ M methotrexate resistant colonies in 1 150mm plates were trypsinized, the cells were confluent, and half of them were re-seeded with 10 μ M methotrexate; so as to further broaden the expression of the zcytor19/Fc4 protein. Samples of conditioned medium from the confluent, expanded cells were tested using SDS-PAGE and Western analysis to determine expression levels.

Individual clones expressing the soluble receptor can also be isolated, screened, and cultured in cell culture media using standard techniques, and then purified. Furthermore, CHO cells are also suitable cells for these purposes.

Example 22

Evaluation of zcytor19 receptor heterodimerization Using the ORIGEN assay

The soluble zcytor19 receptor was biotinylated by reaction with a 5-fold molar excess of Sulfo-NHS-LC-biotin (Pierce, Inc., Rockford, IL) according to the manufacturer's protocol. The soluble zcytor19 receptor and another soluble receptor subunit (e.g., a soluble class II cytokine receptor such as CRF2-4(SEQ ID NO: 40)) were labeled with a 5-fold molar excess of Ru-BPY-NHS (Igen, Inc., Gaithersburg, Md.) according to the manufacturer's protocol. The soluble zcytor19 receptor in biotinylated and Ru-BPY-NHS-labeled forms may be designated Bio-zcytor19 receptor and Ru-zcytor19, respectively; this biotinylated form and the Ru-BPY-NHS labeled form of the other soluble receptor subunit can be similarly named. Assays were performed using conditioned media or using purified ligands.

For initial characterization of soluble receptor binding, the above cytokines or conditioned media were tested to determine whether they could mediate homodimerization of the zcytor19 receptor and whether they could mediate heterodimerization of zcytor19 with the above soluble receptor subunit. To this end, 50. mu.l of conditioned medium or TBS-B containing purified cytokines are mixed with 50. mu.l of TBS-B (20mM Tris, 150mM NaCl, 1mg/ml BSA, pH7.2) containing, for example, 400ng/ml Ru-zcytor19 receptor and Bio-zcytor19, or 400ng/ml Ru-zcytor19 and, for example, Bio-CRF2-4, or 400ng/ml Ru-CRF-4 and Bio-zcytor 19. After 1 hour incubation at room temperature, 30 μ g streptavidin-coated 2.8mm magnetic beads (Dynal, Inc., Oslo, Norway) were added and the reaction was incubated for an additional 1 hour at room temperature. Then 200 μ l of ORIGEN assay buffer (Igen, Inc. gaithersburg, MD) was added and the extent of receptor binding was measured using an M8 ORIGEN analyzer (Igen, Inc.).

Example 23

Constructs for making zcytor19 receptor heterodimers

Vectors expressing secreted human zcytor19 heterodimers are constructed by fusing the extracellular cytokine binding domain of zcytor19 to the IgG γ 1 heavy chain and fusing the extracellular portion of a heteromeric (heteromeric) cytokine receptor subunit (e.g., a class II cytokine receptor such as CRF2-4) to the human kappa light chain.

a. Construction of IgG gamma 1 and human kappa light chain fusion vectors

The IgG γ 1 heavy chain was cloned into Zem229R mammalian expression vector (ATCC accession No. 69447) so that any desired cytokine receptor extracellular domain having 5 'EcoRI and 3' NheI sites can be cloned therein, resulting in the formation of an N-terminal extracellular domain-C-terminal IgG γ 1 fusion. The IgG γ 1 fragment used in this construct was prepared by isolating IgG γ 1 sequences from Clontech human fetal liver cDNA library as a template by PCR. The PCR product was purified using the methods described herein and digested with MluI and EcoRI (Boehringer-Mannheim), ethanol precipitated, and ligated into Zem229R, previously digested with EcoRI, along with oligomers containing the MluI/EcoRI linker using standard molecular biology techniques disclosed herein.

The human kappa light chain was cloned into Zem228R mammalian expression vector (ATCC accession No. 69446) so that any desired cytokine receptor ectodomain having 5 'EcoRI and 3' KpnI sites could be cloned therein, resulting in the formation of an N-terminal cytokine ectodomain-C-terminal human kappa light chain fusion. Since there is one KpnI site located in the human kappa light chain sequence, specific primers were designed to clone the 3' end of the desired cytokine receptor extracellular domain into this KpnI site: the primers were designed so that the resulting PCR product contained the desired cytokine extracellular domain and a stretch of human kappa light chain preceding the KpnI site. The primer preferably comprises a portion of at least 10 nucleotides at the 3 'end of the extracellular domain of the desired cytokine fused in frame to the 5' end of the human kappa light chain. The human kappa light chain fragment used in this construct was prepared by isolating the human kappa light chain sequence by PCR from the same Clontech human fetal liver cDNA library used above. The PCR product was purified using the methods described herein and digested with MluI and EcoRI (Boehringer-Mannheim), ethanol precipitated, and ligated with MluI/EcoRI linker into Zem228R previously digested with EcoRI using standard molecular biology techniques disclosed herein.

b. Insertion of zcytor19 receptor or heterodimerized subunit extracellular domains into fusion vector constructs

Constructs in which zcytor19 was fused to IgG γ 1 were prepared using the above-described construction vectors. This construction is accomplished by amplifying the extracellular domain or cytokine binding domain of the zcytor19 receptor described herein from a prostate cDNA library (Clontech) or an activated lymphocyte cDNA library by PCR using oligomers that provide EcoRI and NheI restriction sites by standard methods. The resulting PCR product was digested with EcoRI and NheI, gel purified, and ligated into the Zem229R/IgG γ 1 described above, previously digested with EcoRI and NheI and purified as described herein. The resulting vector was sequenced to verify that the zcytor19/IgG γ 1 fusion (zcytor19/Ch1 IgG) was correct.

