WO2006074389A2 - Four-helical bundle protein zsig99 - Google Patents

Abstract

A four helical bundle cytokine has been identified and designated zsig99. The polypeptide is 201 amino acids in length with the mature polypeptide starting at residue 43 as is shown in SEQ ID NO:2. When the zsig99 polynucleotide is co-expressed with a polynucleotide encoding for leukemia inhibitory factor (LIF), a heterodimer comprising zsig99 and LIF is secreted. Also disclosed are antibodies to zsig99, methods for producing the polypeptide using expression vectors and host cells. Diagnostic methods for identifying the presence of zsig99 polypeptide are also disclosed.

Description

Description FOUR-HELICAL BUNDLE PROTEIN ZSIG99

BACKGROUND OF THE INVENTION

[1] Cytokines are polypeptide hormones that are produced by a cell and affect cell growth or metabolism in either autocrine, paracrine or endocrine fashion. In multicellular animals, cytokines control cell growth, migration, differentiation, and maturation. Cytokines play a role in both normal development and pathogenesis, including the development of solid tumors.

[2] Cytokines are physicochemically diverse, ranging in size from 5 kDa (TGF-α) to 140 kDa (Mullerian-inhibiting substance). Structurally, cytokines include a group distinguished by their four-helix bundle conformation. They include single polypeptide chains, as well as disulfide-linked homodimers and heterodimers.

[3] Cytokines influence cellular events by binding to cell-surface receptors. Binding initiates a chain of signalling events within the cell, which ultimately results in phenotypic changes such as cell division, protease production, cell migration, expression of cell surface proteins, and production of additional growth factors.

[4] Cell differentiation and maturation are also under control of cytokines. For example, the hematopoietic factors erythropoietin, thrombopoietin, and G-CSF stimulate the production of erythrocytes, platelets, and neutrophils, respectively, from precursor cells in the bone marrow. Development of mature cells from pluripotent progenitors may require the presence of a plurality of factors.

[5] The 1L-12 family of cytokines is involved in immunomodulatory activities. Proteins in the IL-12 family are heterodimers and include 1L-12, IL-23 and IL-27. IL-12 is a heterodimer comprising a p35 and p40 subunit (Kobayasbi et al., J. Exp. Med.170: 827-845, 1989), IL-23 comprises pl9 and p40 subunits (Oppman et al., Immunity 13:715-725, 2000, and IL-27 heterodimer comprises subunits p28 and Epstein Barr virus-induced protein 3 (EBI3; Pflanz et al., Immunity 16:779-790, 2002).

[6] In view of the proven clinical utility of cytokines, there is a need in the art for additional such molecules for use as both therapeutic agents and research tools and reagents. Cytokines are used in the laboratory to study developmental processes, and in laboratory and industry settings as components of cell culture media. BRIEF DESCRIPTION OF THE INVENTION

[7] In one aspect, the present invention provides an isolated polypeptide comprising at least nine contiguous amino acid residues of SEQ ID NO:2. In certain embodiments, the isolated polypeptide comprises from 30 to 201 amino acid residues as shown in SEQ ID NO:2. In another embodiment, the polypeptide is at least nine contiguous amino acid residues of SEQ ID NO:2 which are operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein and an immunoglobulin constant region. Another embodiment provides polypeptides comprising at least 30 contiguous residues of SEQ ID NO:2. In certain embodiments, isolated polypeptides comprising residues 19-201 of SEQ ID NO:2 are provided. In another embodiment, the polypeptides comprise residues 1-201 of SEQ ID NO: 2.

[8] In another aspect of the present invention an isolated polypeptide comprising a sequence of amino acid residues selected from the group consisting of: (a) residues 19-42 of SEQ ID NO:2; (b) residues 58-73 of SEQ ID NO:2; (c) residues 90-106 of SEQ ID NO:2; (d) residues 110-123 of SEQ ID NO:2; and (e) residues 165-180 of SEQ ID NO:2 are provided.

[9] In another aspect, the present invention provides an isolated polynucleotide molecule encoding a polypeptide wherein the encoded polypeptide comprises amino acid sequences selected from the group consisting of: (a) residues 19-42 of SEQ ID NO:2; (b) residues 58-73 of SEQ ID NO:2; (c) residues 90-106 of SEQ ID NO:2; (d) residues 110-123 of SEQ ID NO:2; and (e) residues 165-180 of SEQ ID NO:2.

[10] In another aspect, the present invention provides an isolated polynucleotide molecule encoding a polypeptide wherein the encoded polypeptide comprises an amino acid sequence that is at least nine contiguous amino acid residues of SEQ ID NO:2. _- In certain embodiments, the polynucleotide encodes for a polypeptide comprising residues 43-201 of SEQ ID NO:2. In certain embodiments, the present invention provides an isolated polynucleotide molecule comprising residues 19-201 of SEQ ID NO:2of SEQ ID NO:2. In other embodiments, the present invention provides an isolated polynucleotide molecule comprising residues 1-201 of SEQ ID NO:2.

[11] The present invention also provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a zsig99 polypeptide as described herein; and a transcription terminator, hi other aspects, the invention provides a cultured cell into which has been introduced the expression vector. In another aspect, the cultured cells also comprises a second expression vector which comprises operably linked elements of a transcription promoter, transcription terminator and DNA segment encoding a LIF polypeptide which is shown in SEQ ID NO: 8,. Another aspect of the invention provides a method of making a zsig99 protein comprising: culturing a cell into which has been introduced the expression vector of described herein under conditions whereby the DNA segment is expressed and the polypeptide is produced; and recovering the protein from the cell.

[12] In another aspect, the present invention provides an antibody that specifically binds to the zsig99 polypeptides described herein. Li another embodiment, the invention provides an antibody that specifically binds to a polypeptide consisting of amino acid residues 43-201 of SEQ ID NO:2.

[13] Also included are methods of detecting the presence of a polypeptide as shown in SEQ ID NO:2, or portion thereof, in a biological sample, comprising the steps of: (a) contacting the biological sample with an antibody, or an antibody fragment, wherein the contacting is performed under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibody or bound antibody fragment.

[14] Another aspect provides a method for detecting a genetic abnormality in a patient, comprising: obtaining a genetic sample from a patient; producing a first reaction product by incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO: 1 or the complement of SEQ ID NO: 1, under conditions wherein said polynucleotide will hybridize to complementary polynucleotide sequence; visualizing the first reaction product; and comparing said first reaction product to a control reaction product from a wild type patient, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient.

DESCRIPTION OF THE INVENTION

[15] Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms:

[16] The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzvmol. 198:3, 1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sri. USA 82:7952-4, 1985), substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107, 1991. DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ).

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

[18] The terms "amino-terminal" and "carboxyl-terminal" are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.

[19] The term "cancer" or "cancer cell" is used herein to denote a tissue or cell found in a neoplasm which possesses characteristics which differentiate it from normal tissue or tissue cells. Among such characteristics include but are not limited to: degree of anaplasia, irregularity in shape, indistinctness of cell outline, nuclear size, changes in structure of nucleus or cytoplasm, other phenotypic changes, presence of cellular proteins indicative of a cancerous or pre-cancerous state, increased number of mitoses, and ability to metastasize. Words pertaining to "cancer" include carcinoma, sarcoma, tumor, epithelioma, leukemia, lymphoma, polyp, and scirrus, transformation, neoplasm, and the like.

[20] The term "complement/anti-complement pair" denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair. Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of <10⁹ M^"1.

[21] The term "complements of a polynucleotide molecule" denotes a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. [22] The term "degenerate nucleotide sequence" denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).

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

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

[25] An "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.

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

[27] The term "level" when referring to viral infections refers to a change in the level of viral infection and includes, but is not limited to, a change in the level of CTLs or NK cells (as described above), a decrease in viral load, an increase antiviral antibody titer, decrease in serological levels of alanine aminotransferase, or improvement as determined by histological examination of a target tissue or organ. Determination of whether these changes in level are significant differences or changes is well within the skill of one in the art.

[28] The term "neoplastic", when referring to cells, indicates cells undergoing new and abnormal proliferation, particularly in a tissue where in the proliferation is uncontrolled and progressive, resulting in a neoplasm. The neoplastic cells can be either malignant, i.e. invasive and metastatic, or benign.

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

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

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

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

[33] A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless. [34] The term "receptor" denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell. Membrane-bound receptors are characterized by a multi-peptide structure comprising an extracellular ligand- binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. In general, receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).

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

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

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

[38] The present invention is based in part upon the discovery of a novel four helical bundle cytokine. The cytokine has been designated zsig99, and has properties associated with the IL-12 family of cytokines. Other members of the IL-12 cytokine family include IL-23 and IL-27 (Trinchieri et al., Immunity 19:641-644, 2003; Oppman, et al., Immunity 13:715-725, 2000; Pflanz et al., Immunity 16:779-790, 2002), and are secreted as heterodimers. Previously identified IL-12 family members comprise a four helical bundle cytokine subunit linked with a soluble receptor-like component (Villarino et al., Arthritis Res. Ther. 6:225-233, 2004; Trinchieri, 2003 ibid). Zsig99 is a four helical bundle cytokine-like molecule and that is co-expressed with leukemia inhibitory factor (LIF) which is shown in SEQ ID NO:8, a second four helical bundle cytokine, resulting in a secreted heterodimeric protein. Heterodimeric proteins comprising two four helical cytokine subunits is an expansion of the known structure of the IL-12 family. Co-expression of LIF and zsig99 have been identified in, but are not limited to, muscle, neuronal tissues, hematopoietic tissues such as thymus and fetal liver, and cell lines of the monocyte/macrophage lineage such as U937. Co-expression of LIF and zsig99 from the same tissue type provides additional evidence that a heterodimer is secreted in vivo. In vitro expression of zsig99 required co-expression with LIF in order to be secreted.

[39] Sequence analysis revealed an open reading frame of 201 amino acids, with a secretory signal peptide of 42 amino acids at the N-terminus and a mature protein of 160 amino acids. Cleavage after residue 42 (Thr) results in a mature polypeptide (residues 43-201 of SEQ ID NO:2; with corresponding nucleotide sequence as shown in SEQ ID NO:1) having a calculated molecular weight of approximately 17881 Da. The resulting polypeptide has three cysteine residues, which may form intradisulfide and interdisulfide associations. Within the secretory signal sequence of residues 1 to 42 there are two Met residues, Met 1 and Met 19. In the examples described herein, expression constructs begin at Met 19, and therefore Met 1 may serve as transmembrane domain and anchor the four helical bundle portion of the protein to the cell surface for some period in the protein lifecycle or may serve as an alternative secretory signal sequence initiation point. The invention includes expression constructs, methods and products of those methods where zsig99 is co-expressed with LIF protein. Exemplary methods of co-expressing zsig99 and LIF are described herein.

[40] In general, in a four-alpha helix structure, helices A, C and D are most important in ligand-receptor interactions, containing conserved motifs among members of the family. Those skilled in the art will recognize that predicted domain boundaries are somewhat imprecise and may vary by up to ± 5 amino acid residues. Helical boundaries are shown in SEQ ID NO:2: helix A from amino acid residue 58 (GIy) to amino acid residue 73 (Trp); helix B from amino acid residue 90 (GIu) to amino acid residue 106 (His); helix C from amino acid residue 110 (Ser) to amino acid residue 123 (GIn); helix D from amino acid residue 165 (VaI) to amino acid residue 180 (GIy). Studies using CNTF and IL-6 demonstrated that a CNTF helix can be exchanged for the equivalent helix in IL-6, conferring CTNF-binding properties to the chimera. Thus, it appears that functional domains of four-helical cytokines are determined on the basis of structural homology, irrespective of sequence identity, and can maintain functional integrity in a chimera (Kallen et al., J. Biol. Chem. 274:11859-11867, 1999). Therefore, the helical domains of zsig99 will be useful for preparing chimeric fusion molecules, particularly with other short-helix form cytokines to determine and modulate receptor binding specificity: Of particular interest are fusion proteins engineered with helix A and/or helix D, and fusion proteins that combine helical and loop domains from other short-form cytokines such as EL-2, IL-4, IL-15 and GM-CSF.

[41] Analysis of the tissue distribution of the human mRNA corresponding to this novel DNA showed that expression was found to be restricted to stomach and small intestine, with less abundant expression seen in colon and esophagus. The cDNA is predicted to be about 1.4 kb.

[42] SEQ ID NO: 3 is a degenerate polynucleotide sequence that encompasses all polynucleotides that could encode the zsig99 polypeptide of SEQ ID NO: 2 (amino acids 1 or 43 to 201). Thus, zsig99 polypeptide-encoding polynucleotides ranging from nucleotide 1 or 127 to nucleotide 603 of SEQ ID NO: 1 or nucleotide 1 or 127 to 603 of SEQ ID NO: 3 are contemplated by the present invention. Also contemplated by the present invention are fragments and fusions as described herein with respect to SEQ ID NO: 1, which are formed from analogous regions of SEQ ID NO: 3, wherein nucleotides 77 to 149 of SEQ ID NO: 1 correspond to nucleotides 1 to 126 of SEQ ID NO: 3, for the secretory signal sequence; wherein nucleotides 194 to 241 of SEQ ID NO: 1 correspond to nucleotides 172 to 219 of SEQ ID NO: 3, for helix A; wherein nucleotides 290 to 340 of SEQ ID NO: 1 correspond to nucleotides 268^" to 315 of SEQ ID NO: 3 for helix B; wherein nucleotides 350 to 391 of SEQ ID NO: 1 correspond to nucleotides 370 to 492 of SEQ ID NO: 3, for helix C; and wherein nucleotide 515 to nucleotide 562 of SEQ ID NO: 1 correspond to nucleotide 493 to nucleotide 540 of SEQ ID NO: 3 for helix D. Table 1 sets forth the one-letter codes used within SEQ ID NO: 3 to denote degenerate nucleotide positions. "Resolutions" are the nucleotides denoted by a code letter. "Complement" indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.

TABLE l

Nucleotide Resolution Complement Resolution

A A T T

C C G G

G G C C

T T A A

R A|G Y C|T

Y C|T R A|G

M A|C K G|T K G|T M A|C

S C|G S C|G

W Apr W A|T

H A|C|T D A|G|T

B C|G|T V A|C|G

V A|C|G B C|G|T

D A|G|T H A|C|T

N A|C|G|T N A|C|G|T

[43] The degenerate codons used in SEQ ID NO: 2, encompassing all possible codons for a given amino acid, are set forth in Table 2.

