WO2000050594A2 - Mammalian alpha-helical protein, zsig83 - Google Patents

Abstract

The present invention relates to polynucleotide and polypeptide molecules for zsig83, a novel alpha-helical protein. The polypeptides, and polynucleotides encoding them, are cell growth modulating and may be used for delivery and therapeutics. The present invention also includes antibodies to the zsig83 polypeptides.

Description

Description MAMMALIAN ALPHA-HELICAL PROTEIN, ZSIG83

BACKGROUND OF THE INVENTION

The structural domain of an alpha helix is widely used in protein biology. The secondary structure formed by amino acids of this motif is shaped in a rod-like or spiral held in place by hydrogen bonds between backbone oxygen atoms and hydrogen atoms. Alpha helices and beta sheets represent the major internal supportive elements in proteins.

The function of alpha helices are diverse. Known functional roles of alpha helices include oxygen-binding proteins as those involved in the globin fold of myoglobin and hemoglobin, for example, DNA binding proteins, and in fibrous proteins which are constructed of chains of alpha helical segments.

Many alpha helical proteins are amphipathic and can act as channels or pores in the cell membrane, or can wrap around each other to form "coiled coils" allowing them to form dimers, trimers, or rodlike fibers. Some alpha helical proteins, such as cytokines are known to be involved in proliferation, maintenance, survival and differentiation of multi-cellular organisms. Such proteins are likely to be secreted and act in cell signaling by binding to integral membrane proteins on the cell surface. Other classes of proteins are soluble, such as the transcription factors, and bind to DNA molecules to modulate transcription.

SUMMARY OF THE INVENTION

Within one aspect, the invention provides an isolated polypeptide comprising fifteen contiguous amino acid residues of a polypeptide as shown in SEQ ID NO:2. Within an embodiment, the isolated polypeptide is from 15 to 184 amino acid residues in length. Within another embodiment, the fifteen contiguous amino acid residues are operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein, an immunoglobulin constant region, and a polyhistidine tag. Within another embodiment, the isolated polypeptide comprises at least 30 contiguous residues of SEQ ID NO:2. Within another embodiment, the isolated polypeptide comprises at least 47 contiguous residues of SEQ ID NO:2. Within another embodiment, is provided an isolated polynucleotide comprising the polynucleotides that encode the isolated polypeptide.

Within another aspect, the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding the isolated polypeptide comprising 15 contiguous amino acids of SEQ ID NO:2; and a transcription terminator. Within an embodiment, the invention provides a cultured cell comprising said expression vector. Within another embodiment, the invention provides a method of producing a polypeptide comprising culturing the cell under conditions whereby said sequence of nucleotides is expressed, and recovering said polypeptide. Within another embodiment of the invention is provided the polypeptide produced by said method.

Within another aspect, the invention provides an isolated polynucleotide encoding a fusion protein, said fusion protein comprising a first polypeptide and a second polypeptide wherein the first polypeptide is selected from the group consisting of : SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; and SEQ ID NO: 28, wherein the first portion is operably linked to a second polypeptide.

Within another aspect, the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a fusion protein, said fusion protein comprising the between one and five alpha helices as shown in SEQ ID NO:2, operably linked to a second polypeptide; and a transcription terminator. Within an embodiment, the invention also provides a cultured cell comprising said expression vector, wherein the cell expresses the DNA segment and produces the encoded fusion protein. Within another embodiment, is provided a method of producing a polypeptide comprising culturing said cell under conditions whereby said DNA segment is expressed, and recovering said polypeptide.

Within another aspect, the invention provides a computer-readable medium encoded with a data structure comprising SEQ ID NO:X, wherein X is an integer from 1 to 32.

Within another aspect, the invention provides an antibody that specifically binds to a protein as shown in SEQ ID NO:2.

Within another aspect, the invention provides a polypeptide which contains an amino acid sequence of an epitope-bearing portion of SEQ ID NO:2, said epitope-bearing portion having at least 15 amino acid residues of SEQ ID NO:2. Within another embodiment, the epitope-bearing polypeptide is selected from the group consisting of SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28 or wherein the polypeptide is at least 90% identical to a polypeptide selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.

Within one aspect, the present invention provides an isolated polynucleotide encoding a mammalian protein termed "zsig83". The human zsig83 polypeptide is comprised of a sequence of 184 amino acids long with the initial Met as shown in SEQ ID NO:2. Residues 1-16 comprise the signal sequence, and the mature zsig83 polypeptide is represented by the amino acid sequence comprised by residues 17, an arginine, through residue 184, a leucine. The mature zsig83 is also defined by SEQ ID NO:4. Within an additional embodiment, the polypeptide further comprises an affinity tag. Within a further embodiment, the polynucleotide is DNA or RNA.

Also provided are polypeptides which are at least 90% identical to SEQ ID NO:2 or SEQ ID NO:4 and poly-nucleotides which encode the polypeptides.

Within a second aspect of the invention there is provided an expression vector comprising (a) a transcription promoter; (b) a DNA segment encoding a zsig83 polypeptide, and (c) a transcription terminator, wherein the promoter, DNA segment, and terminator are operably linked. Within a third aspect of the invention there is provided a cultured yeast, fungal, bacterial, plant or mammalian cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses a zsig83 polypeptide encoded by the DNA segment, and a method for purifying and using the polypeptide. Within a further aspect of the invention there is provided a chimeric polypeptide consisting essentially of a first portion and a second portion joined by a peptide bond. The first portion of the chimeric polypeptide is either (a) a zsig83 polypeptide as shown in SEQ ID NO: 2 or SEQ ID NO:4 or (b) protein polypeptides that are at least 90% identical to SEQ ID NO:2 or SEQ ID NO:4 The second portion of the chimeric polypeptide consists essentially of another polypeptide such as an affinity tag. Within one embodiment the affinity tag is an immunoglobulin F_c polypeptide.

The invention also provides expression vectors encoding the chimeric polypeptides and host cells transfected to produce the chimeric polypeptides.

An additional embodiment of the present invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a zsig83 polypeptide having an amino acid sequence described above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a zsig83 polypeptide of the present invention include portions of such polypeptides with at least nine, preferably at least 15 and more preferably at least 30 to 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the present invention described above are also included in the present invention. Also claimed are any of these polypeptides that are fused to another polypeptide or carrier molecule. Antibodies produced from these epitope- bearing portions of zsig83 can be used in purifying zsig83 from cell culture medium. Examples of such epitope-bearing polypeptides are the polypeptides of SEQ ID NOs: 6,

7, 8, 9, 10, 11, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28. Also claimed are proteins or polypeptide which contain a sequence which is at least 90% identical to an epitope- bearing polypeptide described above.

Within an additional aspect of the invention there is provided an antibody or an antibody fragment that specifically binds to a zsig83 polypeptide as disclosed above, and also an anti-idiotypic antibody which neutralizes the antibody to a zsig83 polypeptide.

These and other aspects of the invention will become evident upon reference to the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a Hopp/Woods hydrophilicity profile of the zsig83 protein sequence shown in SEQ ID NO:2. 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. These residues are indicated in the figure by lower case letters.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms: 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 apoly- histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952- 4, 1985), substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-1210, 1988), streptavidin binding peptide,maltose binding protein (Guan et al., Gene 67:21-30, 1987), cellulose binding protein, thioredoxin, ubiquitin, T7 polymerase, 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 and other reagents are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ; New England Biolabs, Beverly, MA; Eastman Kodak, New Haven, CT). 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.

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.

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 < 10^ M" 1.

The term "complements of a polynucleotide molecule" is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'. The term "contig" denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5'-ATGGAGCTT-3' are 5'- AGCTTgagt-3 ' and 3 '-tcgacTACC-5 ' . The term "degenerate nucleotide sequence" denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).

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

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

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

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

"Paralogs" are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, a- globin, b-globin, and myoglobin are paralogs of each other.

A "polynucleotide" is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized 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. Such unpaired ends will in general not exceed 20 nt in length.

A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides". The term "promoter" is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.

A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless. The term "receptor" denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell. Membrane-bound receptors are characterized by a multi-domain or 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).

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. A "segment" is a portion of a larger molecule (e.g., polynucleotide or polypeptide) having specified attributes. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, that, when read from the 5' to the 3' direction, encodes the sequence of amino acids of the specified polypeptide.