An additional independent structure was also constructed as described above with the extracellular domain of heterodimerized cytokine receptor subunit (i.e., CRF2-4) fused to a kappa light chain. Construction of cytokine receptor/human kappa light chain was performed as described above by PCR using standard methods and oligo providing EcoRI and KpnI restriction sites amplified from, for example, a lymphocyte cDNA library (Clontech). The obtained PCR product was digested with EcoRI and KpnI, and then ligated into the above Zem 228R/human kappa light chain vector previously digested with EcoRI and KpnI and purified. The resulting vector was sequenced to confirm that the cytokine receptor subunit/human kappa light chain fusion was correct.

co-expression of zcytor19 and the extracellular domain of heterodimeric cytokine receptor subunits

Using Lipofectamineplus^TMThe reagent (Gibco/BRL), according to the manufacturer's instructions, is used to the above each vector approximately 15 u g transfection of mammalian cells, such as BHK-570 cells (ATCC No. CRL-10314). Transfected cells were selected for 10 days in DMEM + 5% FBS (Gibco/BRL) containing 1. mu.M Methotrexate (MTX) (Sigma, St. Louis, Mo.) and 0.5mg/ml G418 (Gibco/BRL). The resulting transfectant pools were again selected in 10. mu.M MTX and 0.5mg/ml G418 for 10 days.

The protein was prepared using the obtained secondary selected cell pool. 10L serum-free conditioned medium was prepared using 3 factories (Nunc. Denmark) of this library. The conditioned medium was passed through a 1ml protein A column and then eluted in about 10,750. mu.l fractions. Fractions with the highest protein concentration were pooled and dialyzed against PBS (10kD MW cut-off). Finally, Amino Acid Analysis (AAA) was performed on the dialysate using conventional methods.

d. In vitro reconstitution of the zcytor19 receptor

To identify components involved in the zcytor19 signaling complex, receptor reconstitution studies were performed as follows. For example, BHK 570 cells (ATCC No. crl-10314) transfected with a luciferase reporter mammalian expression vector plasmid using standard methods described herein can be used as a bioanalytical cell line to measure the signal transduction response from the transfected zcytor19 receptor complex to a luciferase reporter in the presence of zcytor19 ligand. BHK cells may be used because BHK cells do not endogenously express the zcytor19 receptor. Other cell lines may also be used. One exemplary luciferase reporter mammalian expression vector is the KZ134 plasmid, which is constructed using complementary oligonucleotides containing STAT transcription factor binding elements from 4 genes. Modified c-fos Sis inducible elements (m67SIE or hSIE) (Sadowski, H et al, Science 261: 1739-. These oligonucleotides contain Asp718-XhoI compatible ends and are ligated into the receptor firefly luciferase reporter vector digested with the same enzyme, having the c-Fos promoter and containing the neomycin selection marker using standard methods (Poulsen, L.K. et al, J.biol.chem.273; 6229-6232, 1998). BHK or BaF3 cells were stably transfected with the KZ134 plasmid by standard transfection and selection methods to prepare BHK/KZ134 or BaF3/KZ134 cell lines, respectively.

The bioanalytical cell line was transfected with the zcytor19 receptor alone or co-transfected with zcytor19 together with one of a number of other known receptor subunits. Receptor complexes include, but are not limited to, various combinations of the simple zcytor19 receptor, zcytor19 and class II cytokine receptors such as interferon gamma, alpha and beta chains and interferon alpha/beta receptor alpha and beta chains, zcytor11 (commonly owned U.S. patent 5,965,704), CRF2-4, DIRS1, zcytor7 (commonly owned U.S. patent 5,945,511) receptors. Each independent receptor complex cell line was then analyzed in the presence of cytokine conditioned media or purified cytokine and luciferase activity was measured using conventional methods. Untransfected bioanalytical cell lines were used as controls to obtain background luciferase activity, thus serving as a baseline to facilitate comparison of signal transduction for different receptor complex combinations. Conditioned media or cytokines that bind to the zcytor19 receptor in the presence of the correct receptor complex are expected to give luciferase readings of approximately 5-fold or more against background.

Alternatively, a similar assay can be performed in which a Baf3/zcytor19 cell line is co-transfected as described herein and proliferation is measured using a known assay such as the standard Alamar Blue proliferation assay.

Example 24

Binding of ligands to soluble receptors

The binding of ligands (Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25) to soluble receptors can be tested using the iodobead labeling method. For example,¹²⁵i-labeled zcyto21-CEE is labeled (1.2X 10)⁷CPM/ml; 1.5 ng/. mu.l; and 8.6X 10⁶CPM/μg)。

50ng of the powder¹²⁵I-labeled zcyto21-CEE (see example 3) (399,600CPM) was mixed with 1000ng cold zcytor19/Fc4 homodimer receptor, 1000ng cold zcytor19/CRF2-4 heterodimer receptor or 1000ng control class II cytokine receptor/Fc 4 receptor and about 10,000ng cold zcyto21 as competitors. Samples were incubated at 4 ℃ for 2 hours, after which 30. mu.l of protein G (Zymed San Francisco. CA) was added to each sample. The samples were incubated at 4 ℃ for 1 hour and then washed 3 times with PBS. Radioactivity of washed G-protein was measured using a gamma counter (packard instruments, Downers Grove, IL).