TABLE 2

One Letter

Amino Code Codons Degenerate

Acid Codon

Cys C TGC TGT TGY

Ser S AGC AGT TCA TCC TCG TCT WSN

Thr T ACAACCACGACT ACN

Pro P CCACCCCCGCCT CCN

Ala A GCA GCC GCG GCT GCN

GIy G GGA GGC GGG GGT GGN

Asn N AACAAT AAY

Asp D GAC GAT GAY

GIu E GAAGAG GAR

GIn Q CAACAG CAR

His H CAC CAT CAY

Arg R AGA AGG CGA CGC CGG CGT MGN

Lys K AAAAAG AAR

Met M ATG ATG

He I ATA ATC ATT ATH

Leu L CTA CTC CTG CTT TTA TTG YTN

VaI V GTAGTCGTGGTT GTN

Phe F TTC TTT TTY

Tyr Y TAC TAT TAY

Trp W TGG TGG

Ter . TAATAGTGA TRR

Asn|Asp B RAY

Glu|Gln Z SAR

Any X NNN [44] One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY). A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO: 2. Variant sequences can be readily tested for functionality as described herein.

[45] One of ordinary skill in the art will also appreciate that different species can exhibit "preferential codon usage." In general, see, Grantham, et al., Nuc. Acids Res., 8:1893- 912, 1980; Haas, et al. Curr. Biol.. 6:315-24, 1996; Wain-Hobson, et al., Gene, 13:355-64, 1981; Grosjean and Fiers, Gene, 18:199-209, 1982; Holm, Nuc. Acids Res., 14:3075-87, 1986; Ikemura, J. MoI. Biol., 158:573-97, 1982. As used herein, the term "preferential codon usage" or "preferential codons" is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2). For example, the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential. Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NO: 3 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.

[46] The present invention further provides variant polypeptides and nucleic acid molecules that represent counterparts from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are zsig99 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. The orthologs from dog and chimpanzee have also been identified. A DNA sequence and corresponding amino acid sequence of dog zsig99 are shown in SEQ ID NOS: 4 and 5, respectively and chimpanzee DNA and corresponding amino acid sequence are shown in SEQ E) NOS: 6 and 7, respectively. Percent identity between the chimpanzee and human zsig99 amino acid sequence is high at 97.5% The dog and chimpanzee amino acid sequences have 83% identity. The highest regions of conservation are found in the C-terminus of the polypeptide, which is predicted to be the function region of the polypeptide.

[47] Additional orthologs of human zsig99 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zsig99 as disclosed herein. Suitable sources of mRNA can be identified by probing northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line.

[48] An zsig99-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction with primers designed from the representative human zsig99 sequences disclosed herein. Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to zsig99 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.

[49] The present invention provides polynucleotide molecules including DNA and RNA molecules that encode the zsig99 polypeptides disclosed above.

[50] Zsig99 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a zsig99 gene. In view of the tissue-specific expression observed for zsig99 by Northern blotting, this gene region is expected to provide for stomach and small intestine-specific expression. Promoter elements from a zsig99 gene could thus be used to direct the tissue-specific expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy. Cloning of 5' flanking sequences also facilitates production of zsig99 proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous zsig99 gene in a cell is altered by introducing into the zsig99 locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site. The targeting sequence is a zsig99 5' non-coding sequence that permits homologous recombination of the construct with the endogenous zsig99 locus, whereby the sequences within the construct become operably linked with the endogenous zsig99 coding sequence. In this way, an endogenous zsig99 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue- specific, or otherwise regulated expression. [51] Those skilled in the art will recognize that the sequence disclosed in SEQ ID NO:1 represents a single allele of human zsig99 and that allelic variation and .alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the nucleotide sequence shown in SEQ ID NO:1, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO:2. cDNA molecules generated from alternatively spliced mRNAs, which retain the properties of the zsig99 polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.

[52] Within an embodiment of the invention, the isolated nucleic acid molecules can hybridize under stringent conditions to nucleic acid molecules having the nucleotide sequence of SEQ ID NO: 1 from nucleotide 127 to 603, or the full length sequence, to nucleic acid molecules having a nucleotide sequence complementary to SEQ ID NO: 1. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.

[53] A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA- RNA, can hybridize if the nucleotide sequences have some degree of complementarity. Hybrids can tolerate mismatched base pairs in the double helix, but the stability of the hybrid is influenced by the degree of mismatch. The Tm of the mismatched hybrid decreases by I⁰C for every 1-1.5% base pair mismatch. Varying the stringency of the hybridization conditions allows control over the degree of mismatch that will be present in the hybrid. The degree of stringency increases as the hybridization temperature increases and the ionic strength of the hybridization buffer decreases.

[54] It is well within the abilities of one skilled in the art to adapt these conditions for use with a particular polynucleotide hybrid. The Tm for a specific target sequence is the temperature (under defined conditions) at which 50% of the target sequence will hybridize to a perfectly matched probe sequence. Those conditions which influence the Tm include, the size and base pair content of the polynucleotide probe, the ionic strength of the hybridization solution, and the presence of destabilizing agents in the hybridization solution. Numerous equations for calculating Tm are known in the art, and are specific for DNA, RNA and DNA- RNA hybrids and polynucleotide probe sequences of varying length (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual. Second Edition (Cold Spring Harbor Press 1989); Ausubel et al., (eds.), Current Protocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Grit. Rev. Biochem. MoI. Biol. 26:227 (1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake, MN) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto, CA), as well as sites on the Internet, are available tools for analyzing a given sequence and calculating Tm based on user defined criteria. Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences. Typically, hybridization of longer polynucleotide sequences, >50 base pairs, is performed at temperatures of about 20-25⁰C below the calculated Tm. For smaller probes, <50 base pairs, hybridization is typically carried out at the Tm or 5-1O⁰C below the calculated Tm. This allows for the maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids.

[55] Following hybridization, the nucleic acid molecules can be washed to remove non-hybridized nucleic acid molecules under stringent conditions, or under highly stringent conditions. Typical stringent washing conditions include washing in a solution of 0.5x - 2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 55 - 65°C. That is, nucleic acid molecules encoding a variant zsig99 polypeptide hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement) under stringent washing conditions, in which the wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, including 0.5x SSC with 0.1% SDS at 55°C, or 2x SSC with 0.1% SDS at 65⁰C. One of skill in the art can readily devise equivalent conditions, for example, by substituting SSPE for SSC in the wash solution.

[56] Typical highly stringent washing conditions include washing in a solution of O.lx - 0.2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 50 - 65⁰C. In other words, nucleic acid molecules encoding a variant zsig99 polypeptide hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to O.lx - 0.2x SSC with 0.1% SDS at 50 - 65⁰C, including O.lx SSC with 0.1% SDS at 50°C, or 0.2x SSC with 0.1% SDS at 65°C.

[57] The precise knowledge of a gene's position can be useful for a number of purposes, including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have.

[58] Analysis of chromosomal DNA using the zsig99 polynucleotide sequence is useful for correlating disease with abnormalities localized to chromosome 11. The human zsig99 gene has been localized to chromosome 1 IpI 1.2, which has several genes associated with prostate cancer in this chromosomal region (see, Online Mendelian Inheritance in Man, maintained by National Center for Biotechnology Information). Chromosome 11 contains six regions where deletions have been identified and correlated to carcinoma, including one at llpll.2 (Chunder et a!.. Diagn. MoI. Pathol. 13:172-182, 2004.) Moreover, LIF expression is considered a putative breast carcinoma marker (Zucchi et al, PNAS 101:18147-18152, 2004; Porter et al., MoI. Cancer Res. 1:362-375, 2003.)

[59] Use as a diagnostic could assist physicians in determining the type of disease and appropriate associated therapy, or could assist in genetic counseling. As such, the inventive anti-zsig99 antibodies, polynucleotides, and polypeptides can be used for the detection of zsig99 polypeptide, mRNA or anti-zsig99 antibodies, thus serving as markers and be directly used for detecting genetic diseases or cancers, as described herein, using methods known in the art and described herein. Further, zsig99 polynucleotide probes can be used to detect abnormalities involving chromosome llpll.2 as described herein. Thus, zsig99 polynucleotide probes can be used to detect abnormalities or genotypes associated with these defects.

[60] Positions of introns in the human zsig99 gene were determined by identification of genomic clones and comparison to of the cDNA to the genomic sequence. The coding regions for the zsig99 molecule are contained in three exons. The exons following each of the two introns have phases of 2-0, which is shared with other genes encoding leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neutroptrophic factor 1 (CNTFl), interleukin 31 (IL-31).

[61] Polypeptides of the present invention comprise at least 6, preferably at least 9, more preferably at least 15 contiguous amino acid residues of SEQ ID NO:2. Within certain embodiments of the invention, the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of SEQ ID NO:2, up to the entire predicted mature polypeptide (residues 43 to 201 of SEQ ID NO:2) or the primary translation product (residues 19 to 201 of SEQ ID NO:2). As disclosed in more detail below, these polypeptides can further comprise additional, non-zsig99, polypeptide sequence(s).

[62] Within the scope of the present invention are polypeptides that comprise an epitope-bearing portion of a protein as shown in SEQ ID NO:2. An "epitope" is a region of a protein to which an antibody can bind. See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002, 1984. Epitopes can be linear or conformational, the latter being composed of discontinuous regions of the protein that form an epitope upon folding of the protein. Linear epitopes are generally at least nine amino acid residues in length. Relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, Sutcliffe et al., Science 219:660-666, 1983. Antibodies that recognize short, linear epitopes are particularly useful in analytic and diagnostic applications that employ denatured protein, such as Western blotting (Tobin, Proc. Natl. Acad. Sci. USA 76:4350-4356. 1979), or in the analysis of fixed cells or tissue samples. Antibodies to linear epitopes are also useful for detecting fragments of zsig99, such as might occur in body fluids or cell culture media.

[63] The present invention also provides isolated zsig99 polypeptides that have a substantially similar sequence identity to the polypeptides of SEQ ID NO:2, or their orthologs. The term "substantially similar sequence identity" is used herein to denote polypeptides comprising at least 70% to 80%, and in certain embodiments at least 90% to 95%, or in other embodiments greater than 95% sequence identity to the sequences shown in SEQ ID NO:2, or their orthologs. The present invention also includes polypeptides that comprise an amino acid sequence having at least 70% to 80%, and in certain embodiments at least 90% to 95%, or in other embodiments greater than 95% sequence identity to the sequence of amino acid residues 1 or 43 to 201 of SEQ ID NO:2. The present invention further includes nucleic acid molecules that encode such polypeptides. Methods for determining percent identity are described below.

[64] Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by the standard one-letter codes).

Total number of identical matches : x 100

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

[65] The level of identity between amino acid sequences can be determined using the "FASTA" similarity search algorithm disclosed by Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988) and by Pearson (Meth. Enzvmol. 183:63. 1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO:2) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. Jf there are several regions with scores greater than the "cutoff' value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. MoI. Biol. 48:444, 1970; Sellers, SIAM J. Appl. Math. 26:787, 1974), which allows for amino acid insertions and deletions. Preferred parameters for FASTA analysis are: ktuρ=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, 1990 (ibid.).

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

[67] The present invention includes polypeptides having one or more conservative amino acid changes as compared with the amino acid sequence of SEQ ID NO:2. The BLOSUM62 matrix (Table 1) is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, ibjd.). Thus, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. As used herein, the term "conservative amino acid substitution" refers to a substitution represented by a BLOSUM62 value of greater than 1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least one 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3). [68] Variant zsig99 polypeptides or polypeptides with substantially similar sequence identity are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and amino- or carboxyl- terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. The present invention thus includes polypeptides of from about 28 to 354 amino acid residues that comprise a sequence that is at least 70% to 80%, and in certain embodiments at least 90% to 95%, or in other embodiments greater than 95% or more identical to the corresponding region of SEQ ID NO:2. In particular, peptides and polypeptides corresponding to regions of the zsig99 molecules as shown in SEQ ID NO: 2 include the helix A (residues 64-89), helix B (residues 115-130), helix C (comprising residues 140-154) and helix D (residues 186-200) are within the scope of the present invention. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zsig99 polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.

Table 4

Conservative amino acid substitutions

Basic: arginine lysine histidine

Acidic: glutamic acid aspartic acid

Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine

Aromatic: phenylalanine tryptophan tyrosine

Small: glycine alanine serine threonine methionine

[69] The proteins of the present invention can also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, trans-3- methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occuring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzvmol. 202:301, 1991; Chung et al., Science 259:806-809, 1993; and Chung et al., Proc. Natl. Acad. ScL USA 90:10145-10149, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-19998, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non- naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[70] Amino acid sequence changes are made in zsig99 polypeptides so as to minimize disruption of higher order structure essential to biological activity. Amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can identify specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule. Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity, secondary structure propensities, binary patterns, complementary packing, and buried polar interactions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al., Current Qpin. Struct. Biol. 6:3-10, 1996). In general, determination of structure will be accompanied by evaluation of activity of modified molecules. For example, changes in amino acid residues will be made so as not to disrupt the helical- bundle structure of the protein family. The effects of amino acid sequence changes can be predicted by, for example, computer modeling using available software (e.g., the Insight II® viewer and homology modeling tools; MSI, San Diego, CA) or determined by analysis of crystal structure (see, e.g., Lapthorn et al, Nature 369:455-461, 1994; Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995). Protein folding can be measured by circular dichroism (CD). Measuring and comparing the CD spectra generated by a modified molecule and standard molecule are routine in the art (Johnson, Proteins 7:205-214, 1990). Crystallography is another well known and accepted method for analyzing folding and structure. Nuclear magnetic resonance (NMR), digestive peptide mapping and epitope mapping are other known methods for analyzing folding and structural similarities between proteins and polypeptides (Schaanan et al., Science 257:961-964, 1992). Mass spectrometry and chemical modification using reduction and alkylation can be used to identify cysteine residues that are associated with disulfide bonds or are free of such associations (Bean et al., Anal. Biochem. 201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Patterson et al., Anal. Chem. 66:3727- 3732, 1994). Alterations in disulfide bonding will be expected to affect protein folding. These techniques can be employed individually or in combination to analyze and compare the structural features that affect folding of a variant protein or polypeptide to a standard molecule to determine whether such modifications would be significant.