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

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

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

The present invention relates to polynucleotide molecules, including DNA and RNA molecules that encode the polypeptides disclosed herein. The polynucleotides (SEQ ID NOs:l; and SEQ ID NO:32) and polypeptides (SEQ ID

NO:2) of the present invention have been designated as zsig83. The discovery of zsig83 as a novel alpha-helical polypeptide is based on its homology to another mammalian helical protein, zalphal (SEQ ID NO:5). See international patent application number PCT/US98/26273. Zalphal has also been identified by Nagasaki, K. et al., as a human breast cancer-associated gene, BG-X (Genebank accession number ABO 19527, 1998; Nagasaki, K. et al., Cancer Letters 140 :227-234, 1999). Another member of this alpha-helical cancer associated group of proteins has been identified: Human myometrium tumour EST encoded protein 7 by Rosenthal, A., et al, World Patents Index; 1999-602380/52 and WO 99/54448, 1999. The novel polypeptide of the present invention was first identified by searching the unfinished genomic section of the Genbank database for polynucleotide sequences having homology to zalphal. Zsig83 is 184 amino acids long and consists of one exon. A signal cleavage site at amino acid 16, an alanine, resulting in signal sequence of 16 amino acids (SEQ ID NO:6) and a mature protein (SEQ ID NO:4) of 168 residues, residues 17 to residue 184. There are five alpha helices: Helix A of zsig83 includes the amino acid residues 57 of SEQ ID NO:2, a leucine, through amino acid residue 71, a glutamine. Helix A is also defined by SEQ ID NO:7. Helix B of zsig83 includes amino acid 79 of SEQ ID NO: 2, an alanine, through amino acid residue 97, an alanine. Helix B is also defined by SEQ ID NO:8. Helix C of zsig83 includes amino acid 113 of SEQ ID NO: 2, a glycine, through amino acid residue 121, a threonine residue. Helix C is also defined by SEQ ID NO:9. Helix D of zsig83 includes amino acid 126 of SEQ ID NO: 2, a leucine, through amino acid residue 135, an arginine residue. Helix D is also defined by SEQ ID NO: 10. Helix E of zsig83 includes amino acid 144 of SEQ ID NO: 2, a valine, through amino acid residue 154, a leucine residue. Helix E is also defined by SEQ ID NO:l 1. Additionally, the region of amino acid from residue 38 to residue 54 of SEQ ID NO:2 show similarity to an SH3-binding domain. Those skilled in the art will recognize that predicted domain boundaries are approximations based on primary sequence content, and may vary slightly; however, such estimates are generally accurate to within ±5 amino acid residues.

The present invention also provides post translationally modified polypeptides or polypeptide fragments. Potential N-linked glycosylation sites can be found at amino acid residue 34 and 169 of SEQ ID NO:2. Other examples of post translational modifications include proteolytic cleavage, disulfide bonding and hydroxylation.

Analysis of the tissue distribution of zsig83 can be performed by the Northern blotting technique using Human Multiple Tissue and Master Dot Blots. Such blots are commercially available (Clontech, Palo Alto, CA) and can be probed by methods known to one skilled in the art. Also see, for example, Wu W. et al., Methods in Gene Biotechnology, CRC Press LLC, 1997.

Regions of amino acid identity or high similarity can be used to identify new family members. The region of highest amino acid identity 72% of zsig83 to zalphal is between residues 89 and 99 of SEQ ID NO:2 (See also SEQ ID NO: 12). Thus a degenerate primer to this region is useful to identify new family members. For instance, a degenerate oligonucleotide made to this region can be used to probe cDNA libraries, by nucleic acid hybridization. Specifically, cDNA libraries made from tissues known to be rich for transcription factors and small ligands, as well as cDNA libraries made from cancerous tissues would be useful. An example of a degenerate oligonucleotide is shown in SEQ ID NO:29.

The present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the zsig83 polypeptides disclosed herein. Upon isolation of the polynucleotide encoding a zsig83 polypeptide, a corresponding degenerate polynucleotide sequence may be obtained. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. SEQ ID NO:3 is a degenerate DNA sequence that encompasses all DNAs that encode the zsig83 polypeptide of SEQ ID NO:2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO:3 also provides all RNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus, zsig83 polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 552 of SEQ ID NO:3 and their RNA equivalents are contemplated by the present invention. 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 1

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 A|T 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

The degenerate codons used in SEQ ID NO:3, encompassing all possible codons for a given amino acid, are set forth in Table 2.

TABLE 2

One

Amino Letter Codons Degenerate

Acid Code Codon

Cys C TGC TGT TGY

Ser S AGC AGT TCA TCC TCG TCT WSN

Thr T ACA ACC ACG ACT ACN

Pro P CCA CCC CCG CCT CCN

Ala A GCA GCC GCG GCT GCN

Gly G GGA GGC GGG GGT GGN

Asn N AAC AAT AAY

Asp D GAC GAT GAY

Glu E GAA GAG GAR

Gin Q CAA CAG CAR

His H CAC CAT CAY

Arg R AGA AGG CGA CGC CGG CGT MGN

Lys K AAA AAG AAR

Met M ATG ATG

He I ATA ATC ATT ATH

Leu L CTA CTC CTG CTT TTA TTG YTN

Val V GTA GTC GTG GTT GTN

Phe F TTC TTT TTY

Tyr Y TAC TAT TAY

Trp W TGG TGG

Ter TAA TAG TGA TRR

Asn|Asp B RAY

Glu|Gln Z SAR

Any X NNN

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.

One of ordinary skill in the art will also appreciate that different species can exhibit "preferential codon usage." 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.

Within embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:l, other polynucleotide probes, primers, fragments and sequences recited herein or sequences complementary thereto.

Polynucleotide hybridization is well known in the art and widely used for many applications, see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987; Berger and Kimmel, eds., Guide to Molecular Cloning Techniques, Methods in Enzymology, volume 152, 1987 and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227-59, 1990. Polynucleotide hybridization exploits the ability of single stranded complementary sequences to form a double helix hybrid. Such hybrids include DNA-DNA, RNA-RNA and DNA-RNA. Hybridization will occur between sequences which contain 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 T_m of the mismatched hybrid decreases by 1°C for every 1-1.5% base pair mismatch. Varying the stringency of the hybridization conditions allows control over the degree of mismatch that will be present in the hybrid. The degree of stringency increases as the hybridization temperature increases and the ionic strength of the hybridization buffer decreases. Hybridization buffers generally contain blocking agents such as Denhardt's solution (Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA, milk powders (BLOTTO), heparin or SDS, and a Na⁺ source, such as SSC (IX SSC: 0.15 M NaCl, 15 mM sodium citrate) or SSPE (IX SSPE: 1.8 M NaCl, 10 mM NaH₂PO₄, 1 mM EDTA, pH 7.7). By decreasing the ionic concentration of the buffer, the stability of the hybrid is increased. Typically, hybridization buffers contain from between 10 roM-1 M Na⁺. Premixed hybridization solutions are also available from commercial sources such as Clontech Laboratories (Palo Alto, CA) and Promega Corporation (Madison, WI) for use according to manufacturer's instruction. Addition of destabilizing or denaturing agents such as formamide, tetralkylammonium salts, guanidinium cations or thiocyanate cations to the hybridization solution will alter the T_m of a hybrid. Typically, formamide is used at a concentration of up to 50% to allow incubations to be carried out at more convenient and lower temperatures. Formamide also acts to reduce non-specific background when using RNA probes. Stringent hybridization conditions encompass temperatures of about 5-

25°C below the thermal melting point (T_m) of the hybrid and a hybridization buffer having up to 1 M Na⁺. Higher degrees of stringency at lower temperatures can be achieved with the addition of formamide which reduces the T_m of the hybrid about 1°C for each 1% formamide in the buffer solution. Generally, such stringent conditions include temperatures of 20-70°C and a hybridization buffer containing 5X to 6X SSC and 0-50%) formamide. A higher degree of stringency can be achieved at temperatures of from 40-70°C with a hybridization buffer having 3X to 4X SSC and from 0-50% formamide. Highly stringent conditions typically encompass temperatures of 42-70°C with a hybridization buffer having up to 2X SSC and 0-50% formamide. Different degrees of stringency can be used during hybridization and washing to achieve maximum specific binding to the target sequence. Typically, the washes following hybridization are performed at increasing degrees of stringency to remove non- hybridized polynucleotide probes from hybridized complexes.

The above conditions are meant to serve as a guide and it is well within the abilities of one skilled in the art to adapt these conditions for use with a particular polypeptide hybrid. The T_m 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 that influence the T_m 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 T_m are known in the art, see for example (Sambrook et al., ibid.; Ausubel et al., ibid.; Berger and Kimmel, ibid, and Wetmur, ibid.) and are specific for DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences of varying length. Sequence analysis software such as Oligo 4.0 and Primer Premier, as well as sites on the Internet, are available tools for analyzing a given sequence and calculating T_m based on user defined criteria. Such programs can also analyze a given sequence under defined conditions and suggest suitable probe sequences. Typically, hybridization of longer polynucleotide sequences, >50 bp, is done at temperatures of about 20-25°C below the calculated T_m. For smaller probes, <50 bp, hybridization is typically carried out at the T_m or 5-10°C below. This allows for the maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids.

As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of zsig83 RNA. Such tissues and cells can be identified by Northern blotting

(Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980).