Example 25

Staining of human monocytes with zcyto20 and zcyto 21-Biotin flow cytometry

Peripheral Blood Leukocytes (PBL) were isolated from heparinized human blood using the Ficoll Hypaque (Amersham, Sweden) isolation procedure. PBLs were cultured at 37 ℃ in standard medium at a density of 1 × 10e6 cells/ml in 6-well tissue culture plates. After overnight incubation, PBLs were harvested and stained with biotinylated zcyto20-cee and zcyto21-cee (see example 18) at a concentration of 10. mu.g/ml. Staining was detected with phycoerythrin-labeled streptavidin (Pharmingen, CA, USA) prepared at a dilution of 1: 1000. After staining PBLs were fixed in 2% paraformaldehyde and read on a Facscaliber (Becton Dickinson, San Diego, CA). Data were analyzed using Cellquest software (Becton Dickinson). The results show that both biotinylated zcyto20-cee and zcyto21-cee stained cells in the myeloid gate of peripheral blood leukocytes. Cells in the lymphatic gate (lymphoid gate) do not bind to zcyto20-cee and zcyto 21-cee.

Example 26

Effect of zcyto21-CEE on expression of activation markers on PBLs

Peripheral Blood Leukocytes (PBLs) were isolated from heparinized human blood by the Ficoll Hypaque isolation procedure. PBLs were then stimulated with purified protein or media controls from: 1) zcyto21-CEE (2. mu.g/ml); 2) zcyto21-cee (1. mu.g/ml); 3) a simple culture medium; 4) A141F negative control protein (2. mu.g/ml); or 5) IFN alpha-A (1ng/ml) (PBLBioformal NJ, USA). Stimulated PBL at 37 ℃ and 5% CO₂The cells were cultured at a cell density of 1 × 10e6 cells/ml. Cultures were harvested at 24 and 48 hours and stained for activation markers.

PBL were washed with PBS, then blocked with normal mouse IgG in Facs buffer (HBSS + 2% normal goat serum, 2% BSA, 0.2% NaN3), followed by staining with antibodies to the following markers: CD19, CD14, CD3, HLA-DR, CD54, HLA-ABC (Pharmingen, CA, USA and Immunotech, France). Cells were washed, then fixed in 2% paraformaldehyde, and then analyzed on a Facscaliber (Becton Dickinson, Calif., USA). The data obtained were analyzed using Cellquest software (Becton Dickinson, CA, USA).

The results indicate that surface CD54(ICAM) expression on monocytes stimulated with zcyto21-cee increased at 24 and 48 hours compared to the medium-only control. Stimulation with zcyto21-cee also resulted in increased expression of major histocompatibility complex I on B cells at 24 hours and increased expression of MHCI on B cells and monocytes at 48 hours.

Example 27

Biotinylation of ligands

The zcyto21CEE was biotinylated with Sulfo-NHS-LC-biotin as set forth in the following modification by Pierce Chemical. Mu.g of zcyto21CEE (in 0.5ml PBS) was added to 8.4. mu. l S-NHS-biotin stock (84. mu.g) and incubated at room temperature for 2 hours with shaking. After the incubation step, 20. mu.l of 2M TrisHCl (pH8) was added and the mixture was incubated at room temperature for 20 minutes with shaking. The biotinylated ligand mixture was then stored at 4 ℃. The zcyto20CEE, zcyto22CEE and zcyto24CEE were prepared in substantially the same manner.

Example 28

Northern analysis of the expression of zcytor19

Northern blots were probed to determine the tissue distribution of zcytor 19. A human zcytor19cDNA fragment was obtained using PCR and gene-specific primers, 5 'ZC 40285 (shown in SEQ ID NO: 21) and 3' ZC40286 (shown in SEQ ID NO: 22). The template was a cloned human zcytor19cDNA (SEQ ID NO: 23). Gel purification of the PCR fragment with P³²alpha-dCTP and Prime-ItRmT random primer labeling kit (Stratagene, LaJolla, Calif.) labeled approximately 25 ng.

The following Northern blots (Clontech, Pal Alto, CA) were probed to detect the expression of zcytor19 mRNA: (1) human cancer cell line blot C containing RNA samples from each of the following cancer cell lines: promyelocytic leukemia HL-60, HELA S3, chronic myelogenous leukemia k-562, lymphoblastic leukemia MOLT-4, Burkitt' S lymphoma RAJI, colorectal adenocarcinoma SW480, lung carcinoma A549, and melanoma G-361; (2) human MIN H blot containing mRNA from the following tissues: heart, whole brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas; (3) human MTN H3, containing mRNA from the following tissues: stomach, thyroid, spinal cord, lymph nodes, trachea, adrenal glands and bone marrow; and (4) human MTN H4, which contains mRNA from: spleen, thymus, prostate, testis, uterus, small intestine, colon and peripheral blood leukocytes. Except that an additional 0.2mg/ml salmon sperm DNA was added to the hybridization and prehybridization buffer Washing to reduce non-specific hybridization in ULTRAhyb^TMUltrasensitive hybridization buffer (Ambion, Austin, TX) according to the manufacturer's recommendations. After hybridization, non-specific emission signals were removed by treatment with 0.1 XSSC/0.5% SDS at 50 ℃. The blot was exposed for 3 days using BioMaxMR film and an intensifying screen (Eastman Kodak, Rochester, N.Y.) according to the manufacturer's recommendations.

One-4.5 kb transcript was expressed in the greatest amount in cardiac, skeletal muscle, pancreatic and prostate tissues, as well as in Burkitt's lymphoma (RAJI) cell lines. Lower levels were observed in various other tissues. In addition, there is a 2kb transcript, which is generally less abundant than the larger transcript described above, but is also present in many tissues and cell lines. In addition to having these 2 and 4.5kb transcripts, there may be about 4kb and 1.4kb transcripts in testis tissue. The adrenal glands showed equal expression levels of the 4.5kb and 2kb transcripts.