[71] Those skilled in the art will recognize that hydrophilicity will be taken into account when designing alterations in the amino acid sequence of a zsig99 polypeptide, so as not to disrupt the overall profile. Residues within the core of the helical bundle can be replaced with a hydrophobic residue selected from the group consisting of Leu, He, VaI, Met, Phe, Trp, GIy. The residues predicted to be on the exposed surface of the helical bundle will be relatively intolerant of substitution. The length and amino acid composition of the interdomain loops are also expected to be important for receptor binding (and therefore biological activity); conservative substitutions and relatively small insertions and deletions are thus preferred within the loops, and the insertion of bulky amino acid residues (e.g., Phe) will in general be avoided.

[72] Essential amino acids in the polypeptides of the present invention can be identified experimentally according to procedures known in the art, such as site-directed mutagenesis or alanine- scanning mutagenesis (Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule.

[73] The present invention also includes "functional fragments" of zsig99 polypeptides and nucleic acid molecules encoding such functional fragments. As previously described herein, zsig99 is characterized by a four-helical bundle. Thus, the present invention further provides fusion proteins encompassing (a) polypeptide molecules comprising one or more of the regions described above, and (b) biologically active fragments comprising portions of one or more of the domains. The other polypeptide may be another regions from another cytokine, a non-native and/or an unrelated secretory signal peptide to facilitate secretion of the fusion protein. Thus, as described herein functional domains of zsig99 will be useful for preparing chimeric fusion proteins that combine helical loop domains from other short form cytokines. Such chimeras will have specificity determined by the component regions used (Kallen et al, ibid., 1999). Chimeric molecules can be prepared using one or more helices of secretory signal sequence from zsig99 in combination with corresponding regions from other cytokines.

[74] Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes an zsig99 polypeptide. As an illustration, DNA molecules having the nucleotide sequence of SEQ ID NO:1 can be digested with Bal31 nuclease to obtain a series of nested deletions. The fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for zsig99, or for the ability to bind anti-zsig99 antibodies. One alternative to exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired fragment. Alternatively, particular fragments of an zsig99 gene can be synthesized using the polymerase chain reaction.

[75] Standard methods for identifying functional domains are well-known to those of skill in the art. For example, studies on the truncation at either or both termini of interferons have been summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover, standard techniques for functional analysis of proteins are described by, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993), Content et al., "Expression and preliminary deletion analysis of the 42 kDa 2 5A synthetase induced by human interferon," in Biological Interferon Systems. Proceedings of ISIR TNO Meeting on Interferon Systems. Cantell (ed.), pages 65 72 (Nijhoff 1987), Herschman, "The EGF Receptor," in Control of Animal Cell Proliferation, Vol. 1, Boynton et al., (eds.) pages 169-199 (Academic Press 1985), Coumailleau et al., J. Biol. Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995), and Meisel et al., Plant Molec. Biol. 30:1 (1996).

[76] A Hopp/Woods hydrophilicity profile of the zsig99 protein sequence as shown in SEQ ID NO:2 was generated (Hopp et al., Proc. Natl. Acad. Sci. USA 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. Buried G, S, and T residues and exposed H, Y, and W residues were ignored. Hydrophilicity can be used to determine regions that have the most antigenic potential. Residues 60-65, 86-92, 94-100, 110-115, 123-129, 153-161, and 183-188 as shown in SEQ ID NO:2 were identified as hydrophilic.

[77] Polypeptides of the present invention comprise at least 6, preferably at least 9, more preferably at least 15 contiguous amino acid residues of SEQ ID NO:2. Within certain embodiments of the invention, the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of SEQ ID NO:2, up to the entire mature polypeptide (residues 1 to 201 of SEQ ID NO:2) or the primary translation product (residues 43 to 201 of SEQ ID NO: 2). As disclosed in more detail below, these polypeptides can further comprise additional, non-zsig99 polypeptide sequence(s).

[78] Within the polypeptides of the present invention are polypeptides that comprise an epitope-bearing portion of a protein as shown in SEQ ID NO:2. An "epitope" is a region of a protein to which an antibody can bind. See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998- 4002, 1984. Epitopes can be linear or conformational, the latter being composed of discontinuous regions of the protein that form an epitope upon folding of the protein. Linear epitopes are generally at least 6 amino acid residues in length. Relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, Sutcliffe et al., Science 219:660-666. 1983. Antibodies that recognize short, linear epitopes are particularly useful in analytic and diagnostic applications that employ denatured protein, such as Western blotting (Tobin, Proc. Natl. Acad. Sci. USA 76:4350-4356, 1979), or in the analysis of fixed cells or tissue samples. Antibodies to linear epitopes are also useful for detecting fragments of zsig99 such as might occur in body fluids or cell culture media.

[79] Antigenic, epitope-bearing polypeptides of the present invention are useful for raising antibodies, including monoclonal antibodies, that specifically bind to a zsig99 protein. Antigenic, epitope-bearing polypeptides contain a sequence of at least six, preferably at least nine, more preferably from 15 to about 30 contiguous amino acid residues of a zsig99 protein (e.g., SEQ ID NO:2). Polypeptides comprising a larger portion of a zsig99 protein, i.e. from 30 to 50 residues up to the entire sequence, are included. It is preferred that the amino acid sequence of the epitope-bearing polypeptide is selected to provide substantial solubility in aqueous solvents, that is the sequence includes relatively hydrophilic residues, and hydrophobic residues are substantially avoided.

[80] For any zsig99 polypeptide, including variants and fusion proteins, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above.

[81] The present invention further provides a variety of other polypeptide fusions (and related multimeric proteins comprising one or more polypeptide fusions). For example, a zsig99 polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in this regard include immunoglobulin constant region domains. Immunoglobulin- zsig99 polypeptide fusions can be expressed in genetically engineered cells (to produce a variety of multimeric zsig99 analogs). Auxiliary domains can be fused to zsig99 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen). For example, a zsig99 polypeptide or protein could be targeted to a predetermined cell type by fusing a zsig99 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell, or fused to an antibody directed to an antigen expressed on a specific target cell type. In this way, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. A zsig99 polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain. Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34: 1-9, 1996.

[82] The zsig99 polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides can be produced according to conventional techniques using cells into which have been introduced an expression vector encoding the polypeptide. As used herein, "cells into which have been introduced an expression vector" include both cells that have been directly manipulated by the introduction of exogenous DNA molecules and progeny thereof that contain the introduced DNA. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al, eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

[83] Ih general, a DNA sequence encoding a zsig99 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.

[84] A wide variety of suitable recombinant host cells includes, but is not limited to, gram- negative prokaryotic host organisms. Suitable strains of E. coli include W3110, K12-derived strains MM294, TG-I, JM-107, BL21, and UT5600. Other suitable strains include: BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, DHl, DH4I, DH5, DH5I, DH5IF, DH5MCR, DHlOB, DH10B/p3, DHIlS, C600, HBlOl, JMlOl, JM105, JM109, JMHO, K38, RRl, Y1088, Y1089, CSH18, ER1451, ER1647, E. coli K12, E. coli K12 RV308, E. coli K12 C600, E. coli HBlOl, E. coli K12 C600 /?._k-M_k-, E. coli K12 RRl (see, for example, Brown (ed.), Molecular Biology Labfax (Academic Press 1991)). Other gram-negative prokaryotic hosts can include Serratia, Pseudomonas, Caulobacter. Prokaryotic hosts can include gram-positive organisms such as Bacillus, for example, B. subtilis and B. thuringienesis, and B. thuringienesis var. israelensis, as well as Streptomyces, for example, S. lividans, S. ambofaciens, S. fradiae, and S. griseofiiscus. Suitable strains of Bacillus subtilus include BR151, YB886, Mil 19, MI120, and B 170 (see, for example, Hardy, "Bacillus Cloning Methods," in DNA Cloning: A Practical Approach, Glover (ed.) (JJRL Press 1985)). Standard techniques for propagating vectors in prokaryotic hosts are well-known to those of skill in the art (see, for example, Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3rd Edition (John Wiley & Sons 1995); Wu et al., Methods in Gene Biotechnology (CRC Press, Inc. 1997)).

[85] When large scale production of zsig99 using the expression system of the present invention is required, batch fermentation can be used. Generally, batch fermentation comprises that a first stage seed flask is prepared by growing E. coli strains expressing zsig99 in a suitable medium in shake flask culture to allow for growth to an optical density (OD) of between 5 and 20 at 600 run. A suitable medium would contain nitrogen from a source(s) such as ammonium sulfate, ammonium phosphate, ammonium chloride, yeast extract, hydrolyzed animal proteins, hydrolyzed plant proteins or hydrolyzed caseins. Phosphate will be supplied from potassium phosphate, ammonium phosphate, phosphoric acid or sodium phosphate. Other components would be magnesium chloride or magnesium sulfate, ferrous sulfate or ferrous chloride, and other trace elements. Growth medium can be supplemented with carbohydrates, such as fructose, glucose, galactose, lactose, and glycerol, to improve growth. Alternatively, a fed batch culture is used to generate a high yield of zcyto32 protein. The zsig99 producing E. coli strains are grown under conditions similar to those described for the first stage vessel used to inoculate a batch fermentation.

[86] Following fermentation the cells are harvested by centrifugation, re-suspended in homogenization buffer and homogenized, for example, in an APV-Gaulin homogenizer (Invensys APV, Tonawanda, New York) or other type of cell disruption equipment, such as bead mills or sonicators. Alternatively, the cells are taken directly from the fermentor and homogenized in an APV-Gaulin homogenizer. The washed inclusion body prep can be solubilized using guanidine hydrochloride (5-8 M) or urea (7 - 8 M) containing a reducing agent such as beta mercaptoethanol (10 - 100 mM) or dithiothreitol (5-50 mM). The solutions can be prepared in Tris, phopshate, HEPES or other appropriate buffers. Inclusion bodies can also be solubilized with urea (2-4 M) containing sodium lauryl sulfate (0.1-2%). In the process for recovering purified zsig99 from transformed E. coli host strains in which the zsig99 is accumulates as retractile inclusion bodies, the cells are disrupted and the inclusion bodies are recovered by centrifugation. The inclusion bodies are then solubilized and denatured in 6 M guanidine hydrochloride containing a reducing agent. The reduced zsig99 is then oxidized in a controlled renaturation step. Refolded zsig99 can be passed through a filter for clarification and removal of insoluble protein. The solution is then passed through a filter for clarification and removal of insoluble protein. After the zsig99 protein is refolded and concentrated, the refolded zsig99 protein is captured in dilute buffer on a cation exchange column and purified using hydrophobic interaction chromatography.

[87] To direct a zsig99 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of zsig99, or may be derived from another secreted protein (e.g., t-PA; see, U.S. Patent No. 5,641,655) or synthesized de novo. The secretory signal sequence is operably linked to the zsig99 DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).

[88] Cultured mammalian cells are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-5, 1982), DEAE-dextran mediated transfection (Ausubel et al., ibid.), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90, 1989; Wang and Finer, Nature Med. 2:714-6, 1996). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Patent No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. Suitable cultured mammalian cells include the COS-I (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-Kl; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, VA. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

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

[90] Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POTl vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-65, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533. The use of Pichia methanolica as host for the production of recombinant proteins is disclosed in U.S. Patent Nos. 5,955,349, 5,888,768 and 6,001,597, U.S. Patent No. 5,965,389, U.S. Patent No. 5,736,383, and U.S. Patent No. 5,854,039.

[91] It is preferred to purify the polypeptides of the present invention to >80% purity, more preferably to >90% purity, even more preferably >95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.

[92] Expressed recombinant zsig99 proteins (including chimeric polypeptides and multimeric proteins) are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See, in general, Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice, Springer- Verlag, New York, 1994. Proteins comprising a polyhistidine affinity tag (typically about 6 histidine residues) are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bio/Technol. 6: 1321-1325, 1988. Proteins comprising a glu-glu tag can be purified by immunoaffinity chromatography according to conventional procedures. See, for example, Grussenmeyer et al., ibid. Maltose binding protein fusions are purified on an amylose column according to methods known in the art.

[93] Zsig99 polypeptides can also be prepared through chemical synthesis according to methods known in the art, including exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, EL, 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, ERL Press, Oxford, 1989. La vitro synthesis is particularly advantageous for the preparation of smaller polypeptides.

[94] Using methods known in the art, zsig99 proteins can be prepared as monomers, heterodimers (particularly with LEF as the heterodimeric partner) or multimers; glycosylated or non- glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.

[95] Target cells for use in zsig99 activity assays include, without limitation, vascular cells (especially endothelial cells and smooth muscle cells), hematopoietic (myeloid, erythroid and lymphoid) cells, liver cells (including hepatocytes, fenestrated endothelial cells, Kupffer cells, and Ito cells), fibroblasts (including human dermal fibroblasts and lung fibroblasts), fetal lung cells, articular synoviocytes, pericytes, chondrocytes, osteoblasts, and prostate epithelial cells. Endothelial cells and hematopoietic cells are derived from a common ancestral cell, the hemangioblast (Choi et al., Development 125:725-732. 1998).

[96] Zsig99 proteins of the present invention are characterized by their activity, that is, modulation of the proliferation, differentiation, migration, adhesion, or metabolism of responsive cell types. Biological activity of zsig99 proteins is assayed using in vitro or in vivo assays designed to detect cell proliferation, differentiation, migration or adhesion; or changes in cellular metabolism (e,g., production of other growth factors or other macromolecules). Many suitable assays are known in the art, and representative assays are disclosed herein. Assays using cultured cells are most convenient for screening, such as for determining the effects of amino acid substitutions, deletions, or insertions. However, in view of the complexity of developmental processes (e.g., angiogenesis, wound healing), in vivo assays will generally be employed to confirm and further characterize biological activity. 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 assay histological effects. Assays can be performed using exogenously produced proteins, or may be carried out in vivo or in vitro using cells expressing the polypeptide(s) of interest. Assays can be conducted using zsig99 proteins alone or in combination with other growth factors, such as members of the VEGF family or hematopoietic cytokines (e.g., EPO, TPO, G-CSF, stem cell factor). Representative assays are disclosed below.

[97] Activity of zsig99 proteins can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to an appropriate animal model. Assays measuring cell proliferation or differentiation are well known in the art. For example, assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354. 1990), incorporation of radiolabeled nucleotides (as disclosed by, e.g., Raines and Ross, Methods Enzvmol.109:749-773, 1985; Wahl et al., MoI. Cell Biol. 8:5016- 5025, 1988; and Cook et al., Analytical Biochem. 179:1-7, 1989), incorporation of 5-bromo-2'- deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J. Immunol. Methods 82:169-179, 1985), and use of tetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res. 48:4827-4833, 1988). Differentiation can be assayed using suitable precursor cells that can be induced to differentiate into a more mature phenotype. Assays measuring differentiation include, for example, measuring cell-surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses. 161-171, 1989).