Total RNA can be prepared using guanidine isothiocyante extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al.,

Biochemistry 18:52-94, 1979). Poly (A)⁺ RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69: 1408-12, 1972). Complementary DNA (cDNA) is prepared from poly(A)⁺ RNA using known methods. In the alternative, genomic DNA can be isolated. Polynucleotides encoding zsig83 polypeptides are then identified and isolated by, for example, hybridization or PCR.

A full-length cDNA clone encoding zsig83 can be obtained by conventional cloning procedures. Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron. Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library. Expression libraries can be probed with antibodies to zsig83, or fragments thereof, or other specific binding partners.

Zsig83 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a zsig83 gene. Promoter elements from a zsig83 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 zsig83 proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous zsig83 gene in a cell is altered by introducing into the zsig83 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 zsig83 5' non- coding sequence that permits homologous recombination of the construct with the endogenous zsig83 locus, whereby the sequences within the construct become operably linked with the endogenous zsig83 coding sequence. In this way, an endogenous zsig83 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.

The polynucleotides of the present invention can also be synthesized using DNA synthesizers. Currently the method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 bp) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longer genes (>300 bp), however, special strategies must be invoked, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%. To overcome this problem, synthetic genes (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length. See Glick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA, (ASM Press, Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984 and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-7, 1990. The present invention further provides counterpart polypeptides and polynucleotides 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 zsig83 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human zsig83 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 zsig83 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. A zsig83-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, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human zsig83 sequence 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 zsig83 polypeptide. Similar techniques can also be applied to the isolation of genomic clones. Those skilled in the art will recognize that the sequence disclosed in

SEQ ID NO:l represents a single allele of human zsig83 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 DNA sequence shown in SEQ ID NO:l, 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. cDNAs generated from alternatively spliced mRNAs, which retain the properties of the zsig83 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.

The present invention also provides isolated zsig83 polypeptides that are substantially homologous to the polypeptides of SEQ ID NO:2 and their orthologs. The term "substantially homologous" is used herein to denote polypeptides having about 60%), 70%), or 80%), sequence identity to the sequences shown in SEQ ID NO:2 or their orthologs. Such polypeptides will more preferably be about 90%> or 95%) or more identical to SEQ ID NO:2 or its orthologs. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9, 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 "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by the standard one-letter codes). The percent identity is then calculated as:

Total number of identical matches x lOO

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

r- rH 1

3s rH CM 00 rH 1

En U0 CM CM o 1 1 ω "T 00 CM CM 1 1 1

Ol r- rH rH 00 CM 1 1 1 1 1

S LO O CM H rH rH rH rH 1 1 1 1 1 oo W LO rH 00 o oo CM CM

1 1 1 1 1 1 1 rH "f CM CM o 00 CM rH CM rH rH A 1 1 1 1 t 1

H ^«* CM ro o 00 CM rH 00 rH 00 1 1 1 1 1

3, 00 00 00 CM rH CM rH CM CM CM 00 1 1 1 1 1 1 1 1 1

VD CM <* 00 00 CM o CM CM 00 00 1 1 1 1 1 1 1 1 1 1 w uD CM O 00 00 CM 00 rH o r 00 CM CM 1 1 1 1 1 1 1 1 1 1 ex uO CM CM O 00 CM O 00 rH o rH CM rH CM 1 1 1 1 1 1 1 1 1 u σ\ 00 00 00 rH 00 rH CM 00 rH rH CM CM rH I 1 1 1 1 1 1 1 1 1 1 1 1 α O 00 o CM rH rH 00 rH 00 00 rH o rH 00 00 I 1 1 1 1 1 1 1 1 1 1

2 VD oo o O O 00 ro o CM 00 CM rH o CM 00

1 1 1 1 1 1 1 1

0, LO O CM ro rH o CM O 00 CM CM rH 00 CM rH rH 00 CM 00

I I 1 1 1 1 1 1 1 1 1 1 1 sC ^ CM CM O H rH O CM rH rH rH CM rH O 00 CM o

I I 1 1 1 1 1 1 1 1 1 1 sC Pi Z Q O α ω CJ ffi H H-l X S U_J to s >

Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above.

Those skilled in the art appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant MAPP. The FASTA algorithm is described by Pearson and Lipman, Proc. Nat'I 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. If there are several regions with scores greater than the "cutoff value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions. Illustrative parameters for FASTA analysis are: ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from four to six. Variant zsig83 polypeptides or substantially homologous zsig83 polypeptides 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 binding activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small 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 160 to 200 amino acid residues that comprise a sequence that is at least 70%), 80%>, 90%o, 95%>, or 99%> or more identical to the corresponding region of

SEQ ID NO:2. Such polypeptide variants can be used to generate antibodies which are useful in purifying zsig83 molecules. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zsig83 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 Table 4 cont. Small: glycine alanine serine threonine methionine

The present invention further provides a variety of other polypeptide fusions and related multimeric proteins comprising one or more polypeptide fusions.

For example, a zsig83 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 heavy chain constant region domains.

Immunoglobulin-zsig83 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric zsig83 analogs wherein the Fc portions are disulfide bonded to each other and two non-Ig polypeptides are arrayed in closed proximity to each other. Fusions of this type can be used to evaluate specific donor/acceptor molecules, affinity purify ligands, or use as an in vitro assay tool. This fusion can also be used to determine the homodimerization potential for zsig83. For use in assays, the chimeras are bound to a support via the F_c region and used in an

ELISA format. Additional fusions would include the fusion of an alpha helix of zsig83

(for example, Helices A, B, C, D, and or E, or a combination of these are represented by SEQ ID NOs: 18 through 28) fused to alpha helical domains of other alpha-helical proteins. The native zsig83 signal sequence may also be recombinantly exchanged with the signal sequence of other alpha-helical protein family members, for example, zalphal .

Transcription factors are composed of separable functional domains: a DNA-binding domain which interacts with specific DNA sequences and an activation domain which interacts with other proteins to stimulate transcription from a nearby promoter. Transcription factors may contain more than one activation domain. Alpha helices in the DNA binding domain of eukaryotic transcription factors are oriented so that they lie in the major groove of DNA where protein atoms make specific hydrogen bonds and van der Waals interactions with atoms in the DNA. Thus, the alpha helices of zsig83 can be fused individually or in combination with one or more activation domains of other transcription factors resulting in a novel transcription factor. This methodology is known to one skilled in the art and further discussed in Darnell, J. et al., Molecular Cell Biology, Third Edition, Scientific American Books, 1996.

Polypeptide fusions can be expressed in genetically engineered cells to produce a variety of zsig83 fusion analogs. Such alpha-helical polypeptides 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.

The proteins of the present invention can also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, trαra^,-3-methylproline, 2,4-methanoproline, cw-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, //ø-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRΝAs. Methods for synthesizing amino acids and aminoacylating tRΝA 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 chrornatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al, Science 259:806-9, 1993; and Chung et al., Proc. Νatl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRΝA and chemically aminoacylated suppressor tRΝAs (Turcatti et al., J. Biol. Chem. 271 :19991-8, 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 incoφorated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 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).

A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for zsig83 amino acid residues.

Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-502, 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. See also, Hilton et al., J. Biol. Chem. 271:4699-708, 1996. Sites of activity can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899- 904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related alpha-helical, cytokine-like, or transcription factor molecules.

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241 :53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region- directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

Variants of the disclosed zsig83 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91_.: 10747-51, 1994 and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.

Mutagenesis methods as disclosed herein can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells. Mutagenized DNA molecules that encode active polypeptides (e.g., alpha-helical or cytokine-like activity as evidenced by signal transduction or transcription) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure. Using the methods discussed herein, one of ordinary skill in the art can identify and/or prepare a variety of polypeptide fragments or variants of SEQ ID NO:2 that retain the alpha-helical, signal transduction or transcriptional interactions of the wild-type zsig83 protein. Such polypeptides may include additional amino acids from, for example, an activation domain of another member of the alpha-helical or cytokine- like family as well as part of or the entire signal sequence domain. Additional amino acids from affinity tags and the like may also be included. For any zsig83 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. The zsig83 polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides, can be produced in genetically engineered host cells according to conventional techniques. 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. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. 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. In general, a DNA sequence encoding a zsig83 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. To direct a zsig83 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 zsig83, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo. The secretory signal sequence is operably linked to the zsig83 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 secretory 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). The native secretory signal sequence of the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway. The present invention provides for such fusion polypeptides. A signal fusion polypeptide can be made wherein a secretory signal sequence derived from a zsig83 polypeptide is be operably linked to another polypeptide using methods known in the art and disclosed herein. The secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway. Such constructs have numerous applications known in the art. For example, these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non- secreted protein, such as a receptor. Such fusions may be used in vivo or in vitro to direct peptides through the secretory pathway.