Example 29

In situ analysis of the expression of zcytor19

Specific human tissues were isolated and screened for expression of zcytor19 by in situ hybridization. A variety of human tissues were prepared, sectioned and hybridized in situ, including normal and cancerous colon, cervical cancer, endometrial cancer, normal and cancerous ovary, normal and neoplastic skin, fetal liver, lung, heart and MFH (muscle sarcoma). Tissues were fixed in 10% buffered formaldehyde using standard techniques and paraffin embedded. The tissue was cut into 4 to 8 micron sections. Tissues were prepared using standard protocols. In short, by (National Diagnostics, Atlanta, GA) deparaffinized tissue sections, followed by dehydration with ethanol. Next, the sections were digested with proteinase K (50. mu.g/ml) (Boehringer Diagnostics, Indianapolis, IN) for 2 to 7 minutes at 37 ℃. This step is followed by acetylation and rehydration of the tissue.

Against the human zcytor19 (variant X1) sequence (INC 7) using standard methods128744, see SEQ ID NO: 25) an in situ probe was designed (3' UTR containing zcytor 19). Antisense probes were generated using T7 polymerase. Using an in vitro transcription system (In vitro transcription system, Promega, Madison, WI), probes were labeled according to the manufacturer's instructions except that digoxigenin octant probes were used instead of radiolabeled rCTP and water was adjusted to accommodate the reduced volume of rNTP. In situ hybridization was performed using a digoxigenin-labeled zcytor19 probe (described above). The probe was applied to the slide at 1 to 5pmol/ml and at 60 ℃ for 12 to 16 hours. The slides were then washed in 2 XSSC and 0.1 XSSC at 55 ℃. Use of TSA^TM(typhomide signalling; PerkinElmer Life Sciences inc., Boston, MA) amplified the signal and developed using the VECTOR red substrate kit (VECTOR Laboratories, Burlingame, CA) as directed by the manufacturer. The slides were then counterstained with hematoxylin.

Signals were observed in several tissues tested: in colon cancer tissues, weak signals were observed in cancer cells and several immunoinfiltrated zones. However, no positive signals were observed in the normal colon and small intestine, including the lamina propria, epithelium, cells in the immune node and peripheral ganglion nerve cells. In cervical cancer tissues, there is a weak signal in cancer cells and some cells of the immune node. In endometrial cancer tissue, there is a weak signal in the cancer cells. In normal uterine tissue, no positive signal was observed. In ovarian cancer samples, some cancer cells were weakly positive. In normal ovarian samples, some capillary endothelium and epithelium of large follicles may be weakly positive. In skin cancer samples, the cancerous granular epithelium (granular epithelium) is strongly positive, whereas no positive signal is observed in normal skin. In fetal liver, signals are observed in a mixed population of mononuclear cells within the sinusoids. In the lung, zcytor19 appears to be positive in type II alveolar epithelium. Occasionally bronchial epithelium may also be weakly positive. Macrophage-like mononuclear cells in the interstitial tissue were also positive. In the heart, myocytes are negative, while some circulating mononuclear cells are zcytor19 positive. In one of the samples, the vascular endothelium may be weakly positive. Other tissues tested included MFH (muscle sarcoma) samples and kaposi's sarcoma skin samples. There was no clear positive signal in these tissues.

Example 30

RT-PCR based analysis of human zcytor19 in stimulated versus unstimulated cells Expression of

The following cell types were analyzed using RT-PCR to detect gene expression of zcytor 19: hela, 293, Daudi, CD14+, U937 and HL-60.

First strand cDNA was synthesized from total RNA using a commercial first strand synthesis system for RT-PCR (Invitrogen life technologies, Carlsbad, CA). The use of zcytor 19X 1(SEQ ID NO: 23) and zcytor 19X 2(SEQ ID NO: 28) and specific oligomeric primers ZC40288(SEQ ID NO: 58) and ZC40291(SEQ ID NO: 59), Qiagen HotStarTaq DNA polymerase and buffer (Qiagen, Inc., Valencia, CA), GeneAmp dNTP (applied biosystems, Foster City, CA), Redload, respectively, to produce 806bp and 892bp products^TMThe subsequent PCR reaction was set up with dye (Research Genetics, inc., Huntville, AL) and 2 μ l of first strand cDNA from each cell type (10% first strand reaction). The PCR cycling conditions were as follows: initial 1 cycle: denaturation at 95 ℃ for 15 min; 35 cycles: denaturation at 94 ℃ for 45 seconds, annealing at 63 ℃ for 1 minute and extension at 72 ℃ for 15 seconds; the last 1 cycle follows: extension at 72 ℃ for 7 minutes. The reactions were separated electrophoretically on a 2% agarose gel (EM Science, Gibbstown, NJ) and visualized by ethidium bromide staining.

Bands of the correct size were observed in Hela + -IFN- β (892 bp band only), 293+ parental Adv, Daudi + -IFN- β, Daudi + -IFN- α, activated CD14+, activated HL-60. No bands were observed in resting CD14+, resting and activated U937, and resting HL-60. These results show that zcytor19 expression is induced when monocytes or monocyte cell lines are activated or differentiated.

Example 31

Stimulation of NFKB reporter in RAW cells

The ability of Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 to transmit signals through the NF κ β signal transduction pathway was tested using a mouse monocyte/macrophage reporter cell line. The cell line was prepared by transducing RAW264.7 cells with a KZ170 retroviral reporter containing an NF κ β responsive element driving transcription of a luciferase reporter gene.