[98] Zsig99 activity may also be detected using assays designed to measure zsig99- induced production of one or more additional growth factors or other macromolecules. Preferred such assays include those for determining the presence of hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor alpha (TGFα), interleukin-6 (IL-6), VEGF, acidic fibroblast growth factor (aFGF), angiogenin, and other macromolecules produced by the liver. Suitable assays include autogenesis assays using target cells responsive to the macromolecule of interest, receptor-binding assays, competition binding assays, immunological assays (e.g., ELISA), and other formats known in the art. Metalloprotease secretion is measured from treated primary human dermal fibroblasts, synoviocytes and chondrocytes. The relative levels of collagenase, gelatinase and stromalysin produced in response to culturing in the presence of a zsig99 protein is measured using zymogram gels (Loita and Stetler-Stevenson, Cancer Biology 1:96-106, 1990). Procollagen/collagen synthesis by dermal fibroblasts and chondrocytes in response to a test protein is measured using ³H-proline incorporation into nascent secreted collagen. 3H-labeled collagen is visualized by SDS-PAGE followed by autoradiography (Unemori and Amento, J. Biol. Chem. 265: 10681-10685, 1990). Glycosaminoglycan (GAG) secretion from dermal fibroblasts and chondrocytes is measured using a 1,9-dimethylmethylene blue dye binding assay (Farndale et al., Biochim. Biophys. Acta 883:173-177, 1986). Collagen and GAG assays are also carried out in the presence of IL-lα or TGF-α to examine the ability of zsig99 protein to modify the established responses to these cytokines.

[99] Monocyte activation assays are carried out (1) to look for the ability of zsig99 proteins to further stimulate monocyte activation, and (2) to examine the ability of zsig99 proteins to modulate attachment-induced or endotoxin-induced monocyte activation (Fuhlbrigge et al., J₁ Immunol. 138: 3799-3802, 1987). IL-lαand TNFα levels produced in response to activation are measured by ELISA (Biosource, Inc. Camarillo, CA). Monocyte/macrophage cells, by virtue of CD 14 (LPS receptor), are exquisitely sensitive to endotoxin, and proteins with moderate levels of endotoxin-like activity will activate these cells. W 2

32

[100] Hematopoietic activity of zsig99 proteins can be assayed on various hematopoietic cells in culture. Preferred assays include primary bone marrow colony assays and later stage lineage- restricted colony assays, which are known in the art (e.g., Holly et al., WIPO Publication WO 95/21920). Marrow cells plated on a suitable semi-solid medium (e.g., 50% methylcellulose containing 15% fetal bovine serum, 10% bovine serum albumin, and 0.6% PSN antibiotic mix) are incubated in the presence of test polypeptide, then examined microscopically for colony formation. Known hematopoietic factors are used as controls. Mitogenic activity of zsig99 polypeptides on hematopoietic cell lines can be measured as disclosed above.

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

[102] Cell adhesion activity is assayed essentially as disclosed by LaFleur et al. (J. Biol. Chem. 272:32798-32803, 1997). Briefly, microtiter plates are coated with the test protein, nonspecific sites are blocked with BSA, and cells (such as smooth muscle cells, leukocytes, or endothelial cells) are plated at a density of approximately 104 - 105 cells/well. The wells are incubated at 37°C (typically for about 60 minutes), then non-adherent cells are removed by gentle washing. Adhered cells are quantitated by conventional methods (e.g., by staining with crystal violet, lysing the cells, and determining the optical density of the lysate). Control wells are coated with a known adhesive protein, such as fibronectin or vitronectin.

[103] The activity of zsig99 proteins can be measured with a silicon-based biosensor microphysiometer that measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses. An exemplary such device is the Cytosensor™ Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA. A variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell et al., Science 257:1906-1912, 1992; Pitchford et al., Meth. Enzvmol. 228:84-108, 1997; Arimilli et al., J. Immunol. Meth. 212:49-59, 1998; and Van Liefde et al., Eur. J. Pharmacol. 346:87- 95, 1998.

[104] One in vivo approach for assaying proteins of the present invention utilizes viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acids. For review, see Becker et al., Meth. Cell Biol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine 4:44-53, 1997. The adenovirus system offers several advantages. Adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.

[105] Transgenic mice, engineered to express a zsig99 gene, and mice that exhibit a complete absence of zsig99 gene function, referred to as "knockout mice" (Snouwaert et al., Science 257:1083, 1992), can also be generated (Lowell et al., Nature 366:740-742, 1993). These mice can be employed to study the zsig99 gene and the protein encoded thereby in an in vivo system. Transgenic mice are particularly useful for investigating the role of zsig99 proteins in early development in that they allow the identification of developmental abnormalities or blocks resulting from the over- or underexpression of a specific factor. See also, Maisonpierre et al., Science 277:55-60, 1997 and Hanahan, Science 277:48-50. 1997. Preferred promoters for transgenic expression include promoters from metallothionein and albumin genes.

[106] Another approach uses a hydrodynamic push for in vivo transient expression. Similarly, direct measurement of zsig99 polypeptide, or its loss of expression in a tissue can be determined in a tissue or cells as they undergo tumor progression. Increases in invasiveness and motility of cells, or the gain or loss of expression of zsig99 in a pre-cancerous or cancerous condition, in comparison to normal tissue, can serve as a diagnostic for transformation, invasion and metastasis in tumor progression. As such, knowledge of a tumor's stage of progression or metastasis will aid the physician in choosing the most proper therapy, or aggressiveness of treatment, for a given individual cancer patient. Methods of measuring gain and loss of expression (of either mRNA or protein) are well known in the art and described herein and can be applied to zsig99 expression. For example, appearance or disappearance of polypeptides that regulate cell motility can be used to aid diagnosis and prognosis of prostate cancer (Banyard, J. and Zetter, B.R., Cancer and Metast. Rev. 17:449-458, 1999). As an effector of cell motility, or as a liver-specific marker, zsig99 gain or loss of expression may serve as a diagnostic for liver, neuroblastoma, endothelial, brain, and other cancers. [107] Moreover, the activity and effect of zsig99 on tumor progression and metastasis can be measured in vivo. Several syngeneic mouse models have been developed to study the influence of polypeptides, compounds or other treatments on tumor progression. In these models, tumor cells passaged in culture are implanted into mice of the same strain as the tumor donor. The cells will develop into tumors having similar characteristics in the recipient mice, and metastasis will also occur in some of the models. Appropriate tumor models for our studies include the Lewis lung carcinoma (ATCC No. CRL-1642) and B 16 melanoma (ATCC No. CRL-6323), amongst others. These are both commonly used tumor lines, syngeneic to the C57BL6 mouse, that are readily cultured and manipulated in vitro. Tumors resulting from implantation of either of these cell lines are capable of metastasis to the lung in C57BL6 mice. The Lewis lung carcinoma model has recently been used in mice to identify an inhibitor of angiogenesis (O'Reilly MS, et al. Cell 79: 315-328,1994). C57BL6/J mice are treated with an experimental agent either through daily injection of recombinant protein, agonist or antagonist or a one-time injection of recombinant adenovirus. Three days following this treatment, 10⁵ to 10⁶ cells are implanted under the dorsal skin. Alternatively, the cells themselves may be infected with recombinant adenovirus, such as one expressing zsig99, before implantation so that the protein is, synthesized at the tumor site or intracellularly, rather than systemically. The mice normally develop visible tumors within 5 days. The tumors are allowed to grow for a period of up to 3 weeks, during which time they may reach a size of 1500 - 1800 mm³ in the control treated group. Tumor size and body weight are carefully monitored throughout the experiment. At the time of sacrifice, the tumor is removed and weighed along with the lungs and the liver. The lung weight has been shown to correlate well with metastatic tumor burden. As an additional measure, lung surface metastases are counted. The resected tumor, lungs and liver are prepared for histopathological examination, immunohistochemistry, and in situ hybridization, using methods known in the art and described herein. The influence of the expressed polypeptide in question, e.g., zsig99, on the ability of the tumor to recruit vasculature and undergo metastasis can thus be assessed. In addition, aside from using adenovirus, the implanted cells can be transiently transfected with zsig99. Use of stable zsig99 transfectants as well as use of induceable promoters to activate zsig99 expression in vivo are known in the art and can be used in this system to assess zsig99 induction of metastasis. Moreover, purified zsig99 or zsig99-conditioned media can be directly injected in to this mouse model, and hence be used in this system. For general reference see, O'Reilly MS, et al. Cell 79:315-328, 1994; and Rusciano D, et al. Murine Models of Liver Metastasis. Invasion Metastasis .14:349-361, 1995.

[108] Antisense methodology can be used to inhibit zsig99 gene transcription to examine the effects of such inhibition in vivo. Polynucleotides that are complementary to a segment of a zsig99-encoding polynucleotide (e.g., a polynucleotide as set forth in SEQ ID NO:1) are designed to bind to zsig99-encoding mRNA and to inhibit translation of such mRNA. Such antisense oligonucleotides can also be used to inhibit expression of zsig99 polypeptide-encoding genes in cell culture.

[109] LIF is presently being tested in patients with advanced cancer (Gunawardana et al., Clinical Cancer Res. 9:2056-2065, 2003.) In a phase I clinical report, a faster platelet and neutrophil recovery was seen in some patients after chemotherapy. LIF knockout mice have shown that LIF plays a non-redundant role in blastocyst implantation and is necessary for establishing pregnancy (Metcalf, Stem Cells 21:5-14, 2003.) Higher levels of LIF during pregnancy is associated with reduced maternal to fetal transmission of HIV. LlF also inhibits HIV replication in vitro without growth-inhibiting the target cells (Patterson et al., J. Clin. Investig. 107:287-294, 2001.) LIF maintains the totipotency of mouse embryonic stem cells (ES) and is routinely used in culturing such cells. However, this has not been demonstrated in human ES cells and it may be the LIF/zsig99 heterodimer is required. Some cell types that are inhibited by LIF include myeloid leukemias where it acts to block self -renewal and induces differentiation, human breast epithelial cells and breast cancer cells, vascular endotherlial cells; and olfactory receptor neurons. Some cells are stimulated by LIF and include megakaryocytes and blast CFU, some hematopoietic cell lines, sensory and motor neurons, osteoclassts and osteoblasts, muscle satellite cells, and cardiac myocytes (Metcalf, ibid.). LIF has also been described as regulating ACTH production, inhibiting prolactin and growth hormone production (Metcalf, ibid.).

[110] Most four-helix bundle cytokines as well as other proteins produced by activated lymphocytes play an important biological role in cell differentiation, activation, recruitment and homeostasis of cells throughout the body. Zsig99 and inhibitors of zsig99 activity are expected to have a variety of therapeutic applications. These therapeutic applications include treatment of diseases which require immune regulation, including autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, and diabetes. Zsig99 may be important in the regulation of inflammation, and therefore would be useful in treating rheumatoid arthritis, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), asthma, multiple sclerosis, atopic dermatitis and sepsis. There may be a role of zsig99 in mediating tumorgenesis, whereby a zsig99 antagonist would be useful in the treatment of cancer. Zsig99 may be useful in modulating the immune system, whereby zsig99 and zsig99 antagonists may be used for reducing graft rejection, preventing graft-vs-host disease, boosting immunity to infectious diseases, treating immunocompromised patients (e.g., HIV+ patients), or in improving vaccines.

[Ill] Dendritic cells have activities that are specifically associated with the maturity of the cell, i.e., its differentiated state. To identify a cell's maturity, a population of established cells is assayed and analyzed for a set of differentiation markers that are characteristic of the cell's stage in the differentiation pathway. Preferably, this is done by isolating at least a portion of the cells and subjecting this subpopulation to such analysis. [112] A set of differentiation markers is defined as one or more phenotypic properties that can be identified, and that are specific to a particular cell type and stage of maturity. Differentiation markers are transiently exhibited at various stages of the cell's progression toward terminal differentiation. Pluripotent stem cells that can regenerate without commitment to a specific cell lineage express a set of differentiation markers that are diminished when commitment to a particular cell lineage is made. Precursor cells express a set of differentiation markers that may or may not continue to be expressed as the cells progress down the cell lineage pathway toward maturation. Differentiation markers that are expressed exclusively by mature cells usually represent functional properties, such as cell products, enzymes to produce cell products and receptors. It is possible that with exposure to the appropriate factors, the cell line of the present invention can differentiate and mature into other cells of the monocytic cell lineage. Differentiation markers used for identifying dendritic cells include: Mac-1, F4/80, FcγRMII receptor (FcR), MHC class I, MHC class II, B7-1, B7-2, ICAM-I, CD44, N418, and NLDC-145.

[113] Identification of activated dendritic cells is confirmed by the cells' ability to stimulate the proliferation of allogeneic T cells in a MLR. Briefly, activated dendritic cells are incubated with allogeneic T cells in a 96-well microtiter dish (American Scientific Products, Chicago, EL). Stimulation of the T cells to proliferate is measured by incorporation of ³H~thymidine. It is preferred to expose the dendritic cells of the present invention to irradiation to slow the proliferation of the dendritic cells and reduce background in the assay caused by incorporation of ³H-thymidine by the dendritic cells.

[114] The dendritic cells are activated to induce expression of MHC class II molecules on the cell surface, making these mature dendritic cells competent for antigen processing and presentation. These activated cells (i.e., stimulators) are then exposed to antigen for a time sufficient for antigen presentation. One skilled in the art would recognize that the time required for endocytosis, processing and presentation of antigen is dependent upon the proteinaceous antigen being used for this purpose. Methods for measuring antigen uptake and presentation are known in the art. For example, dendritic cells can be incubated with a soluble protein antigen (e.g., ovalbumin or conalbumin) for 3-24 hours then washed to remove exogenous antigen.

[115] These antigen-presenting stimulator cells are then mixed with responder cells, preferably naive or antigen-primed T lymphocytes. After an approximately 72 hour incubation (for primed T lymphocytes) or approximately 4-7 d period (for naive T lymphocytes), the activation of T cells in response to the processed and presented antigen is measured. In a preferred embodiment, T cell activation is determined by measuring T cell proliferation using 3H-thymidine uptake (Crowley et al., J. Immunol. Meth. 133:55-66, 1990). The responder cells in this regard can be PBMN cells, cultured T cells, established T cell lines or hybridomas. Responder cell activation can be measured by the production of cytokines, such as IL-2, or by determining T cell-specific activation markers. Cytokine production can be assayed by the testing the ability of the stimulator + responder cell culture supernatant to stimulate growth of cytokine-dependent cells. T cell-specific activation markers may be detected using antibodies specific for such markers.