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 L5:80, 1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90, 1989; Wang and Finer, Nature Med. 2:714-6, 1996). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Patent No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL

1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1 ; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, 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. Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. ^Bangalore) 1 L47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa calif ornica nuclear polyhedrosis virus (AcNPV). See, King, L.A. and Possee, R.D., The Baculovirus Expression System: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D.R. et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995. A second method of making recombinant zsig83 baculovirus utilizes a transposon-based system described by Luckow (Luckow, V.A, et al., J. Virol. 67:4566-79, 1993). This system, which utilizes transfer vectors, is sold in the Bac-to-Bac™ kit (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBacl™ (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zsig83 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid." The pFastBacl™ transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case zsig83. However, pFastBacl™ can be modified to a considerable degree. The polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins, M.S. and Possee, R.D., J. Gen. Virol. 71:971-6, 1990; Bonning, B.C. et al., J. Gen. Virol. 75:1551-6, 1994; and, Chazenbalk, G.D., and Rapoport, B., J. Biol. Chem. 270:1543-9, 1995. In such transfer vector constructs, a short or long version of the basic protein promoter can be used.

Moreover, transfer vectors can be constructed which replace the native zsig83 secretory signal sequences with secretory signal sequences derived from insect proteins. For example, a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen, Carlsbad, CA), or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native zsig83 secretory signal sequence. In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed zsig83 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Using a technique known in the art, a transfer vector containing zsig83 is transformed into E. coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus that expresses zsig83 is subsequently produced. Recombinant viral stocks are made by methods commonly used in the art. The recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994. Another suitable cell line is the High FiveO™ cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent #5,300,435). Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 II™ (Life Technologies) or ESF 921 ™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa, KS) or Express FiveO™ (Life Technologies) for the T. ni cells. The cells are grown up from an inoculation density of approximately 2-5 x 10⁵ cells to a density of 1-2 x 10⁶ cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3. Procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R.D., ibid.; O'Reilly, D.R. et al., ibid.; Richardson, C. D., ibid.). Subsequent purification of the zsig83 polypeptide from the supernatant can be achieved using methods described herein. 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 WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in transforming R. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. For polypeptide production in R. methanolica, it is preferred that the promoter and terminator in the plasmid be that of a R. methanolica gene, such as a R. methanolica alcohol utilization gene (A UGl or A UG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid flanked at both ends by host DNA sequences. A preferred selectable marker for use in Pichia methanolica is a R. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine. For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (A UGl and A UG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP 4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform R. methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.). When expressing a zsig83 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell. R. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25°C to 35°C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. A preferred culture medium for R. methanolica is YEPD (2% D-glucose, 2% Bacto™ Peptone (Difco Laboratories, Detroit, MI), \% Bacto™ yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

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.

Expressed recombinant zsig83 polypeptides (or chimeric zsig83 polypeptides) can be purified using fractionation and/or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred. Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross- linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor and receptor-like complementary polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated by a combination of procedures including, but not limited to, anion and cation exchange chromatography, size exclusion, and affinity chromatography. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents. Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in EnzymoL, Vol. 182, "Guide to Protein Purification", M. Deutscher, (ed.), Acad. Press, San Diego, 1990, pp.529-39). Within additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (e.g., maltose-binding protein, an immunoglobulin domain) may be constructed to facilitate purification.

To direct the export of a polypeptide from the host cell, the receptor DNA is linked to a second DNA segment encoding a secretory peptide, such as a t-PA secretory peptide or a zsig83 secretory peptide. To facilitate purification of the secreted receptor polypeptide, a C-terminal extension, such as a poly-histidine tag, substance P, Flag peptide (Hopp et al., Bio/Technology 6:1204-1210, 1988; available from Eastman Kodak Co., New Haven, CT) or another polypeptide or protein for which an antibody or other specific binding agent is available, can be fused to the receptor polypeptide. Moreover, using methods described in the art, polypeptide fusions, or hybrid zsig83 proteins, are constructed using regions or domains of the inventive zsig83 in combination with those of other human alpha-helical or cytokine-like family proteins (e.g. zalphaL¹, or heterologous proteins (Sambrook et al., ibid., Altschul et al., ibid., Picard, Cur. Opin. Biology, 5:511-5, 1994, and references therein). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest. Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure.

Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. For example, part or all of a domain(s) conferring a biological function may be swapped between zsig83 of the present invention with the functionally equivalent domain(s) from another family member, such as zalphal or other alpha-helices, cytokine-like molecule, or transcription factors, etc. Such domains include, but are not limited to, the signal sequence (residues 1 to 16 of SEQ ID NO:2), and other conserved motifs such as the alpha-helical homology region, (residues 17 to 184 of SEQ ID NO:2, as well as individual helices, i.e., Helices A, B, C, D, E, or a combination of these as represented by SEQ ID NOs: 18 to 28), and significant domains or regions in this family. Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known alpha-helical, cytokine-like or transcription factor family proteins (e.g. zalphal), depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein. Zsig83 polypeptides or fragments thereof may also be prepared through chemical synthesis. Zsig83 polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.

Zsig83 polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. The polypeptides are preferably prepared by solid phase peptide synthesis, for example as described by Merrifield, J. Am. Chem. Soc. 85:2149, 1963. Preparations of resins used for solid phase synthesis are commercially available and have been described by Stewart et al., "Solid Phase Peptide Synthesis" (2nd Edition), (Pierce Chemical Co., Rockford, IL, 1984) and Bayer & Rapp Chem. Pept. Prot. 3:3 (1986); and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989.

Various activating agents can be used for the coupling reactions and the extent of completion of the coupling reaction can be monitored at each stage, e.g., by the ninhydrin reaction as described by Kaiser et al., Anal. Biochem. 34:595, 1970. Evaluation of the zsig83 polypeptide, fragments thereof, fusion proteins containing zsig83, such as zsig83-Fc constructs, antibodies, agonists or antagonists for activity in the growth, differentiation, maintenance or survival of tissues can be carried out using cell cultures or animal systems. When administered to murine models, proteins of the present invention are formulated for parental, particularly intravenous or subcutaneous delivery according to standard methods. Delivery to animals would also include the use of viral systems such as the adenovirus, adeno-associated virus and retrovirus systems. Dosing regimen is determined empirically taking into account protein stability and other pharmacokinetic parameters known in the art. The effects of the present invention on growth, differentiation, maintenance or survival of connective and other tissues or organs can be assessed by the examination of histological sections taken from the recipient animals. Particular attention will be paid to tissues or organs in which zsig83 is expressed at high levels. Evaluations would include abnormal cell proliferation or cell death. Masson trichrome stain for collagen; orcein and Verhoeff- van Gieson stains for elastin and collagen; and Hale colloidal iron stain for acid mucopolysaccharide. The direct effect of the present invention on skin elasticity and other effects on skin may be assessed by the use of transdermal delivery systems known in the art.

An assay of interest measures or detects changes in proliferation, differentiation, and development. Proliferation can be measured using cultured cells, ex plant tissues, or in vivo by administering molecules of the claimed invention to the appropriate cells, tissues, or animal models. Generally, proliferative effects are observed as an increase in cell number and therefore, may include inhibition of apoptosis, as well as mitogenesis. Likewise, a decrease in cell number and cell migration could be analyzed. Assays measuring cell proliferation are well known in the art. For example, assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990,), incorporation of radiolabelled nucleotides (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).

Proliferation of bone marrow and peripheral blood lymphocyte cells can be assayed by harvesting these cells from mice, suspending the mononuclear cells in a base medium, and measuring proliferation in the presence of zsig83 protein. Similarly, clonogenic assays can be performed.

To determine if zsig83 is a chemotractant in vivo, zsig83 can be given by intradermal or intraperitoneal injection. Characterization of the accumulated leukocytes at the site of injection can be determined using lineage specific cell surface markers and fluorescence immunocytometry or by immunohistochemistry (Jose, J. Exp. Med. 179:881-87, 1994). Release of specific leukocyte cell populations from bone marrow into peripheral blood can also be measured after zsig83 injection.

Differentiation is a progressive and dynamic process, beginning with pluripotent stem cells and ending with terminally differentiated cells. Pluripotent stem cells that can regenerate without commitment to a lineage express a set of differentiation markers that are lost when commitment to a cell lineage is made. Progenitor 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 are usually functional properties such as cell products, enzymes to produce cell products and receptors and receptor-like complementary molecules. The stage of a cell population's differentiation is monitored by identification of markers present in the cell population. For example, myocytes, osteoblasts, adipocytes, chrondrocytes, fibroblasts and reticular cells are believed to originate from a common mesenchymal stem cell (Owen et al., Ciba Fdn. Symp. 136:42-46, 1988). Markers for mesenchymal stem cells have not been well identified (Owen et al., J. of Cell Sci. 87:731-738, 1987), so identification is usually made at the progenitor and mature cell stages.