Initial reporter assays to test Zcyto20, Zcyto21, Zcyto22, Zcyto24 and Zcyto25 activity were performed using conditioned media describing transient transfection 293 for antiviral assays. RAW264.7/KZ170 cells were harvested and seeded at a density of 50,000 cells/well in 96-well plates. Cells were incubated overnight in RPMI + 10% FBS at 37 ℃. The next day, media was removed from adherent cells, and then undiluted zcyto20-25 conditioned media or dilutions of zcyto20-25 conditioned media (diluted in RPMI + 0.1% BSA) were added to the cells. After 5 hours incubation at 37 ℃, cells were lysed and read on a luminometer after addition of luciferase substrate. The results were analyzed by comparing the Relative Light Units (RLU) of zcyto20-25 conditioned medium with the relative light units of conditioned medium of untransfected cells. Undiluted zcyto20-25 conditioned medium induced 4-9 fold higher luciferase expression compared to undiluted, untransfected cell conditioned medium. These results indicate that zcyto20-25 is capable of signaling through the NF-. kappa.beta.signaling pathway in the mouse monocyte/macrophage cell line.

As a further study, conditioned medium from Sf9 cells infected with baculoviruses expressing zcyto20, zcyto21 or zcyto22 was used in the reporter assay. Wild-type baculovirus was used as a negative control. Baculovirus constructs and conditioned media were prepared as described above.

The results of the RAW264.7 NFkb-receptor assay using baculovirus-derived conditioned medium were similar to those obtained using transient transfection 293 conditioned medium. Baculovirus-derived conditioned media containing zcyto20-22 induced luciferase expression in a dose-dependent manner, whereas corresponding control conditioned media did not.

Example 32

In vivo results

The toxicity and biological activity of zcyto24 was compared to another class II cytokine, the parental adenoviral vector, and the non-injected mice. 4 groups (8 per group) of C57B16 mice (female, 9 weeks old) were injected as follows:

group 1: injection of 1X 10 per mouse¹¹An Adzcyto24 particle;

group 2: by 1 × 10¹¹Each particle was injected with class II cytokine (Adzcyto);

group 3: by 1 × 10¹¹Individual particle injection of parental adenovirus vectors (Adzpar); and

group 4: untreated.

The temperature transmitter was placed on the mice on day-1 and the virus was injected on day 0. Cage facilitates food intake, body weight was measured every 5 days, and day 10 blood (0.25 ml maximum) was collected for testing, including CBC and Abbot blood analyses. All mice were sacrificed on day 20.

Blood from mice treated with Adzcyto24 (group 1) was taken on day 10 and sera tested to determine the presence or absence of zcyto24 biological activity. Viral experiments demonstrated significant antiviral activity at 1:500 maximal dilution for each mouse in the Adzcyto24 group. This corresponds to about 160ng/ml purified zcyto24 CEE. The antiviral assay used to detect mouse interferon did not detect activity in the Adzcyto24 group. Bioassays using reporter molecules with IRSE also detected significant zcyto24 activity in the sera of the Adzcyto24 injected mice, but not in other groups.

The temperature probes showed that the average body temperature of group 2 decreased by more than 5 ℃ by day 10, while the average body temperature of the Adzcyto24 (group 1) group decreased by more than 2 ℃. The body temperature of the control group showed less than 1 ℃ change throughout the experiment.

The mice of Adzcyto24 (group 1) showed a slight increase in body weight in the 20-day experiment, as in the control groups (groups 3 and 4). Group 2 mice weight loss: by day 10 the mean body weight of group 2 was reduced by about 8%.

Analysis of Abbot hematology analyzers on blood taken on days 10 and 20 revealed significant changes in white blood cell counts in groups 1 and 2. Figure 1 shows that the monocytic cell count of the Adzcyto 24-treated mice was significantly increased relative to the other groups. The mice injected with Adzcyto24 had 2.77-fold higher monocyte counts than the mice injected with the parental vector (Adzpar). By day 20, the monocyte count decreased slightly, but was still significantly higher than that of the parental vector injected mice. Figure 2 shows that injection with adenovirus encoding class II cytokines (Adzcyto) but not with adenovirus encoding zcyto24 resulted in an increase in neutrophil counts at day 10.

To verify the nature of the cell types detected by the Abbot hematology analyzer, flow cytometry analysis was performed on blood taken on day 20 of the experiment using lineage specific mabs (monoclonal antibodies). Figure 3 shows the percentage of CD11b positive cells, i.e. monocytes, in the peripheral blood of each mouse. The average values for each group were plotted and figure 4 shows that the percentage of monocytes was significantly increased in the group injected with the adenovirus with zcyto24 compared to the group infected with the parental vector (p 0.05). This is consistent with the changes previously observed using an Abbot hematology analyzer.

Analysis of the same blood samples with a specific MAb against granulocytes (GR-1) showed no significant increase in the percentage of granulocytes (i.e. predominantly neutrophils) in mice injected with Adzcyto 24. PBLs were also stained with B cell (B220) specific Mabs and no significant difference was observed for the Adzcyto24 injected mice relative to the other groups.

Notably, the chills and weight loss associated with the Adzcyto injection were not evident with Adzcyto 24. The significant increase in monocytes detected by Abbot hematology analyzer on days 10 and 20 was confirmed by flow cytometry analysis of day 20 blood with lineage specific MAbs. The significant increase in the percentage of monocytes in PBL of mice injected with Adzcyto24 appears to be a unique activity mediated directly or indirectly by zcyto24 in the case of adenoviral infection. Significant levels of zcyto24 expressed in mice injected with Adzcyto24 may promote an antiviral response by recruiting and mobilizing monocytes from surrounding tissues or by stimulating the production of monocytes from bone marrow or liver-derived progenitors. This evidence suggests that monocytes/macrophages are activated by zcyto24 and zcyto 21.