[116] For T cell proliferation assays, it is preferred to inhibit the proliferation of dendritic cells prior to mixing with T responder cells. This inhibition may be achieved by exposure to gamma irradiation or to an anti-mitotic agent, such as mitomycin C.

[117] Alternatively, activated dendritic cells can be used to induce non-responsiveness in T lymphocytes. In addition to MHC class II recognition, T cell activation requires co-receptors on the antigen-presenting cell (APC; e.g., the dendritic cell) that have been stimulated with co-stimulatory molecules. By blocking or eliminating stimulation of such co-receptors (for instance, by blocking with anti-receptor or anti-ligand antibodies, or by "knocking out" the gene(s) encoding such receptors), presentation of antigen by co-receptor-deficient dendritic cells can be used to render T lymphocytes non-responsive to antigen.

[118] Zsig99 proteins may be used either alone or in combination with other hematopoietic factors such as IL-3, G-CSF, GM-CSF, IL-4, M-CSF, IL-12 or stem cell factor to enhance expansion and mobilization of hematopoietic or mesenchymal stem cells, including precursor stem cells. Cells that can be expanded in this manner include cells isolated from bone marrow, cells isolated from blood, neonatal heart or liver. Zsig99 proteins may also be given directly to an individual to enhance stem cell production and differentiation within the treated individual. In particular, zsig99 can be used for expansion of dendritic cell and dendritic cell precursor populations.

[119] Administration of a zsig99 polypeptide to a subject can be topical, inhalant, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection. When administering therapeutic proteins by injection, the administration may be by continuous infusion or by single or multiple boluses.

[120] Additional routes of administration include oral, mucosal-membrane, pulmonary, and transcutaneous. Oral delivery is suitable for polyester microspheres, zein microspheres, proteinoid microspheres, polycyanoacrylate microspheres, and lipid-based systems (see, for example, DiBase and Morrel, "Oral Delivery of Microencapsulated Proteins," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)). In general, pharmaceutical formulations will include a zsig99 polypeptide in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water, or the like. Other suitable vehicles are well-known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995). A formulation is said to be a "pharmaceutically acceptable vehicle" if its administration can be tolerated by a recipient patient. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995. Zsig99 will preferably be used in a concentration of about 10 to 100 μg/ml of total volume, although concentrations in the range of 1 ng/ml to 1000 μg/ml may be used. For topical application, such as for the promotion of wound healing, the protein will be applied in the range of 0.1-10 μg/cm² of wound area, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art. Dosing is daily or inteπnittently over the period of treatment. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. Sustained release formulations can also be employed. In general, a "therapeutically effective amount" of zsig99 is an amount sufficient to produce a clinically significant change in the treated condition, such as a clinically significant change in hematopoietic or immune function, a significant reduction in morbidity, or a significantly increased histological score.

[121] A pharmaceutical formulation comprising zsig99 (or zsig99 analog or fusion protein) can be furnished in liquid form, in an aerosol, or in solid form. Liquid forms, are illustrated by injectable solutions, aerosols, droplets, topological solutions and oral suspensions. Exemplary solid forms include capsules, tablets, and controlled-release forms. The latter form is illustrated by miniosmotic pumps and implants (Bremer et al., Pharm. Biotechnol. 10:239 (1997); Ranade, "Implants in Drug Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al., "Protein Delivery with Infusion Pumps," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997); Yewey et al., "Delivery of Proteins from a Controlled Release Injectable Implant," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)). Other solid forms include creams, pastes, other topological applications, and the like.

[122] Zsig99 proteins, agonists, and antagonists are useful for modulating the expansion, proliferation, activation, differentiation, migration, or metabolism of responsive cell types, which include both 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, smooth muscle cells, fibroblasts, hepatocytes, neural cells and embryonic stem cells. Zsig99 polypeptides are added to tissue culture media for these cell types at a concentration of about 10 pg/ml to about 100 ng/ml. Those skilled in the art will recognize that zsig99 proteins can be advantageously combined with other growth factors in culture media.

[123] Within the laboratory research field, zsig99 proteins can also be used as molecular weight standards or as reagents in assays for determining circulating levels of the protein, such as in the diagnosis of disorders characterized by over- or under-production of zsig99 protein or in the analysis of cell phenotype.

[124] As used herein, the term "antibodies" includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and Fab fragments, single chain antibodies, and the like, including genetically engineered antibodies. Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered" antibody). In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. One skilled in the art can generate humanized antibodies with specific and different constant domains (i.e., different Ig subclasses) to facilitate or inhibit various immune functions associated with particular antibody constant domains/ Antibodies are defined to be specifically binding if they bind to a zsig99 polypeptide or protein with an affinity at least 10-fold greater than the binding affinity to control (non- zsig99) polypeptide or protein. The affinity of a monoclonal antibody can be readily determined by one of ordinary skill in the art (see, for example, Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949). Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see for example, Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982).

[125] As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats. The immunogenicity of a zsig99 polypeptide may be increased through the use of an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of a zsig99 polypeptide or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

[126] Alternative techniques for generating or selecting antibodies include in vitro exposure of lymphocytes to zsig99 polypeptides, and selection of antibody display libraries in phage or similar vectors (e.g., through the use of immobilized or labeled zsig99 polypeptide). Human antibodies can be produced in transgenic, non-human animals that have been engineered to contain human immunoglobulin genes as disclosed in WIPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination.

[127] A variety of assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to zsig99 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: concurrent immunoelectrophoresis, radioimmunoassays, radio-immunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, Western blot assays, inhibition or competition assays, and sandwich assays.

[128] Antibodies to zsig99 may be used for affinity purification of the protein, within diagnostic assays for determining circulating levels of the protein; for detecting or quantitating soluble zsig99 polypeptide as a marker of underlying pathology or disease; for immunolocalization within whole animals or tissue sections, including immunodiagnostic applications; for immunohistochemistry; and as antagonists to block protein activity in vitro and in vivo. Antibodies to zsig99 may also be used for tagging cells that express zsig99; for affinity purification of zsig99 polypeptides and proteins; in analytical methods employing FACS; for screening expression libraries; and for generating anti-idiotypic antibodies. Antibodies can be linked to other compounds, including therapeutic and diagnostic agents, using known methods to provide for targeting of those compounds to cells expressing receptors for zsig99. For certain applications, including in vitro and in vivo diagnostic uses, it is advantageous to employ labeled antibodies. Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti-complement pairs as intermediates. Antibodies of the present invention may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications (e.g., inhibition of cell proliferation). See, in general, Ramakrishnan et al., Cancer Res. 56:1324-1330, 1996.

[129] Polypeptides and proteins of the present invention can be used to identify and isolate receptors. Zsig99 receptors may be involved in growth regulation in the liver, blood vessel formation, and other developmental processes. For example, zsig99 proteins and polypeptides can be immobilized on a column, and membrane preparations run over the column (as generally disclosed in Immobilized Affinity Ligand Techniques, Hermanson et al., eds., Academic Press, San Diego, CA, 1992, pp.195-202). Proteins and polypeptides can also be radiolabeled (Methods Enzvmol., vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Academic Press, San Diego, 1990, 721-737) or photoaffinity labeled (Brunner et al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedan et al., Biochem. Pharmacol. 33:1167-1180, 1984) and used to tag specific cell-surface proteins. In a similar manner, radiolabeled zcyto32 proteins and polypeptides can be used to clone the cognate receptor in binding assays using cells transfected with an expression cDNA library. [130] Polynucleotides encoding zsig99 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zsig99 activity. If a mammal has a mutated or absent zsig99 gene, a zsig99 gene can be introduced into the cells of the mammal.

[131] Zsig99 polypeptides and anti-zsig99 antibodies can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. For instance, polypeptides or antibodies of the present invention may be used to identify or treat tissues or organs that express a corresponding anticomplementary molecule (receptor or antigen, respectively, for instance). More specifically, zsig99 polypeptides or anti-zsig99 antibodies, or bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues, or organs that express the anti-complementary molecule.

[132] Suitable detectable molecules can be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles, and the like. Suitable cytotoxic molecules can be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin, saporin, and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90. These can be either directly attached to the polypeptide or antibody, or indirectly attached according to known methods, such as through a chelating moiety. Polypeptides or antibodies can also be conjugated to cytotoxic drugs, such as adriamycin. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule may be conjugated with a member of a complementary/anticomplementary pair, where the other member is bound to the polypeptide or antibody portion. For these purposes, biotin/streptavidin is an exemplary complementary/anticomplementary pair.

[133] Polypeptide-toxin fusion proteins or antibody/fragment-toxin fusion proteins may be used for targeted cell or tissue inhibition or ablation, such as in cancer therapy. Of particular interest in this regard are conjugates of a zsig99 polypeptide and a cytotoxin, which can be used to target the cytotoxin to a tumor or other tissue that is undergoing undesired angiogenesis or neovascularization. Target cells (i.e., those displaying the zsig99 receptor) bind the zsig99-toxin conjugate, which is then internalized, killing the cell. The effects of receptor-specific cell killing (target ablation) are revealed by changes in whole animal physiology or through histological examination. Thus, ligand-dependent, receptor-directed cyotoxicity can be used to enhance understanding of the physiological significance of a protein ligand. A preferred such toxin is saporin. Mammalian cells have no receptor for saporin, which is non-toxic when it remains extracellular.

[134] In another embodiment, zsig99-cytokine fusion proteins or antibody/fragment- cytokine fusion proteins may be used for enhancing in vitro cytotoxicity (for instance, that mediated by monoclonal antibodies against tumor targets) and for enhancing in vivo killing of target tissues (for example, blood and bone marrow cancers). See, generally, Hornick et al., Blood 89:4437-4447, 1997). In general, cytokines are toxic if administered systemically. The described fusion proteins enable targeting of a cytokine to a desired site of action, such as a cell having binding sites for zsig99, thereby providing an elevated local concentration of cytokine. Suitable cytokines for this purpose include, for example, interleukin-2 and granulocyte-macrophage colony-stimulating factor (GM- CSF). Such fusion proteins may be used to cause cytokine-induced killing of tumors and other tissues undergoing angiogenesis or neovascularization.

[135] The bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intra-arterially or intraductally, or may be introduced locally at the intended site of action.

[136] The invention is further illustrated by the following non-limiting examples. EXAMPLES

Example 1 Expression constructs

[137] . Constructs for the expression of zsig99 starting at methionine 1 (zsig99XlMl) or methionine 19 (zsig99XlM2) were made in pAMP41zeo. The zZMP41zeo is derived from plasmid pZMP40, where the zeocin resistance gene has been substituted for the DHFR gene and the CD8 gene was replaced with CD4. Zmp40 was cut with BgIII, was used in a three-way recombination with both of the PCR insert fragments. Plasmid pZMP40 is a mammalian expression vector containing an expression cassette having the MPSV promoter, and multiple restriction sites for insertion of coding sequences; an E. coli origin of replication; a mammalian selectable marker expression unit comprising an SV40 promoter, enhancer and origin of replication, a DHFR gene, and the SV40 terminator; and URA3 and CEN-ARS sequences required for selection and replication in S. cerevisiae. Plasmid pZMP40 was constructed from pZMP21 (deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, and designated No. PTA-5266) by addition of several restriction enzyme sites to the polylinker.

[138] Furthermore, constructs for the expression of either zsig99XlMl or zsig99XlM2 with either a C-terminal FLAG tag (SEQ ID NO:9) or a C-terminal 6xHis tag (SEQ ID NO: 10) were prepared. Using the cDNA encoding zsig99XlMl as a templated, PCR-amplified cDNAs for zsig99XlMl-Cflag or C-His were prepared using the oligonucleotides zc49178 (SEQ ID NO: 11) and zc49569 (SEQ ID NO: 12), or zc49178 (SEQ ID NO: 11) and zc49570 (SEQ ID NO: 13) as primers. Using the cDNA encoding zsig99XlMl as a templated, PCR-amplified cDNAs for zsig99XlM2- Cflag or C-His were prepared using the oligonuclotides zc49177 (SEQ ID NO: 14) and zc49569 (SEQ ID NO: 12), or zc49177 (SEQ ID NO: 14) and zc49570 (SEQ ID NO: 13) as primers. Following agarose gel purification the cDNAs were inserted into EcoRI/Bgiπ cut pzmp41zeo using the MFusion reaction (BD Biosciences, Palo Alto, CA) as per manufacturers protocol. Plasmid DNA was prepared in Ecoli, DHlOB and purified using QIAFILTER Maxi-prep kit (Qiagen, Valencia, CA ) as described by manufacturer. All constructs were sequence verified. B. Expression in HEK293T cells

[139] HEK293T cells (ATCC No. CRL 11268) were transfected with expression constructs for zsig99XlMl-Cflag, zsig99XlMl-Chis, zsig99XlM2-Cflag or zsig99XlM2-Chis. Lipofectamine 2000 (12uL) was combined with 3 ug of construct DNA and allowed to complex at 25oC for 20 min. 2 x 106 293T cells were added to the Lipofectamine 2000 complex and incubated at 37oC for 30 min. Transfected cells were then plated into 6-well collagen coated plates for 24 hrs. Cells were then switched to serum-free media and incubated for an additional 48hrs. The conditioned media (CM) was collected (5mLs) and spun down to remove debris. The transfected cells were lysed in 1.5 RIPA lysis buffer (20 mM Tris:HCL, pH 7.4, 150 mM NaCl, 2mM EGTA, 1% TX-100,and complete protease inhibitors (Roche Diagnostics, Mannheim, Germany )) and spun down to remove debris. The CM was incubated overnight at 4oC with either 50 μl Anti-FlagM2-Agarose (Sigma Chemical Co., St. Louis, MO) or 50 μl NiNTA (Qiagen, Valencia, CA). The affinity resin was collected, washed with PBS and the bound proteins were eluted in 50 μl 2X reducing loading buffer (InVitrogen, Carlsbad, CA) at 80oC. The samples were then analyzed by western blot using Anti- FlagM2 antibody (Sigma Chemical Co., St. Louis, MO) or Anti-His antibody (R&D Systems, Minneapolis, MN). AU of the zsig99XlMl-Cflag, zsig99XlMl-Chis, zsig99XlM2-Cflag or zsig99XlM2-Chis protein expressed in HEK293T cells that were transfected with the respective expression vectors, was cell associated, and no zsig99XlMl-Cflag, zsig99XlMl-Chis, zsig99XlM2- Cflag or zsig99XlM2-Chis protein was found in the CM.