There is evidence to suggest that factors that stimulate specific cell types down a pathway towards terminal differentiation or dedifferentiation affect the entire cell population originating from a common precursor or stem cell. Thus, zsig83 polypeptides may stimulate inhibition or proliferation of cells which have differentiated from a common precursor.

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).

Methods to study effects of the present invention on proliferation or differentiation generally comprise incubating cells derived from these tissues in the presence and absence of zsig83 polypeptide, monoclonal antibody, agonist or antagonist thereof and observing changes in cell proliferation or differentiation. As a ligand or transcription factor, the activity of zsig83 polypeptide can be measured by a silicon-based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with cell signaling and subsequent physiologic cellular responses. An exemplary 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, H.M. et al., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol. 228:84-108, 1997; Arimilli, S. et al., J. Immunol. Meth. 212:49-59, 1998; Van Liefde, I. et al, Eur. J. Pharmacol. 346:87-95, 1998. The microphysiometer can be used for assaying adherent or non- adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including zsig83 polypeptide, its agonists, or antagonists. Preferably, the microphysiometer is used to measure responses of a zsig83-responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to zsig83 polypeptide. ZSIG83-responsive eukaryotic cells comprise cells into which a receptor for zsig83 has been transfected creating a cell that is responsive to zsig83; or cells naturally responsive to zsig83 such as cells derived from tissues identified by Northern analysis. Differences, measured by a change, for example, an increase or diminution in extracellular acidification, in the response of cells exposed to zsig83 polypeptide, relative to a control not exposed to zsig83, are a direct measurement of zsig83- modulated cellular responses. Moreover, such zsig83-modulated responses can be assayed under a variety of stimuli. Using the microphysiometer, there is provided a method of identifying agonists of zsig83 polypeptide, comprising providing cells responsive to a zsig83 polypeptide, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test compound, and detecting a change, for example, an increase or diminution, in a cellular response of the second portion of the cells as compared to the first portion of the cells. The change in cellular response is shown as a measurable change extracellular acidification rate. Moreover, culturing a third portion of the cells in the presence of zsig83 polypeptide and the absence of a test compound can be used as a positive control for the zsig83-responsive cells, and as a control to compare the agonist activity of a test compound with that of the zsig83 polypeptide. Moreover, using the microphysiometer, there is provided a method of identifying antagonists of zsig83 polypeptide, comprising providing cells responsive to a zsig83 polypeptide, culturing a first portion of the cells in the presence of zsig83 and the absence of a test compound, culturing a second portion of the cells in the presence of zsig83 and the presence of a test compound, and detecting a change, for example, an increase or a diminution in a cellular response of the second portion of the cells as compared to the first portion of the cells. The change in cellular response is shown as a measurable change extracellular acidification rate. Antagonists and agonists, for zsig83 polypeptide, can be rapidly identified using this method. Moreover, zsig83 can be used to identify cells, tissues, or cell lines which respond to a zsig83-stimulated pathway. The microphysiometer, described above, can be used to rapidly identify ligand-responsive cells, such as cells responsive to zsig83 of the present invention. Cells can be cultured in the presence or absence of zsig83 polypeptide. Those cells which elicit a measurable change in extracellular acidification in the presence of zsig83 are responsive to zsig83. Such cell lines, can be used to identify antagonists and agonists of zsig83 polypeptide as described above.

Upon identification of tissues which have high quantities of zsig83 mRNA, one skilled in the art would know how to order and establish cell lines from these tissues, and perform assays as described herein. Such cell lines can be identified and obtained from a cell culture vendor such as, for example, American Type Culture

Collection, Manassas, VA.

Other assays to measure the effects of zsig83 include proliferation assays by testing tissue and cells from healthy volunteers with zsig83 protein, or a zsig83-free negative control for the ability of the tissue and cells to proliferate.

An in vivo approach for assaying proteins of the present invention involves viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, lentivirus, 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 acid (for a review, see T.C. Becker et al., Meth. Cell

Biol. 43:161-89, 1994; and J.T. Douglas and D.T. 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 a large number of available vectors containing different promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.

By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co- transfected plasmid. In an exemplary system, the essential El gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary). When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.

The adenovirus system can also be used for protein production in vitro. By culturing adenovirus-infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division. Alternatively, adenovirus vector infected 293 S cells can be grown in suspension culture at relatively high cell density to produce significant amounts of protein (see Gamier et al., Cytotechnol. 15:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant. Within the infected 293 S cell production protocol, non- secreted proteins may also be effectively obtained. Zsig83, agonists (including the natural ligand/ substrate/ cofactor/ etc.) and antagonists have enormous potential for both in vitro and in vivo applications. Compounds identified as zsig83 agonists are useful for studying alpha-helical and cytokine mediated activities as well as transcription in vitro and in vivo. For example, zsig83 and agonist compounds are useful as components of defined cell culture media, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture. Agonists are thus useful in specifically modulating the growth and/or development of tissue in culture. Alternatively, zsig83 polypeptides and zsig83 agonist polypeptides are useful as a research reagent, particularly for the growth and expansion of cells. Zsig83 polypeptides are added to tissue culture media for this purpose. Antagonists are also useful as research reagents for characterizing sites of interactions between member of complement/anti-complement pairs.

Inhibitors of zsig83 activity (zsig83 antagonists) include anti-zsig83 antibodies and soluble zsig83 ligands and transcription factors as well as other peptidic and non-peptidic agents (including ribozymes).

Zsig83 can also be used to identify inhibitors (antagonists) of its activity. Test compounds are added to the assays disclosed herein to identify compounds that inhibit the activity of zsig83. In addition to those assays disclosed herein, samples can be tested for inhibition of zsig83 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of zsig83-dependent cellular responses. For example, zsig83-responsive cell lines can be transfected with a reporter gene construct that is responsive to a zsig83-stimulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a zsig83-DNA response element operably linked to a gene encoding an assayable protein, such as luciferase. DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE) insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) and serum response elements (SRE) (Shaw et al. Cell 56: 563-72, 1989). Cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem. 263 (19):9063-6; 1988 and Habener, Molec. Endocrinol. 4 (8): 1087-94; 1990. Hormone response elements are reviewed in Beato, Cell 56:335-44; 1989. Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of zsig83 on the target cells as evidenced by a decrease in zsig83 stimulation of reporter gene expression. Assays of this type will detect compounds that directly block zsig83 binding to cell-surface receptors, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding. In the alternative, compounds or other samples can be tested for direct blocking of zsig83 binding to receptor using zsig83 tagged with a detectable label (e.g., ¹²⁵I, biotin, horseradish peroxidase, FITC, and the like). Within assays of this type, the ability of a test sample to inhibit the binding of labeled zsig83 to the receptor is indicative of inhibitory activity, which can be confirmed through secondary assays. Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors, or receptor-like complementary molecules.

Also, zsig83 polypeptides, agonists or antagonists thereof may be therapeutically useful for promoting wound healing. To verify the presence of this capability in zsig83 polypeptides, agonists or antagonists of the present invention, such zsig83 polypeptides, agonists or antagonists are evaluated with respect to their ability to facilitate wound healing according to procedures known in the art. If desired, zsig83 polypeptide performance in this regard can be compared to growth factors, such as EGF, NGF, TGF-α, TGF-β, insulin, IGF-I, IGF-II, fibroblast growth factor (FGF) and the like. In addition, zsig83 polypeptides or agonists or antagonists thereof may be evaluated in combination with one or more growth factors to identify synergistic effects.

Zsig83 polypeptides can also be used for purification of ist binding partner. The polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross- linked polyacrylamide, or like materials that are stable under the conditions of use. Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation. The resulting medium will generally be configured in the form of a column or chip, and fluids containing ligand are passed through the column or chip one or more times to allow ligand to bind to the receptor or receptor-like complementary polypeptide. The binding partner is then eluted using changes in salt concentration, chaotropic agents (guanidine HC1), or pH to disrupt ligand-receptor binding. An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/ anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed. Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed by Karlsson, J. Immunol. Methods

145:229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993. A receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell. A test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding. Ligand-binding receptor polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51 : 660-72, 1949) and calorimetric assays (Cunningham et al., Science 253:545-48, 1991; Cunningham et al, Science 245:821-25, 1991). Soluble forms of zsig83 polypeptides can be generated by removing the hydrophobic region between residues 1 and 16 of SEQ ID NO: 2. Soluble zsig83 polypeptides are useful in studying the effects of the present invention in vivo and in vitro.

Another embodiment of the present invention provides for a peptide or polypeptide comprising an epitope-bearing portion of a zsig83 polypeptide of the invention. The epitope of the this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention. A region of a protein to which an antibody can bind is defined as an "antigenic epitope". See for instance, Geysen, H.M. et al, Proc. Natl. Acad Sci. USA 81.3998-4002 (1984). As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in the art that 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, J.G. et al. Science 219:660-666 (1983). Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer soluble peptides, especially those containing proline residues, usually are effective.