It will be appreciated from the foregoing that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

The application also discloses the following:

1. an isolated polypeptide having at least 80% identity to a polypeptide selected from the group consisting of:

(a) comprises the amino acid sequence of SEQ ID NO: 2 from amino acid residue 22 to amino acid residue 205;

(b) comprises the amino acid sequence of SEQ ID NO: 7 from amino acid residue 22 to amino acid residue 205;

(c) comprises the amino acid sequence of SEQ ID NO: 9 from amino acid residue 29 to amino acid residue 202; and

(d) comprises the amino acid sequence of SEQ ID NO: 11 from amino acid residue 29 to amino acid residue 202.

2. The isolated polypeptide of item 1, wherein the polypeptide specifically binds to a receptor as follows: SEQ ID NOS: 24. 27 or 29, or a monomeric or homodimeric receptor of SEQ ID NOS: 24. 27 or 29 in combination with SEQ ID NO: 41.

3. An isolated polypeptide having at least 95% identity to a polypeptide selected from the group consisting of:

(a) Comprises the amino acid sequence of SEQ ID NO: 2 from amino acid residue 22 to amino acid residue 205;

(b) comprises the amino acid sequence of SEQ ID NO: 7 from amino acid residue 22 to amino acid residue 205;

(c) comprises the amino acid sequence of SEQ ID NO: 9 from amino acid residue 29 to amino acid residue 202; and

(d) comprises the amino acid sequence of SEQ ID NO: 11 from amino acid residue 29 to amino acid residue 202.

4. The isolated polypeptide of item 3, wherein the polypeptide specifically binds to a receptor as follows: SEQ ID NOS: 24. 27 or 29, or a monomeric or homodimeric receptor of SEQ ID NOS: 24. 27 or 29 in combination with SEQ ID NO: 41.

5. An isolated polypeptide comprising SEQ ID NO: 2 from amino acid residue 22 to amino acid residue 205 or SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 205.

6. An isolated polypeptide comprising SEQ ID NO: 7 from amino acid residue 22 to amino acid residue 205 or SEQ ID NO: 7 from amino acid residue 1 to amino acid residue 205.

7. An isolated polypeptide comprising SEQ ID NO: 9 from amino acid residue 29 to amino acid residue 202 or SEQ ID NO: 9 from amino acid residue 1 to amino acid residue 202.

8. An isolated polypeptide comprising SEQ ID NO: 11 from amino acid residue 29 to amino acid residue 202 or SEQ ID NO: 11 from amino acid residue 1 to amino acid residue 202.

9. An isolated polypeptide which is SEQ ID NO: 2 from amino acid residue 22 to amino acid residue 205 or SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 205, wherein the polypeptide stimulates an antigenic response in a mammal.

10. An isolated polypeptide which is SEQ ID NO: 7 from amino acid residue 22 to amino acid residue 205 or SEQ ID NO: 7 from amino acid residue 1 to amino acid residue 205, wherein the polypeptide stimulates an antigenic response in a mammal.

11. An isolated polypeptide which is SEQ ID NO: 9 from amino acid residue 29 to amino acid residue 202 or SEQ ID NO: 9 from amino acid residue 1 to amino acid residue 202, wherein the polypeptide stimulates an antigenic response in a mammal.

12. An isolated polypeptide which is SEQ ID NO: 11 from amino acid residue 29 to amino acid residue 202 or SEQ ID NO: 11 from amino acid residue 1 to amino acid residue 202, wherein the polypeptide stimulates an antigenic response in a mammal.

13. A pharmaceutical composition comprising SEQ ID NO: 2 from amino acid residue 22 to amino acid residue 205 or SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 205.

14. A pharmaceutical composition comprising SEQ ID NO: 7 from amino acid residue 22 to amino acid residue 205 or SEQ ID NO: 7 from amino acid residue 1 to amino acid residue 205.

15. A fusion protein comprising at least two polypeptides, wherein at least one polypeptide comprises a polypeptide selected from the group consisting of:

(a) comprises the amino acid sequence of SEQ ID NO: 2 from amino acid residue 22 to amino acid residue 205;

(b) comprises the amino acid sequence of SEQ ID NO: 7 from amino acid residue 22 to amino acid residue 205;

(c) Comprises the amino acid sequence of SEQ ID NO: 9 from amino acid residue 29 to amino acid residue 202; and

(d) comprises the amino acid sequence of SEQ ID NO: 11 from amino acid residue 29 to amino acid residue 202.

16. An isolated polynucleotide encoding a polypeptide, wherein the nucleic acid molecule is selected from the group consisting of:

(a) comprises the amino acid sequence of SEQ ID NO: 3;

(b) after stringent washing conditions still compared to the sequence defined by SEQ ID NO: 1 from nucleotide 64 to 618, or SEQ ID NO: 1 from nucleotide 64 to 618.

17. An isolated polynucleotide encoding a polypeptide, wherein the nucleic acid molecule is selected from the group consisting of:

(a) comprises the amino acid sequence of SEQ ID NO: 36;

(b) after stringent washing conditions still compared to the sequence defined by SEQ ID NO: 6 from nucleotide 64 to 618, or SEQ ID NO: 6 from nucleotide 64 to 618.