Example 2

Co-expression with IL-6 and IL- 12 family members

[140] Expression constructs for zsig99XlMl-Cflag, zsig99XlMl-Chis, zsig99XlM2-Cflag or zsig99XlM2-Chis were transfected in combination with expression constructs for IL23A (Oppman et al., Immunity 13:715-725, 2000) IL12p40 (Koybayaski et al., J. Exp. Med. 170:827-845, 1989), EBI3 (Pflanz et al., Immunity 16:779-790, 2002), soluble IL-6 receptor (!L-6Sr ; Lust, et al., Cytokine, 4(2):, 96-100, 1992), Ciliary Neurotrophic Factor Receptor (CNTFR ;Panayotaros, et. al,. Biochemistry, 33(19): 5813-5818, 1994), Cardiotrophin-Like Cytokine or CLF-Cytokine-Like Factor. (CLC and CLF; Elson, et. al, Nature Neuroscience. 3(9): 867-872, 2000), or LIF (SEQ ID NO: 8) into HEK293T cells. Lipofectamine 2000 (InVitrogen, Carlsbad, CA) was combined with 3 μg of each construct DNA and allowed to complex at 25°C for 20 min. 2 x 10⁶ HEK293T cells were added to the Lipofectamine 2000 (Invitrogen, Carlsbad, CA) complex and incubated at 37°C for 30 min. Transfected cells were then plated into 6-well collagen coated plates for 24 hrs. Culture medium was W 2

44 removed and replaced with serum-free media, and the cell were incubated for and additional 48hrs. After 48hrs., the conditioned media was collected and cleared of cell debris by centrifuge. The cells were lysed with RIPA lysis buffer (1.5 mLs) and the lysate was cleared of cell debris by centrifuge. Both conditioned media and whole cell lysates were combined with 50 μl Ni NTA-agarose (Qiagen, Valencia, CA). Conditioned medium and lysate from the cells transfected with zsig99 alone were combined with 50 μl of anti-FLAG agarose (Sigma Chemical Co., St. Louis, MO). Following an overnight incubation, the resins from the immunoprecipitation reactions were pelleted and washed once with PBS and then analyzed by SDS-PAGE and western blot. Blots were incubated with an anti-FLAG-bio M2 antibody (1:3000) overnight at 4⁰C with agitation. Blots were then washed and then avidin-HRP (1:5000) was added for 1 hr. at 25⁰C. After a final wash, ECL was used to visualize the Western blots. The western blots show that when zsig99XlM2-Cflag is expressed alone, the majority of the zsig99XlM2-Cflag protein is retained in the whole cell lysate fraction. Co-expression of zsig99XlM2-Cflag with LEF-C-His, resulted in the secretion of both zsig99XlM2-Cflag and Lif- CHis. In contrast, co-expression of zsig99XlM2-Cflag with the other members of the IL-6 and IL- 12 family members did not lead to secretion of zsig99XlM2-Cflag. These data show that LIF, but none of the other proteins tested, could stimulate the secretion of zsig99XlM2-Cflag.

[141] In a subsequent experiment when zsig99XlM2-Cflag and LIF-C-His were co- expressed in the same cell, either Ni NTA-agar (Qiagen, Valencia, CA) or an anti-LIF antibody (R&D Systems, Minneapolis, MN) were able to immunoprecipitate , zsig99XlM2-Cflag from 293T conditioned media. In addition, anti-FLAG-agarose (sigma Chemical Co., St Louis, MO) was able to capture LIF-C-His from the same conditioned medium. These data demonstrate a close association of LIF with zsig99XlM2-Cflag.

[142] The results of these experiments show that secretion of zsig99XlM2-Cflag is dependent on the co-expression of LIF, illustrated by the lack of secretion of zsig99XlM2-Cflag when paired with other proteins of the 11-6 or IL 12 family and the robust secretion in the presence of LIF. Furthermore, the immunoprecipitation experiments showed that there is a close association of zsig99XlM2-Cflag, and LIF-Chis.

Example 3

Tissue Distribution in cDNA Library Panels

[143] A panel of DNAs from cDNA libraries made in-house was screened for zalpha99 expression using PCR. The panel contained 45 DNA samples from cDNA libraries made from various human tissues (normal, cancer, and diseased) and resting or stimulated cell lines. The in-house cDNA libraries were QC tested by PCR with vector oligos for average insert size, PCR for alpha tubulin or G3PDH for full length cDNA using 5' vector oligonucleotide and 3' gene specific oligonucleotide and sequencing for ribosomal or mitochondrial DNA contamination. The panel was set up in a 96- well format that included a lOOpg human genomic DNA (Clonetech, Palo Alto, CA) positive control sample. Each well contained 5ul of cDNA library DNA and 10.5 ul of water. Expression of the DNA in the human Library cDNA panel for zsig99 was assayed by PCR with sense oligonucleotide zc49542 (SEQ ID NO:21) and antisense oligonucleotide zc49541 (SEQ ID NO:22) under these PCR conditions per sample: 2.5 μl 1OX buffer and 0.5 μl advantage 2 cDNA polymerase mix (BD Biosciences Clontech, Palo Alto, CA), 2 μl 2.5mM dNTP mix (Applied Biosystems, Foster City, CA), 2.5 μl 1OX Rediload (Invitrogen, Carlsbad, CA), and 0.5 μl 2OuM each sense and antisense primer. Cycling conditions were 94⁰C for 2 minutes, 35 cycles of 94°C for 30 seconds, 66.5°C for 30 seconds, 72⁰C for 30 seconds and one cycle of 72⁰C for 5 minutes. 10 μl of each reaction was subjected to agarose gel electrophoresis and gels were scored for positive or negative expression of zsig99. See Table 5 below for expression profile and tissues screened. Oligonucleotides generate a 350bp cDNA sized band and a 569bρ genomic sized band. The DNA fragments for brain was excised and purified using a Gel Extraction Kit (Qiagen, Chatsworth, CA) according to manufacturer's instructions. The fragment was confirmed by sequencing to show that it was indeed zsig99. Table 5 cDNA's Zsig99

Brain . YES

Fetal brain NO

Islet YES

Prostate 0.5-1.6KB NO

Prostate >1.6KB NO

RPMI (B-cells) NO

Kidney NO

Thyroid YES

Spinal cord YES

CD4+ NO

CD4+ YES

HaCat NO

REH NO

K562 NO

Prostate SM NO

CD3+ NO

SKLU-I NO

Pancreas NO

Pituitary NO

THP-I NO

THP-I YES

Fetal thymus NO

HL60 + PMA NO

HL60 + PMA NO

Fetal liver YES

Tonsil NO

Inflamed tonsil YES

KG-I NO

CaCO-2 NO PBMC-I NO

PBMC-2 NO

U-937 PMA NO

U-937 NO

CD19 NO

CD 19 PMA/IONO NO

CD14+ NO

CD 14+ IFNγ/LPS NO

Dendritic cell NO

Dendritic Cell, stim NO

NK PMA/IONO NO

HL-60 vitD, 12, 36, NO

96hrs

HL-60 Ret. Acid, 12, NO

36, 96hrs

HL-60 Butyric Acid, NO

12, 36, 96hrs

THP-I #2 IFNγ 13, 39 NO hrs

HT-29 NO

Example 4

Tissue Distribution in Blood Fraction Panel

[144] A panel of 1st strand cDNAs from human cells and tissues was screened for zsig99 expression using PCR. The panel was purchased from BD Bioscience (Palo Alto, CA) and contained 9 cDNA samples from various human blood cells and tissues. The 1st strand cDNAs were QC tested by PCR with G3PDH control primers by BD BioScience (Palo Alto, CA). The panel was set up in a 96-well format that included 1 positive control sample, human genomic DNA BD Bioscience (Palo Alto, CA). A dilution series was performed. Each well contained either 5 μl of cDNA and 6.25 μl of water, 1 μl of cDNA and 10.25 μl of water or 1 μl of a 1:5 dilution of cDNA and 10.25 μl water. Expression of the DNA in the human Library cDNA panel for zsig99 was assayed by PCR with sense oligonucleotide zc49542 (SEQ ID NO:21) and antisense oligonucleotide zc49541 (SEQ ID NO:22) under these PCR conditions per sample: 2.5 μl 1OX buffer and 0.5 μl advantage 2 cDNA polymerase mix (BD Biosciences Clontech, Palo Alto, CA), 2 μ 2.5mM dNTP mix (Applied Biosystems, Foster City, CA), 2.5 μl 1OX Rediload (Invitrogen, Carlsbad, CA), and 0.5 μl 2OuM each sense and antisense primer. Cycling conditions were 94°C for 2minutes, 35 cycles of 94⁰C for 30 seconds, 66.5⁰C for 30 seconds, 72°C for 30 seconds and one cycle of 72°C for 5 minutes. 10 μl of each reaction was subjected to agarose gel electrophoresis and gels were scored for positive or negative expression of zsig99. Oligonucleotides generate a 350bp cDNA sized band and a 569bp genomic sized band. See Table 6 below for expression profile and tissues screened. Table 6 cDNA's Zsig99

Activated CD4+ NO

Resting CD4+ NO

Activated CD8+ NO

Resting CD8+ NO

Resting CD14+ NO

Activated CD19+ NO

Resting CD 19+ NO

Activated NO

Mononuclear

Mononuclear NO

Control placenta NO

Example 5

Tissue Distribution in cDNA samples

[145] DNA from cDNA libraries, 1st strand cDNAs and marathon cDNAs made in-house were screened for zalpha99 expression using PCR. The PCR experiment contained DNA samples from cDNA made from various human tissues (normal, cancer, and diseased). Each well contained 5 μl of cDNA library DNA and 10.5 μl of water. Expression of for zsig99 was assayed by PCR with sense oligonucleotide zc49542 (SEQ ID NO:21) and antisense oligonucleotide zc49541 (SEQ ID NO:22) under these PCR conditions per sample: 2.5 μl 1OX buffer and 0.5 μl advantage 2 cDNA polymerase mix (BD Biosciences Clontech, Palo Alto, CA), 2 μl 2.5mM dNTP mix (Applied Biosystems, Foster City, CA), 2.5 μl 1OX Rediload (Invitrogen, Carlsbad, CA), and 1.0 μl 20μM each sense and antisense primer. Cycling conditions were 94⁰C for 2minutes, 35 cycles of 94°C for 30 seconds, 66.5°C for 30 seconds, 72⁰C for 30 seconds and one cycle of 72°C for 5 minutes. 10 μl of each reaction was subjected to agarose gel electrophoresis and gels were scored for positive or negative expression of zsig99. See Table 7 below for expression profile and tissues screened. Oligos generate a 350bp cDNA sized band and a 569bp genomic sized band. Table 7 cDNA's Zsig99

Fetal brain library NO

Fetal brain 1^st strand cDNA YES

Fetal brain mcDNA YES

Fetal skin cDNA YES

Skin 1^st strand cDNA YES

Skin 1^st strand cDNA NO

Spinal cord mcDNA YES

Spinal cord library YES

Spinal cord cDNA YES

Example 6

Distribution of mRNA in cell line panels [146] Total RNA was purified from resting and stimulated cell lines grown in-house and purified using a Qiagen (Valencia, CA) RNeasy kit according to the manufacturer's instructions, or an acid-phenol purification protocol (Chomczynski and Sacchi, Analytical Biochemistry, 162:156-9, 1987). The quality of the RNA was assessed by running an aliquot on an Agilent Bioanalyzer. If the RNA was significantly degraded, it was not used for subsequent creation of first strand cDNA. Presence of contaminating genomic DNA was assessed by a PCR assay on an aliquot of the RNA with zc41011 (SEQ ID NO: 15) and zc41012 (SEQ ID NO: 16), primers that amplify a single site of intergenic genomic DNA. The PCR conditions for the contaminating genomic DNA assay were as follows: 2.5 μl 1OX buffer and 0.5 μl Advantage 2 cDNA polymerase mix (BD Biosciences Clontech, Palo Alto, CA), 2 μl 2.5mM dNTP mix (Applied Biosystems, Foster City, CA), 2.5 μl 1OX Rediload (Invitrogen, Carlsbad, CA), and 0.5 μl 20μM zc41011 (SEQ ID NO: 15) and zc41012 (SEQ ID NO: 16), in a final volume of 25 μl. Cycling parameters were 94°C 20", 40 cycles of 94°C 20" 6O⁰C l'2O" and one cycle of 72°C 7'. lOul of each reaction was subjected to agarose gel electrophoresis and gels were examined for presence of a PCR product from contaminating genomic DNA. If contaminating genomic DNA was observed, the total RNA was DNAsed using DNA-free reagents (Ambion, Inc, Austin, TX) according to the manufacturer's instructions, then retested as described above. Only RNAs which appeared to be free of contaminating genomic DNA were used for subsequent creation of first strand cDNA.

[147] 20 μg total RNA from 82 human cell lines were each brought to 98 μl with H2O, then split into two 49 μl aliquots, each containing 10 μg total RNA, and placed in two 96-well PCR plates. To each aliquot was added reagents for first strand cDNA synthesis (Invitrogen First Strand cDNA Synthesis System, Carlsbad, CA): 20 μl 25mM MgCl₂, 10 μl 1OX RT buffer, 10 μl 0.1M DTT, 2 μl oligonucleotide dT, 2 μl RNAseOut. Then, to one aliquot from each cell line 2 μl Superscript II Reverse Transcriptase was added, and to the corresponding cell line aliquot 2 μl H₂O was added to make a minus Reverse Transcriptase negative control. All samples were incubated as follows: 25°C 10', 42⁰C 50', 70⁰C 15'. Samples were arranged in deep well plates and diluted to 1.7 ml with H₂O. A Multipette (Saigan) robot was used to aliquot 16.5 μl into each well of a 96-well PCR plate multiple times, generating numerous one-use PCR panels of the cell lines, which were then sealed and stored at -2O⁰C. Each well in these panels represents first strand cDNA from approximately 100 ng total RNA. The 82 cell lines are spread across two panels, array #118A and #118B. Arrangement and content of the samples on the arrays is detailed below in Tables 8 A and 8B. Quality of first strand cDNA on the panels was assessed by a multiplex PCR assay on one set of the panels using primers to two widely expressed, but only moderately abundant genes, CLTC (clathrin) and TFRC (transferrin receptor C). 0.5 μl each of Clathrin primers zc42901 (SEQ ID NO: 17), zc42902 (SEQ ID NO: 18), and TFRC primers zc42599 (SEQ ID NO: 19), zc42600 (SEQ ID NO:20), were mixed with 2.5 μl 1OX buffer and 0.5 μl Advantage 2 cDNA polymerase mix (BD Biosciences Clontech, Palo Alto, CA), 2 μl 2.5mM dNTP mix (Applied Biosystems,, Foster City, CA), 2.5 μl 1OX Rediload (Invitrogen, Carlsbad, CA), and added to each well of a panel of array#l 18A and array #118B. Cycling parameters were as follows: 94⁰C 20", 35 cycles of 94°C 20", 67⁰C 80", and one cycle of 72°C T. 10 μl of each reaction was subjected to agarose gel electrophoresis and gels were scored for the presence of a robust PCR product for each gene specific to the +RT wells for each cell line.