Zsig83 polypeptides can also be used to prepare antibodies that bind to zsig83 epitopes, peptides or polypeptides. The zsig83 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response. One of skill in the art would recognize that antigenic, epitope-bearing polypeptides contain a sequence of at least 6, preferably at least 9, and more preferably at least 15 to about 30 or about 50 contiguous amino acid residues of a zsig83 polypeptide (e.g., SEQ ID NO:2). Polypeptides comprising a larger portion of a zsig83 polypeptide, i.e., from 30 to 184 residues up to the entire length of the amino acid sequence are included. Antigens or immunogenic epitopes can also include attached tags, adjuvants and carriers, as described herein. Suitable antigens include the mature zsig83 polypeptide encoded by SEQ ID NO:2 from amino acid number 17 to amino acid number 184, or a contiguous 9 to 30 amino acid fragment thereof. Other suitable antigens include the individual helices of zsig83 polypeptides and contiguous combinations thereof as disclosed herein. Specifically, the polypeptides of SEQ ID NOs:6 to 11 and 18 to 28 would be suitable antigens. Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and hydrophobic residues are preferably avoided); and sequences containing proline residues are particularly preferred. Preferred peptides to use as antigens are hydrophilic peptides such as those predicted by one of skill in the art from a hydrophobicity plot

(See Figure: 1). Zsig83 hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: residues 142-147 (SEQ ID NO: 13); residues 119-124 (SEQ ID NO:14); residues 178-183 (SEQ ID NO:15); residues 76-81 (SEQ ID NO: 16); and residues 90-95 (SEQ ID NO: 17); all of SEQ ID NO:2. Antibodies from an immune response generated by inoculation of an animal with these antigens can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982.

The present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of an zsig83 polypeptide described herein. Such fragments or peptides may comprise an "immunogenic epitope," which is a part of a protein that elicits an antibody response when the entire protein is used as an immunogen. Immunogenic epitope-bearing peptides can be identified using standard methods (see, for example, Geysen et al, Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).

In contrast, polypeptide fragments or peptides may comprise an "antigenic epitope," which is a region of a protein molecule to which an antibody can specifically bind. Certain epitopes consist of a linear or contiguous stretch of amino acids, and the antigenicity of such an epitope is not disrupted by denaturing agents. It is known in the art that relatively short synthetic peptides that can mimic epitopes of a protein can be used to stimulate the production of antibodies against the protein (see, for example, Sutcliffe et al, Science 219:660 (1983)). Accordingly, antigenic epitope- bearing peptides and polypeptides of the present invention are useful to raise antibodies that bind with the polypeptides described herein.

Antigenic epitope-bearing peptides and polypeptides contain at least four to ten amino acids, or at least ten to fifteen amino acids, or 15 to 30 amino acids of SEQ ID NO:2. Such epitope-bearing peptides and polypeptides can be produced by fragmenting an zsig83 polypeptide, or by chemical peptide synthesis, as described herein. Moreover, epitopes can be selected by phage display of random peptide libraries (see, for example, Lane and Stephen, Curr. Opin. Immunol. 5:268 (1993), and Cortese et al, Curr. Opin. Biotechnol. 7:616 (1996)). Standard methods for identifying epitopes and producing antibodies from small peptides that comprise an epitope are described, for example, by Mole, "Epitope Mapping," in Methods in Molecular

Biology, Vol. 10, Manson (ed.), pages 105-1 16 (The Humana Press, Inc. 1992), Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies: Production, Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages 60-84 (Cambridge University Press 1995), and Coligan et al. (eds.), Current Protocols in Immunology, pages 9.3.1 - 9.3.5 and pages 9.4.1 - 9.4.11 (John Wiley & Sons 1997).

As an illustration, potential antigenic sites in zsig83 polypeptides (SEQ ID NO:2) were identified using the Jameson- Wolf method, Jameson and Wolf, CABIOS :181, (1988), as implemented by the PROTEAN program (version 3.14) of LASERGENE (DNASTAR; Madison, WI). Default parameters were used in this analysis.

Suitable antigens of zsig83 polypeptides include residue 13 to residue 21 of SEQ ID NO:2; residue 32 to residue 57 of SEQ ID NO:2; residue 73 to residue 81 of SEQ ID NO:2; residue 101 to residue 111 of SEQ ID NO:2; residue 117 to residue 151 of SEQ ID NO:2; residue 155 to residue 161 of SEQ ID NO:2; residue 166 to residue 175 of SEQ ID NO:2; and residue 179 to residue 184 of SEQ ID NO:2; or a portion thereof which contains a 4 to 10 amino acid segment. Hydrophilic peptides, such as those predicted by one of skill in the art from a hydrophobicity plot are also immonogenic. Zsig83 hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: residue 11 to residue 17 of SEQ ID NO:2; residue 33 to residue 56 of SEQ ID NO:2; residue 74 to residue 80 of SEQ ID NO:2; residue 90 to residue 151 of SEQ ID NO:2; residue 162 to residue 173 SEQ ID NO:2; and residue 178 to residue 184 of SEQ ID NO:2; or a portion thereof which contains a 4 to 10 amino acid segment. Additionally, antigens can be generated to portions of the polypeptide which are likely to be on the surface of the folded protein. These antigens include: residue 34 to residue 51 SEQ ID NO:2; residue 101 to residue

111 of SEQ ID NO:2; residue 117 to residue 140 of SEQ ID NO:2; residue 142 to residue 147 of SEQ ID NO:2; and residue 179 to residue 184 of SEQ ID NO:2; or a portion thereof which contains a 4 to 10 amino acid segment. Antigenic epitope- bearing peptides and polypeptides of the present invention are useful to raise antibodies that bind with the polypeptides described herein which then can be used to purify the protein in either a native or denatured form or to detect the zsig83 polypeptide in a Western blot.

Antibodies generated from this immune response can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982.

As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a zsig83 polypeptide or a fragment thereof. The immunogenicity of a zsig83 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of zsig83 or a portion thereof with an immunoglobulin polypeptide or with a 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.

As used herein, the term "antibodies" includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included. Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incoφorating 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. Antibodies are considered to be specifically binding if: 1) they exhibit a threshold level of binding activity, and 2) they do not significantly cross-react with related polypeptide molecules. A threshold level of binding is determined if anti-zsig83 antibodies herein bind to a zsig83 polypeptide, peptide or epitope with an affinity at least 10-fold greater than the binding affinity to control (non-zsig83) polypeptide. It is preferred that the antibodies exhibit a binding affinity (K_a) of

or greater,

preferably 10 7 M-1 or greater, more preferably 108 M-1 or greater, and most preferably

9 -1 10 M or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G., Ann.

NY Acad. Sci. 51 : 660-672, 1949). Whether anti-zsig83 antibodies do not significantly cross-react with related polypeptide molecules is shown, for example, by the antibody detecting zsig83 polypeptide but not known related polypeptides using a standard Western blot analysis

(Ausubel et al., ibid.). Examples of known related polypeptides are those disclosed in the prior art, such as known orthologs, and paralogs, and similar known members of a protein family. Screening can also be done using non-human zsig83, and zsig83 mutant polypeptides. Moreover, antibodies can be "screened against" known related polypeptides, to isolate a population that specifically binds to the zsig83 polypeptides. For example, antibodies raised to zsig83 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to zsig83 will flow through the matrix under the proper buffer conditions. Screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to known closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995). Screening and isolation of specific antibodies is well known in the art. See, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principles and Practice, Goding, J.W. (eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984. Specifically binding anti-zsig83 antibodies can be detected by a number of methods in the art, and disclosed below. A variety of assays known to those skilled in the art can be utilized to detect antibodies which bind to zsig83 proteins or 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, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant zsig83 protein or polypeptide.

Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zsig83 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zsig83 protein or peptide). Genes encoding polypeptides having potential zsig83 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli. Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.

These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al, US Patent NO. 5,223,409; Ladner et al, US Patent NO. 4,946,778; Ladner et al., US Patent NO. 5,403,484 and Ladner et al., US Patent NO. 5,571,698) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Random peptide display libraries can be screened using the zsig83 sequences disclosed herein to identify proteins which bind to zsig83. These "binding polypeptides" which interact with zsig83 polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding polypeptides can also be used in analytical methods such as for screening expression libraries and neutralizing activity, e.g., for blocking interaction between ligand and receptor, or viral binding to a receptor. The binding polypeptides can also be used for diagnostic assays for determining circulating levels of zsig83 polypeptides; for detecting or quantitating soluble zsig83 polypeptides as marker of underlying pathology or disease. These binding polypeptides can also act as zsig83 "antagonists" to block zsig83 binding and signal transduction in vitro and in vivo. These anti-zsig83 binding polypeptides would be useful for inhibiting zsig83 activity or protein-binding.