18. An isolated polynucleotide encoding a polypeptide selected from the group consisting of:

(a) comprises the amino acid sequence of SEQ ID NO: 2 from amino acid residue 22 to amino acid residue 205;

(b) Comprises the amino acid sequence of SEQ ID NO: 7 from amino acid residue 22 to amino acid residue 205;

(c) comprises the amino acid sequence of SEQ ID NO: 9 from amino acid residue 29 to amino acid residue 202; and

(d) comprises the amino acid sequence of SEQ ID NO: 11 from amino acid residue 29 to amino acid residue 202.

19. The isolated polynucleotide of item 18, wherein any differences between the amino acid sequence encoded by said nucleic acid molecule and the corresponding amino acid sequence are due to conservative amino acid substitutions.

20. An isolated polynucleotide comprising the sequence of SEQ ID NO: 1 from nucleotide 64 to nucleotide 618 or SEQ ID NO: 1 from nucleotide 1 to nucleotide 618.

21. An isolated polynucleotide comprising the sequence of SEQ ID NO: 6 from nucleotide 64 to nucleotide 618 or the nucleotide sequence shown in SEQ ID NO: 6 from nucleotide 1 to nucleotide 618.

22. An isolated polynucleotide comprising the sequence of SEQ ID NO: 8 from nucleotide 67 to nucleotide 606 or SEQ ID NO: 1 from nucleotide 1 to nucleotide 606.

23. An isolated polynucleotide comprising the sequence of SEQ ID NO: 10 from nucleotide 67 to nucleotide 606 or SEQ ID NO: 10 from nucleotide 1 to nucleotide 606.

24. An expression vector comprising the isolated nucleic acid molecule of item 20, item 21, item 22, or item 23, a transcriptional promoter, and a transcriptional terminator, wherein the promoter is operably linked to the nucleic acid molecule and the nucleic acid molecule is operably linked to the transcriptional terminator.

25. A recombinant host cell comprising the expression vector of item 24, wherein said host cell is selected from the group consisting of: bacteria, yeast cells, fungal cells, insect cells, mammalian cells, and plant cells.

26. A method of producing a polypeptide, the method comprising the steps of: culturing a recombinant host cell comprising the expression vector of item 24 and producing the polypeptide, and isolating the polypeptide from the cultured host cell.

27. An antibody or antibody fragment that specifically binds to a polypeptide of item 9, item 10, item 11, or item 12.

28. A method for detecting the presence or absence of gene expression in a biological sample comprising the steps of:

(a) contacting under hybridization conditions a nucleic acid probe with (1) a test RNA isolated from the biological sample or (2) a nucleic acid molecule synthesized from the isolated RNA molecule, wherein the probe consists of a nucleic acid molecule comprising SEQ ID NO: 1, SEQ ID NO: 1 a portion of a complementary molecule to the nucleotide sequence; SEQ ID NO: 6 nucleotide sequence, SEQ ID NO: 6 nucleotide sequence, and

(b) Detecting hybrids formed between the nucleic acid probes and the test RNA molecules or the synthesized nucleic acid molecules,

wherein the presence of the hybrid indicates the presence of the RNA in the biological sample,

or

(a') contacting the biological sample with an antibody or antibody fragment of item 27, wherein said contacting is performed under conditions that allow binding of the antibody or antibody fragment to the biological sample, and

(b) detecting all bound antibodies or bound antibody fragments.

29. A method of expanding monocytes or monocyte progenitors comprising culturing bone marrow or peripheral blood cells with a composition comprising a polypeptide of item 9, item 10, item 11, and item 12, wherein the polypeptide is in an amount in the composition sufficient to result in an increase in the number of monocytes or monocyte progenitors in the bone marrow or peripheral blood cells as compared to bone marrow or peripheral blood cells cultured in the absence of administration of the polypeptide.

30. A method of stimulating an immune response in a mammal exposed to an antigen or pathogen comprising:

(1) determining the level of antigen or pathogen specific antibodies;

(2) administering a composition comprising a polypeptide of item 9, item 10, item 11, or item 12 in a pharmaceutically acceptable carrier;

(3) determining the level of antigen or pathogen-specific antibody after administration;

(4) Comparing the level of antibody in step (1) to the level of antibody in step (3), wherein an increase in the level of antibody indicates that the immune response is stimulated.

Sequence listing

<110>ZymoGenetics，InC.

<120> cytokine protein ZCYT020

<130>01-17PC

<150>US 60/285,408

<151>2001-04-20

<150>US 60/286,482

<151>2D01-04-25

<150>US 60/341,050

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<150>US 60/341,105

<151>2001-10-22

<150>US 09/895,834

<151>2001-06-29

<150>US 60/285,424

<151>2001-04-20

<160>59

<170>FastSEQ for Windows Version 4.0

<210>1

<211>618

<212>DNA

<213> human (Homo sapiens)

<220>

<221>CDS

<222>(1)...(618)

<400>1

<210>2

<211>205

<212>PRT

<213> human

<400>2

<210>3

<211>615

<212>DNA

<213> Artificial sequence

<220>

<223> degenerate sequence

<221>misc_feature

<222>(1)...(615)

<223> n ═ A, T, C or G

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<210>4

<211>603

<212>DNA

<213> human

<220>

<221>CDS

<222>(1)...(603)

<400>4

<210>5

<211>200

<212>PRT

<213> human

<400>5

<210>6

<211>615

<212>DNA

<213> human

<220>

<221>CDS

<222>(1)...(615)

<400>6

<210>7

<211>205

<212>PRT

<213> human

<400>7

<210>8

<211>633

<212>DNA

<213> human

<220>

<221>CDS

<222>(22)...(630)

<400>8

<210>9

<211>202

<212>PRT

<213> human

<400>9

<210>10

<211>632

<212>DNA

<213> human

<220>

<221>CDS

<222>(22)...(630)

<400>10

<210>11

<211>202

<212>PRT

<213> human

<400>11

<210>12

<211>49

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC40923

<400>12

<210>13

<211>88

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC40927

<400>13

<210>14

<211>42

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC41932

<400>14

<210>15

<211>42

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC41933

<400>15

<210>16

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> glu glu tag

<400>16

<210>17

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC40240

<400>17

<210>18

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC40241

<400>18

<210>19

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC447

<400>19

<210>20

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC976

<400>20

<210>21

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC40285

<400>21

<210>22

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC40286

<400>22

<210>23

<211>1476

<212>DNA

<213> human

<220>

<221>CDS

<222>(1)...(1473)

<400>23

<210>24

<211>491

<212>PRT

<213> human

<400>24

<210>25

<211>611

<212>DNA

<213> human

<400>25

<210>26

<211>1563

<212>DNA

<213> human

<220>

<221>CDS

<222>(1)...(1563)

<400>26

<210>27

<211>520

<212>PRT

<213> human

<400>27

<210>28

<211>674

<212>DNA

<213> human

<220>

<221>CDS

<222>(1)...(633)

<400>28

<210>29

<211>211

<212>PRT

<213> human

<400>29

<210>30

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC40134

<400>30

<210>31

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC40214

<400>31

<210>32

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC40209

<400>32

<210>33

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC40213

<400>33

<210>34

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC39295

<400>34

<210>35

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC39298

<400>35

<210>36

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC40632

<400>36

<210>37

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC40633

<400>37

<210>38

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC40638

<400>38

<210>39

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC40639

<400>39

<210>40

<211>1013

<212>DNA

<213> human

<220>

<221>CDS

<222>(14)...(991)

<400>40

<210>41

<211>325

<212>PRT

<213> human

<400>41

<210>42

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> FLAG peptide tag

<400>42

<210>43

<211>699

<212>DNA

<213> human

<400>43

<210>44

<211>28

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC37967

<400>44

<210>45

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC37972

<400>45

<210>46

<211>615

<212>DNA

<213> Artificial sequence

<220>

<223> degenerate sequence

<221>misc_feature

<222>(1)...(615)

<223> n ═ A, T, C or G

<400>46

<210>47

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC39339

<400>47

<210>48

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC39393

<400>48

<210>49

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC39340

<400>49

<210>50

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC39341

<400>50

<210>51

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC39295

<400>51

<210>52

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC39298

<400>52

<210>53

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC39687

<400>53

<210>54

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC 397641

<400>54

<210>55

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC 397732

<400>55

<210>56

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC 3977

<400>56

<210>57

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC39688

<400>57

<210>58

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC40288

<400>58

<210>59

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer ZC40291

<400>59

Claims (10)

1. An isolated polypeptide having at least 80% identity to a polypeptide selected from the group consisting of:

(a) comprises the amino acid sequence of SEQ ID NO: 2 from amino acid residue 22 to amino acid residue 205;

(b) comprises the amino acid sequence of SEQ ID NO: 7 from amino acid residue 22 to amino acid residue 205;

(c) comprises the amino acid sequence of SEQ ID NO: 9 from amino acid residue 29 to amino acid residue 202; and

(d) comprises the amino acid sequence of SEQ ID NO: 11 from amino acid residue 29 to amino acid residue 202.

2. The isolated polypeptide of claim 1, wherein the polypeptide specifically binds to a receptor that: SEQ ID NOS: 24. 27 or 29, or a monomeric or homodimeric receptor of SEQ ID NOS: 24. 27 or 29 in combination with SEQ ID NO: 41.

3. An isolated polypeptide having at least 95% identity to a polypeptide selected from the group consisting of:

(a) comprises the amino acid sequence of SEQ ID NO: 2 from amino acid residue 22 to amino acid residue 205;

(b) comprises the amino acid sequence of SEQ ID NO: 7 from amino acid residue 22 to amino acid residue 205;

(c) Comprises the amino acid sequence of SEQ ID NO: 9 from amino acid residue 29 to amino acid residue 202; and

(d) comprises the amino acid sequence of SEQ ID NO: 11 from amino acid residue 29 to amino acid residue 202.

4. The isolated polypeptide of claim 3, wherein the polypeptide specifically binds to a receptor that: SEQ ID NOS: 24. 27 or 29, or a monomeric or homodimeric receptor of SEQ ID NOS: 24. 27 or 29 in combination with SEQ ID NO: 41.

5. An isolated polypeptide comprising SEQ ID NO: 2 from amino acid residue 22 to amino acid residue 205 or SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 205.

6. An isolated polypeptide comprising SEQ ID NO: 7 from amino acid residue 22 to amino acid residue 205 or SEQ ID NO: 7 from amino acid residue 1 to amino acid residue 205.

7. An isolated polypeptide comprising SEQ ID NO: 9 from amino acid residue 29 to amino acid residue 202 or SEQ ID NO: 9 from amino acid residue 1 to amino acid residue 202.

8. An isolated polypeptide comprising SEQ ID NO: 11 from amino acid residue 29 to amino acid residue 202 or SEQ ID NO: 11 from amino acid residue 1 to amino acid residue 202.

9. An isolated polypeptide which is SEQ ID NO: 2 from amino acid residue 22 to amino acid residue 205 or SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 205, wherein the polypeptide stimulates an antigenic response in a mammal.

10. An isolated polypeptide which is SEQ ID NO: 7 from amino acid residue 22 to amino acid residue 205 or SEQ ID NO: 7 from amino acid residue 1 to amino acid residue 205, wherein the polypeptide stimulates an antigenic response in a mammal.