[148] Expression of mRNA in the human first strand cDNA panels for zsig99 was assayed by PCR with sense oligonucleotide zc49542 (SEQ ID NO:21) and antisense oligonucleotide zc49541 (SEQ ID NO:22) under these PCR conditions per sample: 2.5 μl 1OX buffer and 0.5 μl advantage 2 cDNA polymerase mix (BD Biosciences Clontech, Palo Alto, CA), 2 μl 2.5mM dNTP mix (Applied Biosystems, Foster City, CA ), 2.5 μl 1OX Rediload (Invitrogen, Carlsbad, CA), and 0.5 μl 2OuM each sense and antisense primer. Cycling conditions were 94⁰C for 2minutes, 35 cycles of 94°C for 30 seconds, 66.5⁰C for 30 seconds, 72⁰C for 30 seconds and one cycle of 72⁰C for 5 minutes. 10 μl of each reaction was subjected to agarose gel electrophoresis and gels were scored for positive or negative expression of zsig99. Oligonucleotides generate a 350bp cDNA sized band and a 569bp genomic sized band. Samples that were positive for zsig99 were: Array 118A: HCT-15 (HA). Array 118B: C32 (2G), OPM-2 (3E), Granta 519 (5C), RPMI 8226 (6E) and Cess (9A).

Table 8A.

ARRAY #118 A

ROW COL CELL LINE NAME ROW COL CELL LINE NAME

A 1 CTB-I A 7 HOS

B 1 CTB-I -RT B 7 HOS -RT

C 1 L363 C 7 HL-60

D 1 L363 -RT D 7 HL-60 -RT

E 1 HS Sultan E 7 THP-I

F 1 HS Sultan -RT F 7 THP-I -RT

G 1 A375 G 7 SK-MEL-2

H 1 A375 -RT H 7 SK-MEL-2 -RT

A 2 CTB-I + PMA/Iono A 8 CCRF-CEM

B 2 CTB-I + PMA/Iono -RT B 8 CCRF-CEM -RT

C 2 TFI C 8 HL-60 Butyric acid 12,36,96 hr

D 2 TFI -RT D 8 HL-60 Butyric acid 12,36,96 hr -RT

E 2 HUT 78 E 8 THP-I + LPS+ gIFN

F 2 HUT 78 -RT F 8 THP-I + LPS+ gIFN -RT

G 2 C32 G 8 WM-115

H 2 C32 -RT H 8 WM-115 -RT

A 3 MacLLC A 9 Cess

B 3 MacLLC -RT B 9 Cess -RT

C 3 ARH 77 C 9 HL-60 DMSO 96 hr

D 3 ARH 77 -RT D 9 HL-60 DMSO 96 hr -RT

E 3 OPM-2 E 9 HBL-100

F 3 OPM-2 -RT F 9 HBL-IOO -RT

G 3 G-361 G 9 A-431

H 3 G-361 -RT H 9 A-431 -RT

A 4 MacLLC + PMA/Iono A 10 HEL 92.1.7

B 4 MacLLC + PMA/Iono -RT B 10 HEL 92.1.7 -RT

C 4 DOHH-2 C 10 HL-60 PMA 12 hr

D 4 DOHH-2 -RT D 10 HL-60 PMA 12 hr -RT

E 4 REH E 10 MCF7

F 4 REH -RT F 10 MCF7 -RT

G 4 HaCat G 10 AsPC-I

H 4 HaCat -RT H 10 AsPC-I -RT

A 5 Ramos A 11 KG-I

B 5 Ramos -RT B 11 KG-I -RT

C 5 Granta 519 C 11 HL-60 VitD3 36 hr

D 5 Granta 519 -RT D 11 HL-60 VitD3 36 hr -RT

E 5 RL E 11 T-47D

F 5 RL -RT F 11 T-47D -RT

G 5 Hs 294T G 11 BeWo

H 5 Hs 294T -RT H 11 BeWo -RT

A 6 Bjab

B 6 Bjab -RT

C 6 GRANTA-519

D 6 GRANTA-519 -RT

E 6 RPMI 8226

F 6 RPMI 8226 -RT

G 6 Malme 3M

H 6 Mamie 3M -RT Table 8B.

ARRAY #118 B

ROW COL CELL LINE NAME ROW COL CELLLINE NAME

A 1 A-172 A 7 CaCo-2

B 1 A-172 -RT B 7 CaCo-2 -RT

C 1 HepG2 C 7 MRC-5

D 1 HepG2 -RT D 7 MRC-5 -RT

E 1 U-937 + PMA + ionomycin E 7 PC-3

F 1 U-937 + PMA + ionomycin -RT F 7 PC-3 -RT

G 1 TrBMEC G 7 DEL + PMA/Iono

H 1 TrBMEC -RT H 7 DEL + PMA/Iono -RT

A 2 SK-N-SH A 8 Caco-2, diff.

B 2 SK-N-SH -RT B 8 Caco-2, diff. -RT

C 2 HepG2 + IL-6 C 8 NCI-H69

D 2 HepG2 + IL-6 -RT D 8 NCI-H69 -RT

E 2 U-937 PMA 12 hr E 8 FHS74.Int

F 2 U-937 PMA 12 hr -RT F 8 FHS74.Int -RT

G 2 5637 G 8

H 2 5637 -RT H 8

A 3 U-138 MG A 9 DLD-I

B 3 U-138 MG -RT B 9 DLD-I -RT

C 3 HuH7 C 9 SK-Lu-I

D 3 HuH7 -RT D 9 SK-Lu-I -RT

E 3 WSU-NHL E 9 INT407

F 3 WSU-NHL -RT F 9 INT407 -RT

G 3 ME 180 Cytoplasmic B G 9

H 3 ME 180 Cytoplasmic B -RT H 9

A 4 U-373 MG A 10 HCT 116

B 4 U-373 MG -RT B 10 HCT 116 -RT

C 4 Huh7 Cytoplasmic C 10

D 4 Huh7 Cytoplasmic -RT D 10

E 4 NCI-H929 E 10 Tonsil cell line

F 4 NCI-H929 -RT F 10 Tonsil cell line -RT

G 4 ME 180 total G 10

H 4 ME 180 total -RT H 10

A 5 ARPE-19 A 11 HCT-15

B 5 ARPE-19 -RT B 11 HCT-15 -RT

C 5 Huh7 nucleus C 11

D 5 Huh7 nucleus -RT D 11

E 5 NCI-H929 Cytoplasmic E 11 697

F 5 NCI-H929 Cytoplasmic -RT F 11 697 -RT

G 5 ME180 G 11

H 5 ME180 -RT H 11

A 6 Y-79

B 6 Y-79 -RT

C 6 A-549

D 6 A-549 -RT

E 6 U-937

F 6 U-937 -RT

G 6 DEL

H 6 DEL -RT Example 8

Distribution of mRNA in cell line panels using PCR

[149] Total RNA was purified from human digestive system tissues and purified using a Qiagen (Valencia, CA) RNeasy kit according to the manufacturer's instructions, or an acid-phenol purification protocol (Chomczynski and Sacchi, Analytical Biochemistry, 162:156-9, 1987). The quality of the RNA was assessed by running an aliquot on an Agilent Bioanalyzer. If the RNA was significantly degraded, it was not used for subsequent creation of first strand cDNA. Presence of contaminating genomic DNA was assessed by a PCR assay on an aliquot of the RNA with zc41011 (SEQ ID NO: 15) and zc41012 (SEQ ID NO: 16), primers that amplify a single site of intergenic genomic DNA. The PCR conditions for the contaminating genomic DNA assay were as follows: 2.5 μl 1OX buffer and 0.5 μl Advantage 2 cDNA polymerase mix (BD Biosciences Clontech, Palo Alto, CA), 2 μl 2.5mM dNTP mix (Applied Biosystems, Foster City, CA), 2.5 μl 1OX Rediload (Invitrogen, Carlsbad, CA), and 0.5 μl 2OuM zc41011 (SEQ ID NO: 15) and zc41012 (SEQ ID NO: 16), in a final volume of 25 μl. Cycling parameters were 94°C 20", 40 cycles of 94°C 20" 60⁰C l'2O" and one cycle of 72⁰C T . 10 μl of each reaction was subjected to agarose gel electrophoresis and gels were examined for presence of a PCR product from contaminating genomic DNA. If contaminating genomic DNA was observed, the total RNA was DNAsed using DNA-free reagents (Ambion, Inc, Austin, TX) according to the manufacturer's instructions, then retested as described above. Only RNAs which appeared to be free of contaminating genomic DNA were used for subsequent creation of first strand cDNA.

[150] 20 μg total RNA from 92 human digestive system tissues were each brought to 98ul with H₂O, then split into two 49 μl aliquots, each containing 10 μg total RNA, and placed in two 96- well PCR plates. To each aliquot was added reagents for first strand cDNA synthesis (Invitrogen First Strand cDNA Synthesis System, Carlsbad, CA): 20 μ 25mM MgCl₂, 10 μl 1OX RT buffer, 10 μl 0.1M DTT, 2 μl oligo dT, 2 μl RNAseOut. Then, to one aliquot from each cell line 2 μl Superscript II Reverse Transcriptase was added, and to the corresponding cell line aliquot 2 μl H2O was added to make a minus Reverse Transcriptase negative control. All samples were incubated as follows: 25⁰C 10', 42°C 50', 70⁰C 15 '. Samples were arranged in deep well plates and diluted to 1.7 ml with H₂O. A Multipette (Saigan) robot was used to aliquot 16.5 μl into each well of a 96-well PCR plate multiple times, generating numerous one-use PCR panels of the cell lines, which were then sealed and stored at -20⁰C. Each well in these panels represents first strand cDNA from approximately 100 ng total RNA. The 92 human digestive system RNAs were aliquoted to a panel, array #111. Arrangement and content of the samples on the arrays is detailed below in Tables 8. Quality of first strand cDNA on the panels was assessed by a multiplex PCR assay on one set of the panels using primers to two widely expressed, but only moderately abundant genes, CLTC (clathrin) and TFRC (transferrin receptor C). 0.5 μl each of Clathrin primers zc42901 (SEQ ID NO: 17), zc42902 (SEQ ID NO: 18), and TFRC primers zc42599 (SEQ ID NO: 19), zc42600 (SEQ ID NO:20), were mixed with 2.5 μl 1OX buffer and 0.5 μl Advantage 2 cDNA polymerase mix (BD Biosciences Clontech, Palo Alto, CA), 2 μl 2.5mM dNTP mix (Applied Biosystems,, Foster City, CA), 2.5 μl 1OX Rediload (Invitrogen, Carlsbad, CA), and added to each well of a panel of array#lll. Cycling parameters were as follows: 94°C 20", 35 cycles of 94⁰C 20", 67⁰C 80", and one cycle of 72°C 7'. lOul of each reaction was subjected to agarose gel electrophoresis and gels were scored for the presence of a robust PCR product for each gene specific to the +RT wells for each cell line.

[151] Expression of mRNA in the human first strand cDNA panels for zsig99 was assayed by PCR with sense oligonucleotide zc49542 (SEQ ID NO:21) and antisense oligonucleotide zc49541 (SEQ ID NO:22) under these PCR conditions per sample: 2.5 μl 1OX buffer and 0.5 μl advantage 2 cDNA polymerase mix (BD Biosciences Clontech, Palo Alto, CA), 2 μl 2.5mM dNTP mix (Applied Biosystems, Foster City, CA ), 2.5 μl 1OX Rediload (Invitrogen, Carlsbad, CA), and 0.5 μl 2OuM each sense and antisense primer. Cycling conditions were 94⁰C for 2minutes, 35 cycles of 94°C for 30 seconds, 66.5°C for 30 seconds, 72°C for 30 seconds and one cycle of 72⁰C for 5 minutes. 10 μl of each reaction was subjected to agarose gel electrophoresis and gels were scored for positive or negative expression of zsig99. Oligonucleotides generate a 350bp cDNA sized band and a 569bp genomic sized band. Samples that were positive for zsig99 were: Colon, esophagus, small intestine and stomach.

Table 9 biological late * Row Column Tissue In-house lot# System Health

Caco-2, diff.