Antibodies or polypeptides herein can also 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 can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance). More specifically, zsig83 polypeptides or anti-zsig83 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.

Suitable detectable molecules may 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 may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium- 188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance). Polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion. For these puφoses, biotin streptavidin is an exemplary complementary/ anticomplementary pair.

In another embodiment, polypeptide-toxin fusion proteins or antibody- toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues). Alternatively, if the polypeptide has multiple functional domains (i.e., an activation domain or a ligand binding domain, plus a targeting domain), a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest. In instances where the domain only fusion protein includes a complementary molecule, the anti-complementary molecule can be conjugated to a detectable or cytotoxic molecule. Such domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue- specific delivery of generic anti-complementary-detectable/ cytotoxic molecule conjugates.

In another embodiment, zsig83-cytokine fusion proteins or antibody- cytokine fusion proteins can be used for enhancing in vivo killing of target tissues for example, in tissues identified in Northern blotting, if the zsig83 polypeptide or anti- zsig83 antibody targets, for example, the hypeφroliferative tissue (See, generally, Hornick et al., Blood 89:4437-47, 1997). They described fusion proteins enable targeting of a cytokine to a desired site of action, thereby providing an elevated local concentration of cytokine. Suitable zsig83 polypeptides or anti-zsig83 antibodies target an undesirable cell or tissue (i.e., a tumor or a leukemia), and the zsig 83 -cytokine or the anti-zsig83-cytokine fusions mediate improved target cell lysis by effector cells. Suitable cytokines for this puφose include interleukin 2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), for instance.

The bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action. Molecules of the present invention can be used to identify and isolate receptors, ligands, or members of complement/anti-complement pairs involved in cell signaling, transcription, and metastasis. For example, proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column (Immobilized Affinity Ligand Techniques, Hermanson et al., eds., Academic Press, San Diego, CA, 1992, pp.195-202). Proteins and peptides can also be radiolabeled (Methods in Enzymol., vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990, 721-37) or photoaffinity labeled (Brunner et al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedan et al., Biochem. Pharmacol. 33: 1167-80, 1984) and specific cell-surface proteins can be identified.

As a reagent, the polynucleotides of the present invention can be used to identify new family members. An exemplary region of polynucleotide for this use would be that encoding the amino acid residues from residue 89 to 99 of SEQ ID NO: 2, and the corresponding degenerate polynucleotide of SEQ ID NO:3. This would be useful in finding new alpha-helical, cytokine-like peptides from the same or other tissues. The polypeptides, nucleic acid and/or antibodies of the present invention can be used in treatment of disorders associated with abnormal cell growth and metastasis. The molecules of the present invention can be used to modulate cell signaling or transcription or to treat or prevent development of pathological conditions in diverse tissues. The present invention also provides reagents with significant therapeutic value. The zsig83 polypeptide (naturally occurring or recombinant), fragments thereof, antibodies and anti-idiotypic antibodies thereto, along with compounds identified as having binding affinity to the zsig83 polypeptide, should be useful in the treatment of conditions associated with abnormal physiology or development, including abnormal proliferation, e.g., cancerous conditions, or degenerative conditions. For example, a disease or disorder associated with abnormal expression or abnormal signaling by a zsig83 polypeptide should be a likely target for an agonist or antagonist of the zsig83 polypeptide.

Antibodies to the zsig83 polypeptide can be purified and then administered to a patient. These reagents can be combined for therapeutic use with additional active or inert ingredients, e.g., in pharmaceutically acceptable carriers or diluents along with physiologically innocuous stabilizers and excipients. These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations. This invention also contemplates use of antibodies, binding fragments thereof or single-chain antibodies of the antibodies including forms which are not complement binding.

Proteins of the present invention are useful as targets for identifying modulators of transcription or cell signaling activity. More particularly, zsig83 polypeptides are useful for screening and/or identifying new transcription inhibitors. The zsig83 polypeptides may also be used as a basis for rational drug design of inhibitory molecules. These newly identified inhibitory molecules may be more specific and/or more potent than known transcription inhibitors. Zsig83 inhibitors may exhibit a more favorable side effect profile than known transcription factor or cell signaling inhibitors. Inhibitory molecules identified using zsig83 polypeptides as a target may modulate different biological or physiological activities than known transcription inhibitors (i.e., may be effective for disorders other than those related to cancer and metastasis). Zsig83 inhibitors may provide broader inhibition than just transcription inhibition (for instance, these inhibitors may modulate many alpha-helical, cytokine-like and transcription factor family members). Due to the similarity of zsig83 to zalphal, which has been associated with breast cancer, inhibitors may beneficially improve the status of patients with various types of cancer and abnormal cell growth. The effects of zsig83 inhibitors can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to the appropriate animal model.

The quantities of reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medications administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in vivo administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Zsig83 molecules, including alternatively spliced peptides variants, and fragments can be applied in isolation, or in conjunction with other molecules (growth factors, cytokines, etc.) in tissue. Alternative splicing of zsig83 may be cell-type specific and confer activity to specific tissues.

Polynucleotides encoding zsig83 polypeptides (including fragments) are useful within gene therapy applications where it is desired to increase or inhibit zsig83 activity. If a mammal has a mutated or absent zsig83 gene, the zsig83 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zsig83 polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA virus, such as, but not limited to, heφes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. A defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective heφes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30, 1992; and a defective adeno-associated virus vector (Samulski et al., J. Virol. 61 :3096- 101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

In another embodiment, a zsig83 gene can be introduced in a retroviral vector, e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al. Cell

33:153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., J. Virol. 62:1120, 1988; Temin et al., U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al.; and Kuo et al., Blood 82:845, 1993. Alternatively, the vector can be introduced by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. More particularly, directing transfection to particular cells represents one area of benefit. For instance, directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the puφose of targeting. Targeted peptides (e.g., hormones or neurotransmitters), proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically. It is possible to remove the target cells from the body; to introduce the vector as a naked DNA plasmid; and then to re-implant the transformed cells into the body. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem. 267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

Various techniques, including antisense and ribozyme methodologies, can be used to inhibit zsig83 gene transcription and translation, such as to inhibit cell proliferation in vivo. Polynucleotides that are complementary to a segment of a zsig83- encoding polynucleotide (e.g., a polynucleotide as set forth in SEQ ID NO:l) are designed to bind to zsig83-encoding mRNA and to inhibit translation of such mRNA. Such antisense polynucleotides are used to inhibit expression of zsig83 polypeptide- encoding genes in cell culture or in a subject. Zsig83 gene may be useful to as a probe to identify humans who have a defective zsig83 gene. Thus, polynucleotides and polypeptides of zsig83, and mutations to them, can be used a diagnostic indicators of cancer in tissues identified by Northern analysis.

Transgenic mice, engineered to express the zsig83 gene, and mice that exhibit a complete absence of zsig83 gene function, referred to as "knockout mice"

(Snouwaert et al., Science 257:1083, 1992), may also be generated (Lowell et al, Nature 366:740-42, 1993). These mice may be employed to study the zsig83 gene and the protein encoded thereby in an in vivo system.

The protein of the present invention has 40%> identity to zalphal, which has been indicated as a breast cancer associated gene (Nagasaki, K. et al., ibid).

Additionally, as discussed above, it is also related to a protein that has been identified in an uterus myoma. This suggests that zsig83 plays a role in the process of cell growth, differentiation, or proliferation in these and other tissues. Thus, zsig83 polynucleotides and polypeptides are useful in diagnosis, tracking and treatment of diseases associated with cancerous or abnormal growth. Zsig83 polypeptides would be useful as a replacement therapy for pre- cancerous and cancer tissues. To verify the presence of zsig83 activity in normal cell lines and tumor cell lines, such cell lines are evaluated with respect to the presence of the zsig83 polynucleotides and polypeptides according to procedures known in the art.

The polypeptides of the present invention are useful in studying cell adhesion and the role thereof in metastasis and may be useful in preventing metastasis. Similarly, polynucleotides and polypeptides of zsig83 may be used to replace their defective counteφarts in tumor or malignant tissues.

Similarly, a defective zsig83 gene may result in improper cell signaling or transcription of tissues, thus affecting cell-cell interactions and possibly cell cycle regulation. Such cases could be treated by administering polypeptides of zsig83 to mammals with such a defective gene.

Zsig83 polypeptides, variants, fragments thereof, nucleic acid, and/or antibodies of the present invention may be used in treatment of disorders associated with abnormal cell growth stemming from impaired receptor binding, signal transduction and/or transcription. The molecules of the present invention may used to modulate or to treat or prevent development of pathological conditions in the tissues identified by Northern analysis. In particular, certain syndromes or diseases may be amenable to such diagnosis, treatment or prevention.

The polynucleotides of the present invention may also be used in conjunction with a regulatable promoter, thus allowing the dosage of delivered protein to be regulated.

Zsig83 polynucleotides can be used to express zsig83 polypeptides; as probes to identify nucleic acid encoding alpha-helical proteins having signal transducing and receptor binding acitivity; to identify human chromosome 22; to map genes on or identify genes associated with certain diseases, syndromes, or other conditions, associated with human chromosome 22; as single-stranded sense or antisense oligonucleotides to inhibit expression on the polypeptide encoded by the zsig83 gene.

Polynucleotides and polypeptides of the present invention will additionally find use as educational tools as a laboratory practicum kit for courses related to genetics and molecular biology. Due to its unique polynucleotide and polypeptide sequence molecules of zsig83 can be used as standards or as "unknowns" for testing puφoses. For example, zsig83 polynucleotides can be used as an aid, such as, for example, to teach a student how to prepare expression constructs for bacterial, viral, and/or mammalian expression, including fusion constructs, wherein zsig83 is the gene to be expressed; for determining the restriction endonuclease cleavage sites of the polynucleotides; determining mRNA and DNA localization of zsig83 polynucleotides in tissues (i.e., by Northern and Southern blotting as well as polymerase chain reaction); and for identifying related polynucleotides and polypeptides by nucleic acid hybridization. Zsig83 polypeptides can be used educationally as an aid to teach preparation of antibodies; identifying proteins by Western blotting; protein purification; determining the weight of expressed zsig83 polypeptides as a ratio to total protein expressed; identifying peptide cleavage sites; coupling amino and carboxyl terminal tags; as well as, but not limited to monitoring biological activities of both the native and tagged protein (i.e., receptor binding, signal transduction, proliferation, and differentiation) in vitro and in vivo.

The gene for zsig83 is located at the chromosomal locus 22ql3.1-ql3.2. Significant recurrent chromosomal aberrations observed in human neoplasia have been mapped to this locus. (See Mitelman, F. et al., Nature Genetics Special Issue, April, 1997). These include one balanced aberration and five unbalanced aberrations. The balanced aberration is t(l;22)(pl3;ql3) which has been observed in Acute Myeloid

Leukemia, an hematological neoplasm. The unbalanced aberration is del(22)(ql3), which has been observed in five diseases. Of these five, two are hematological neoplasms, Chronic Lymphoproliferative Disorder, and Non-Hodgkins Lymphoma; two are neurological neoplasms, Astrocytoma, and Neuroblastoma; and one is a malignant epithelial neoplasm, Adenocarcinoma of the stomach. Considering the similarity of zsig83 to zalphal, which is a breast cancer-associated gene, the translocation of zsig83 (as would occur in a balanced aberration), or the deletion of zsig83 (as would occur in an unbalanced aberration) may cause unregulated control of zsig83 expression resulting in abnormal cell growth. Thus, zsig83 polynucleotides and polypeptides are useful for treatment of disorders related to such chromosomal aberrations.

The present invention also provides reagents which will find use in diagnostic applications. For example, the zsig83 gene has been mapped on chromosome 22ql3.1-ql3.2. A zsig83 nucleic acid probe could be used to check for abnormalities on chromosome 22. For example, a probe comprising zsig83 DNA or RNA or a sub- sequence thereof can be used to determine if the zsig83 gene is present on chromosome 22q or if a mutation has occurred. Detectable chromosomal aberrations at the zsig83 gene locus include but are not limited to aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymoφhism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art [Sambrook et al, ibid.; Ausubel, et. al, ibid.; Marian, A.J., Chest, 108: 255-265, (1995)].

For pharmaceutical use, the proteins of the present invention can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as powders, ointments, drops or transdermal patch) bucally, or as a pulmonary or nasal inhalant. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. In general, pharmaceutical formulations will include a zsig83 protein in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995. Therapeutic doses will generally be in the range of 0.1 to 100 μg/kg of patient weight per day, preferably 0.5-20 mg/kg per day, 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. The proteins may be administered for acute treatment, over one week or less, often over a period of one to three days or may be used in chronic treatment, over several months or years. In general, a therapeutically effective amount of zsig83 is an amount sufficient to produce a clinically significant change in tissues, such as those tissues identified by a Northern blot analysis. Similarly, a therapeutically effective amount of zsig83 is an amount sufficient to produce a clinically significant change in disorders associated with abnormal cell growth.

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

EXAMPLES Example 1 : Cloning cDNA

One skilled in the art would be able to isolate the full-length polynucleotide in the following manner: Sense and antisense oligonucleotides can be designed to regions of low degeneracy of the polynucleotide sequence. Exemplary oligonucleotides would be a sense primer (SEQ. ID NO:30), and an antisense primer (SEQ. ID NO:31). The PCR product of these oligonucleotides will be 165 nucleotides long. Commercially available cDNA from a library known to be rich in cytokines and transcription factors, such as from liver or kidney tissue, for example, is used as template. The following thermalcycler conditions are used: 94 degrees for 2 minutes; followed by thirty cycles of 94 degrees for 20 seconds, 72 degrees for 1 minute; followed by a final extension of 72 degrees for 7 minutes. The resulting PCR product is subcloned and sequenced. Once the sequence is verified, the PCR product is labeled with a commercially available kit (Mega-prime DNA labeling Kit, Piscataway. NJ ) and used as a probe to screen a cDNA phage library for the full-length gene. Phage screening is known to one skilled in the art. Additional information can be found in Wu, ibid. An exemplary cDNA phage library to screen is from human kidney or human liver (Clontech, Palo Alto, CA). An isolated phage containing the gene of interest is converted to a phagemid using a commercially available kit (Rapid Excision Kit, Stratagene, La Jolla, CA).

Example 2:Tissue Distribution Analysis of tissue distribution is performed by the Northern blotting technique using Human Multiple Tissue and Master Dot Blots (Clontech, Palo Alto, CA). A probe of about 165 base pairs is obtained by PCR as in Example 1. The PCR product is gel-purified and random prime labeled with ³²P using a commercially available kit (Rediprime DNA Labeling System; Amersham Coφ., Arlington Heights, IL) according to the manufacturer's direction. The probe is then purified using a NucTrap® probe purification column (Stratagene, La Jolla, CA). ExpressHyb™ Hybridization Solution (Clontech, Palo Alto, CA) is used for pre-hybridization and hybridization. Hybridization is overnight at 65°C, and the blots are then washed three times in 2X SSC and 0.05% SDS at 55^αC, followed by two washes in 0.1X SSC and 0.1%) SDS at 55°C. The blots are then exposed to autoradiograph film, which is then developed.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for puφoses 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

CLAIMSWhat is claimed is:

1. An isolated polypeptide comprising fifteen contiguous amino acid residues of a polypeptide as shown in SEQ ID NO:2.

2. The isolated polypeptide of claim 1, wherein said at least fifteen contiguous amino acid residues are operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein, an immunoglobulin constant region, and a polyhistidine tag.

3. An isolated polynucleotide comprising the polynucleotides that encode the isolated polypeptide of claim 1.

4. An isolated polynucleotide encoding a fusion protein, said fusion protein comprising a first polypeptide and a second polypeptide wherein the first polypeptide is selected from the group consisting of :

(a) SEQ ID NO:2;

(b) SEQ ID NO:4;

(c) SEQ ID NO: 6;

(d) SEQ ID NO: 7;

(e) SEQ ID NO: 8;

(f) SEQ ID NO: 9;

(g) SEQ ID NO: 10; (h) SEQ ID NO: 11 (i) SEQ ID NO: 18 G) SEQ ID NO: 19 (k) SEQ ID NO: 20 (1) SEQ ID NO: 21 (m) SEQ ID NO: 22; (n) SEQ ID NO: 23 (o) SEQ ID NO: 24 (p) SEQ ID NO: 25 (q) SEQ ID NO: 26 (r) SEQ ID NO: 27; and (s) SEQ ID NO: 28 wherein the first portion is operably linked to a second polypeptide.

5. An expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide selected from the group consisting of:

(a) the isolated polypeptide according to claim 3; and

(b) the isolated polynucleotide according to claim 4; and a transcription terminator.

6. A cultured cell comprising the expression vector of claim 5.

7. A method of producing a polypeptide comprising culturing the cell of claim 6 under conditions whereby said sequence of nucleotides is expressed, and recovering said polypeptide.

8. A polypeptide produced by the method of claim 7.

9. An antibody that specifically binds to a protein as shown in SEQ ID NO:2.

10. A polypeptide which contains an amino acid sequence of an epitope- bearing portion of SEQ ID NO:2, wherein said epitope-bearing portion is selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO.21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28 or wherein the polypeptide is at least 90%) identical to a polypeptide selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,

SEQ ID NO:27, and SEQ ID NO:28.