111 A 1 cell line HKES091201A digestive cancer

111 B 1 Colon HKES091201B digestive cancer

111 C 1 Colon HKES091201C digestive cancer

111 D 1 Colon HKES091201D digestive cancer

111 E 1 Colon HKES091201E digestive Cancer

111 F 1 Colon HKES091201F digestive Cancer

111 G 1 Colon HKES091201G digestive Cancer

111 H 1 Colon HKES091201H digestive Cancer

111 A 2 Colon HKES0912011 digestive Cancer

111 B 2 Colon HKES091201J digestive Cancer

111 C 2 Colon HKES091201K digestive Cancer

111 D 2 Colon HKES091201L digestive Cancer

111 E 2 Colon HKES091201M digestive Cancer

111 F 2 Colon HKES091201N digestive disease

111 G 2 Colon HKES091201O digestive Disease

111 H 2 Colon HKES091201P digestive Disease

111 A 3 Colon HTLG091201A digestive normal

111 B 3 Colon HTLG091201B digestive normal

111 C 3 Colon HTLG091201C digestive normal

111 D 3 Colon HTLG091201D digestive normal 111 E 3 Colon HTLG091201E digestive Normal

111 F 3 Colon HTLG091201F digestive Normal

111 G 3 Colon HTLG091201G digestive Normal

111 H 3 Colon HTLG091201H digestive Normal

111 A 4 Colon HTLG091201I digestive Normal

111 B 4 Colon HTLG091201J digestive Normal

111 C 4 Esophagus HTLG091201K digestive cancer

111 D 4 Esophagus HTLG091201L digestive cancer

111 E 4 Esophagus HTLG091201M digestive cancer

111 F 4 Esophagus HTLG091201N digestive cancer

111 G 4 Esophagus HTLG091201O digestive normal

111 H 4 Esophagus HTLG091201P digestive normal

111 A 5 Esophagus HACF091201A digestive Normal

111 B 5 Esophagus HACF091201B digestive no info

111 C 5 Liver HACF091201C digestive Cancer

111 D 5 Liver HACF091201D digestive cancer

111 E 5 Liver HACF091201E digestive normal

111 F 5 Liver HACF091201F digestive normal

111 G 5 Parotid gland HACF091201G digestive cancer

111 H 5 Parotid gland HACF091201H digestive cancer

111 A 6 Parotid Gland HACF091201I digestive cancer

111 B 6 Parotid gland HACH091201J digestive cancer

111 C 6 Parotid gland HACH091201K digestive Cancer

111 D 6 Parotid gland HACF091201L digestive Cancer

111 E 6 Parotid gland HACF091201M digestive Cancer

111 F 6 Parotid gland HACF091201N digestive Cancer

111 G 6 Parotid gland HACF091201O digestive Cancer

111 H 6 Parotid gland HACF091201P digestive disease

111 A 7 Parotid gland HKES091301A digestive normal

111 B 7 Parotid gland HKESO913O1B digestive normal

111 C 7 Parotid gland HKES091301C digestive Normal

111 D 7 Salivary gland HKES091301D digestive cancer

111 E 7 Salivary gland HKES091301E digestive Cancer

111 F 7 Small intestine HKES091301F digestive cancer

111 G 7 Small intestine HKES091301G digestive cancer

111 H 7 Small intestine HKES091301H digestive cancer

111 A 8 Small intestine HKES091301I digestive cancer

111 B 8 Small intestine HKES091301J digestive cancer

111 C 8 Small intestine KKES091301K digestive cancer

111 D 8 Small intestine HKES091301L digestive cancer

111 E 8 Small intestine HKES091301M digestive Cancer

111 F 8 Small intestine HKES091301N digestive Cancer

111 G 8 Small intestine HKES091301O digestive disease

111 H 8 Small intestine HKES091301P digestive Disease

111 A 9 Small intestine HACF091301A digestive Normal

111 B 9 Small intestine HACF091301B digestive normal

111 C 9 Small intestine HACF091301C digestive normal

111 D 9 Small intestine HACF091301D digestive normal

111 E 9 Small intestine HACF091301E digestive Normal 111 F 9 Small intestine ; HACF091301F digestive Normal

111 G 9 Stomach HACF091301G digestive cancer

111 H 9 Stomach HACF091301H digestive cancer

111 A 10 Stomach HACF091301I digestive cancer

111 B 10 Stomach HACF091301J digestive cancer

111 C 10 Stomach HACF091301K digestive cancer

111 D 10 Stomach HACF091301L digestive cancer

111 E 10 Stomach HACF091301M digestive cancer

111 F 10 Stomach HACF091301N digestive Cancer

111 G . 10 Stomach HACF091301O digestive Cancer

111 H 10 Stomach HACF091301P digestive Cancer

111 A 11 - Stomach HKES091701A digestive Disease

111 B 11 Stomach HKES091701B digestive normal

111 C 11 Stomach HKES091701C digestive normal

111 D 11 Stomach HKES091701D digestive normal

111 E 11 Stomach HKES091701E digestive normal

111 F 11 Stomach HKES091701F digestive normal

111 G 11 Stomach HKES091701G digestive Cancer

111 H 11 Stomach KHES091701H digestive normal

111 A 12 Stomach HKES091701I digestive Normal

111 B 12 Stomach HKES091701J digestive , Normal

111 C 12 Stomach HKES091701K digestive Normal

111 D 12 Stomach HKES091701L digestive Normal

Example 9

Construction of zsig99/LIF tandem polypeptides

A. zcyto34f2CHis - tandem construct of LIF-zsig99.

[152] Constructs for the expression of zcyto34f2 (designation for zsig99 and LDF tandem polypeptides; shown in SEQ ID NO: 28, with corresponding amino acid sequence in SEQ ID NO: 29) were prepared in the expression vector pZMP21. Plasmid pZMP21 is a mammalian expression vector containing an expression cassette having the MPSV promoter, and multiple restriction sites for insertion of coding sequences; an E. coli origin of replication; a mammalian selectable marker expression unit comprising an SV40 promoter, enhancer and origin of replication, a DHFR gene, and the SV40 terminator; and URA3 and CEN-ARS sequences required for selection and replication in S. cerevisiae, (deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, and designated No. PTA-5266).

[153] Furthermore, constructs for the expression of zcyto34f2 a C-terminal 6xHis tag (SEQ ID NO: 10) were prepared. Using the cDNA encoding zsig99 as a template, PCR-amplified cDNAs for zsig99 were prepared using the oligonucleotides zc50209 (SEQ ID NO: 23) and zc50211 (SEQ ID NO: 24) as primers. These cDNAs encode zsig99, beginning at T42, a 5' extension encoding an amino-terminal linker (SEQ ID NO:25)), and a 3' extension encoding a carboxy-terminal histidine tag. Using cDNA encoding LIF as a template, PCR-amplified cDNAs for LIF were prepared using the oligonucleotides zc50212 (SEQ ID NO: 26) and zc50210 (SEQ E) NO: 27) as primers. These cDNAs encode LIF, beginning at Ml, a 3' extension complementary to the 5' extension on the zsig99 cDNAs, encoding an carboxy-terminal linker. Following agarose gel purification the cDNAs were inserted into EcoRIZBglll cut pzmp21 by three-way yeast recombination in vivo. Yeast DNA was isolated and transformed into E.coli for amplification. Plasmid DNA was prepared in Ecoli, DHlOB and purified using QIAFILTER Maxi-prep kit (Qiagen, Valencia, CA ) as described by manufacturer. Finally, a non-canonical mRNA splice site was removed by site directed mutagenesis, this did not change the amino acid sequence. All constructs were sequence verified.

B. Expression in HEK293T cells

[154] HEK293T cells (ATCC No. CRL 11268) were transfected with expression constructs for zcyto34f2CHis. Lipofectamine 2000 (12μL) was combined with 3 ug of construct DNA and allowed to complex at 25°C for 20 min. 2 x 106 293T cells were added to the Lipofectamine 2000 complex and incubated at 37⁰C for 30 min. Transfected cells were then plated into 6-well plates for 24 hrs. Cells were then switched to serum-free media and incubated for an additional 48hrs. The conditioned media (CM) was collected (5mLs) and spun down to remove debris. The transfected cells were lysed in 1.5 RIPA lysis buffer (20 mM Tris:HCL, pH 7.4, 150 mM NaCl, 2mM EGTA, 1% TX-100,and complete protease inhibitors (Roche Diagnostics, Mannheim, Germany )) and spun down to remove debris. The CM was incubated overnight at 4°C with 50 μl NiNTA (Qiagen, Valencia, CA). The affinity resin was collected, washed with PBS and the bound proteins were eluted in 50 μl 2X reducing loading buffer (InVitrogen, Carlsbad, CA) at 80⁰C. The samples were then analyzed by western blot using Anti-His antibody (R&D Systems, Minneapolis, MN). Zcyto34f2- CHis protein was found in the CM.

C. Expression in BHK 570 cells

[155] BHK 570 cells (ATCC No. CRL 10314) were transfected with expression constructs for zcyto34f2-CHis. Lipofectamine 2000 (12 μL) was combined with 3 ug of construct DNA and allowed to complex at 25oC for 20 min. 2 x 10⁶ cells were added to the Lipofectamine 2000 complex and incubated at 37⁰C for 30 min. Transfected cells were then plated into 150 mm plates for 24 hrs. Cells were then switched to culture media containing 1 μM methotrexate and incubated for an additional two weeks. Colonies were picked and transferred to 24 well dishes and grown to confluence. The conditioned media (CM) was collected from the 24 well dishes and spun down to remove debris. The CM was incubated overnight at 4⁰C with 50 μl NiNTA (Qiagen, Valencia, CA). The affinity resin was collected, washed with PBS and the bound proteins were eluted in 50 μl 2X reducing loading buffer (InVitrogen, Carlsbad, CA) at 8O⁰C. The samples were then analyzed by western blot using Anti-His antibody (R&D Systems, Minneapolis, MN). Colonies expressing high levels of zcyto34f2CHis were expanded. D. Purification of zcyto34f2-CHis

[156] A single BHK Factory expressing zcyto34f2-CHis was harvested every 48 hours for six harvests of 1.5 liters each. Sodium Azide added to a final concentration of 0.02%, 0.22 μm filtered (Nalgene) and stored at 4°C until all harvests were delivered.

[157] After the sixth and final harvest was delivered, expression of zcyto34f2-CHis assessed using quantitative western blot analysis. Expression in Ix media was confirmed for harvests 1 through 4, but not seen in harvests 5 or 6. Harvests 1-4 were combined (~4.5L total volume) and -concentrated to 450 mL using a peristaltic pump device with a 5 kDa molecular weight cut off membrane (Millipore 0.1 m²). Concentrate buffer exchanged into 50 mM NaPC>₄, 500 mM NaCl, 25 mM Imidazole pH 7.5 via 8 column volume exchange using the same peristaltic pump system.

[158] 3 mL of Ni-NTA His Bind Superflow Resin (Novagen) equilibrated in 5OmM NaPO₄, 500 mM NaCl, 25 mM Imidazole pH 7.5 (equilibration buffer) and combined with the adjusted media. Slurry allowed to rock overnight at 4⁰C. The following morning, the slurry transferred to a gravity flow column (BioRAD econo-column), and the flow through collected via gravity. Column washed with 50 column volumes of equilibration buffer. Resin eluted via competition using two steps of 50 mM NaPO₄, 500 mM NaCl, 500 mM Imidazole pH 7.5, each step being 8 column volumes in size. Ni-NTA elution pool concentrated to 3 mL using Ultracel 3OkDa MWCO membrane (Millipore). The concentrate was injected over Superdex 200 column (16/60mm GE Healthcare) and eluted isocratically at 1.0 mL/min into 50 mM NaPO₄, 109 mM NaCl pH 7.3. 1.5 mL fractions were collected and analyzed via reducing and non-reducing SDS-PAGE gels stained with silver (Silver Stain Kit from GenoTech) and by RP-HPLC. Pooled fractions, based on SDS-PAGE and RP-HPLC analysis, were concentrated to 1.5 mL using 30 kDa Ultracel membrane (Millipore), and 0.22 μm filtered using Millex syringe driven filter unit (Millipore). The sterile filtered protein was aliquotted, and frozen at -80⁰C.

[159] From the foregoing, it will be appreciated 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.

Claims

CLAIMS What is claimed is:

1. An isolated polypeptide comprising at least nine contiguous amino acid residues of SEQ ID NO:2.

2. The isolated polypeptide of claim 1 having from 30 to 201 amino acid residues.

3. The isolated polypeptide of claim 2, wherein the at least nine contiguous amino acid residues of SEQ K) NO:2 are operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein and an immunoglobulin constant region.

4. The isolated polypeptide of claim 1 comprising at least 30 contiguous residues of SEQ ID NO:2.

5. The isolated polypeptide of claim 1 comprising residues 19-201 of SEQ ID NO:2.

6. The isolated polypeptide of claim 1 comprising residues 1-201 of SEQ ID NO: 2.

7. The isolated polypeptide of claim 1 comprising an amino acid sequence of SEQ ID NO:2.

8. An isolated polypeptide comprising a sequence of amino acid residues selected from the group consisting of:

(a) residues 19-42 of SEQ ID NO:2;

(b) residues 58-73 of SEQ ID NO:2;

(c) residues 90-106 of SEQ ID NO:2;

(d) residues 110-123 of SEQ ID NO:2; and

(e) residues 165-180 of SEQ ID NO:2.

9. An isolated polynucleotide molecule encoding a polypeptide wherein the encoded polypeptide comprises amino acid sequences selected from the group consisting of:

(a) residues 19-42 of SEQ ID NO:2; (b) residues 58-73 of SEQ ID NO:2;

(c) residues 90-106 of SEQ ED NO:2;

(d) residues 110-123 of SEQ ID NO:2; and

(e) residues 165-180 of SEQ ID NO:2

10. An isolated polynucleotide molecule encoding a polypeptide wherein the encoded polypeptide comprises an amino acid sequence that is at least nine contiguous amino acid residues of SEQ ID NO:2.

11. The isolated polynucleotide molecule of claim 10 comprising residues 43-201 of SEQ ID NO:2.

12. The isolated polynucleotide molecule of claim 10 comprising residues 19-201 of SEQ JD NO:2of SEQ ID NO:2.

13. The isolated polynucleotide molecule of claim 10 comprising residues 1-201 of SEQ ID NO:2.

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

15. A cultured cell into which has been introduced the expression vector of claim 14, wherein the cell expresses the DNA segment.

16. The cultured cell of claim 15, wherein a second expression vector is introduced into the cell said second expression comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a leukemia inhibitory factor (LD?) polypeptide shown in SEQ ID NO:8; and a transcription terminator.

17. A method of making a protein comprising: culturing a cell into which has been introduced the expression vector of claim 14 under conditions whereby the DNA segment is expressed and the polypeptide is produced; and recovering the protein from the cell.

18. An antibody that specifically binds to the polypeptide of claim 1.

19. An antibody that specifically binds to a polypeptide consisting of amino acid residues 43-201 of SEQ ID NO:2.

20. A method of detecting the presence of a polypeptide as shown in SEQ ID NO:2, or portion thereof, in a biological sample, comprising the steps of:

(a) contacting the biological sample with an antibody, or an antibody fragment, of claim 17, wherein the contacting is performed under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and

(b) detecting any of the bound antibody or bound antibody fragment.

21. A method for detecting a genetic abnormality in a patient, comprising: obtaining a genetic sample from a patient; producing a first reaction product by incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO: 1 or the complement of SEQ ID NO:1, under conditions wherein said polynucleotide will hybridize to complementary polynucleotide sequence; visualizing the first reaction product; and comparing said first reaction product to a control reaction product from a wild type patient, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient.