CA2355039A1 - Pancreatic polypeptide zsig66 - Google Patents

Abstract

The present invention relates to polynucleotide and polypeptide molecules for zsig66, a novel secreted protein. The polynucleotides encoding zsig66, may, for example, be used to identify a region of the genome associated with human disease states. The present invention also includes methods for producing the protein, uses therefor and antibodies thereto.

Description

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23-12-2000 .
~'~NT SHEBT
WO 00/36104 Ir'CT/IJS99/29669 PATENT AppL~CA~ON
>aoc»,r 98-8lpc es PZT27ITARY POLYPEPT1DE ZSxClb6 BACKGROUND OF THB IIWE.NTION
Proliferation and differentiation of cells of multiceiiular organisms ace controlled by hormones and polypeptide growth factors. These diffusible molecules allow cells to comraunicate with each other and act in concert to regulate cells and form organs, anti to repair and regenerate damaged tissue, Examples of hormones anti growth factors include the steroid hormones (c.g. estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, the interleu3das, platelet derived gmwth factor (F'DGF), epidermal growth factor (EG>~'j, granulocyte-macrophage colony stimulating factor (GM-CS1~, erythropoietin (EP4~ and calcitoain.
Hormones and growth factors influence cellular metabolism by binding to proteins. These proteins raay be integral membrane proteins that are linked to signaling pathways within the cell, such as second messenger systems, Other classes of proteins that hormones and growth factors influence sue soluble molecules, such as the 24 transcription factors.
Thus, there is a contir5uing need to discover new hormones, growth factors and the Like. The ire vivo activities of these cytokines illustrates the enormous clinical potential of, and need foz, other eytolciaes, cytokine agonists, ~d cytoltine antagonists.
The present invention provides such polypepddes for these and other uses that should be apparent to those skilled in the art frara the teaclsings herein.
SUMMAR.y pp ~ ~y~~ION
Within one aspect, the present invention provides an isolated polynucleotide epcoding a zsig66 polypeptide comprising a sequence of amino acid residues that is at least 90~1o identical to au amino acid sequence selected from the AMENDED SHEET

group consisting of: (a) the amino acid sequence as shown in SEQ ID N0:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID N0:2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID N0:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID N0:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID N0:2, wherein the amino acid percent identity is determined using a FASTA program with ktup=l, gap opening penalty=10, gap extension penalty=I, and substitution matrix=BLOSUM62, with other parameters set as default. Within one embodiment, the isolated polynucleotide disclosed above is selected from the group consisting of: (a) a polynucleotide sequence as shown in SEQ ID NO: l from nucleotide 431 to nucleotide 472; (b) a polynucleotide sequence as shown in SEQ ID NO:1 from nucleotide 425 to nucleotide 472; (c) a polynucleotide sequence as shown in SEQ ID NO:I from nucleotide 425 to nucleotide 475; (d) a polynucleotide sequence as shown in SEQ ID NO:I from nucleotide 305 to nucleotide 475; (e) a polynucleotide sequence as shown in SEQ ID NO:1 from nucleotide 224 to nucleotide 475; and (f) a polynucleotide sequence complementary to (a) or (b). Within another embodiment, the isolated polynucleotide disclosed above 2 0 comprises nucleotide I to nucleotide 252 of SEQ ID NO: l l . Within another embodiment, the isolated polynucleotide disclosed above comprises a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as showwin SEQ ID N0:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID N0:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ
ID N0:2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID N0:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID
N0:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ
ID

3 o N0:2.

'WO 00/36104 PCT/US99/29669 Within a second aspect, the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a zsig66 polypeptide comprising an amino acid sequence as shown in SEQ ID N0:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and a transcription terminator. Within one embodiment, the expression vector disclosed above further comprising a secretory signal sequence operably linked to the DNA segment.
Within a third aspect, the present invention provides a cultured cell into which has been introduced an expression vector as disclosed above, wherein the cell 1 o expresses a polypeptide encoded by the DNA segment.
Within a fourth aspect, the present invention provides a DNA construct encoding a fusion protein, the DNA construct comprising: a first DNA segment encoding a polypeptide comprising a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO: 2 from residue number 28 (Ala) to amino acid number 84 (Gly); and at least one other DNA
segment encoding an additional polypeptide, wherein the first and other DNA segments are connected in-frame; and encode the fusion protein.
Within another aspect, the present invention provides a fusion protein produced by a method comprising: culturing a host cell into which has been introduced 2 0 a vector comprising the following operably linked elements: (a) a transcriptional promoter; (b) a DNA construct encoding a fusion protein as disclosed above;
and (c) a transcriptional terminator; and recovering the protein encoded by the DNA
segment.
Within another aspect, the present invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 90%
identical 2 5 to an amino acid sequence selected from the group consisting of (a) the amino acid sequence as shown in SEQ ID N0:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID N0:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ TD N0:2 from amino acid number 69 (Phe) to amino acid number 84 3 0 (Gly); (d) the amino acid sequence as shown in SEQ ID N0:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID N0:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID N0:2, wherein the amino acid percent identity is determined using a FASTA
program with ktup=l, gap opening penalty=10, gap extension penalty=1, and substitution matrix=BLOSUM62, with other parameters set as default. Within one embodiment, the isolated polypeptide disclosed above further comprises motifs through 4 spaced apart from N-terminus to C-terminus in a co~guration AIa28-{7}-M1-{3}-M2-{6}-M3-{6}-M4-{10}- Gly~, wherein Ml is "motif 1," a sequence of amino acids as shown in amino acids 36 to 41 of SEQ ID N0:2, M2 is "motif 2,"
a sequence of amino acids as shown in amino acids 45 to 50 of SEQ ID N0:2, M3 is "motif 3," a sequence of amino acids as shown in amino acids 56 to 6I of SEQ
ID
N0:2, M4 is "motif 4," a sequence of amino acids as shown in amino acids 68 to 73 of SEQ ID N0:2, and Alazg is the Alanine residue at amino acid number 28 in SEQ
ID
N0:2, Gly~ is the Glycine residue at amino acid number 84 in SEQ ID N0:2, and {#}
denotes the number of amino acids between the motifs. Within another embodiment, the isolated polypeptide disclosed above comprises a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID
N0:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID N0:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID N0:2 from 2 0 amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID N0:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID N0:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID N0:2.
Within another embodiment, the isolated polypeptide disclosed above further comprises an 2 5 amidated C-terminal Serine residue.
Within another aspect, the present invention provides a method of producing a zsig66 polypeptide comprising: culturing a cell as disclosed above; and isolating the zsig66 polypeptide produced by the cell.
Within another aspect, the present invention provides a method of 3 0 detecting, in a test sample, the presence of a modulator of zsig66 protein activity, comprising: transfecting a zsig66-responsive cell, with a reporter gene construct that is responsive to a zsig66-stimulated cellular pathway; and producing a zsig66 polypeptide by the method as disclosed above; and adding the zsig66 polypeptide to the cell, in the presence and absence of a test sample; and comparing levels of response to the zsig66 polypeptide, in the presence and absence of the test sample, by a biological or 5 biochemical assay; and determining from the comparison, the presence of the modulator of zsig66 activity in the test sample.
Within another aspect, the present invention provides a method of producing an antibody to zsig66 polypeptide comprising the following steps in order:
inoculating an animal with a polypeptide selected from the group consisting of: (a) a 10 polypeptide consisting of 9 to 57 amino acids, wherein the polypeptide has a contiguous sequence of amino acids as shown within SEQ ID N0:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); (b) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 from residue number 28 (Ala) to amino acid number 84 (Gly); (c) a polypeptide as disclosed above; (d) a polypeptide consisting of 15 amino acid number 1 (Met) to amino acid number 6 (Glu) of SEQ ID N0:2; (e) a polypeptide consisting of amino acid number 7 (Val) to amino acid number 12 (Ile) of SEQ ID N0:2; (f) a polypeptide consisting of amino acid number 26 (Ser) to amino acid number 32 (Ser) of SEQ ID N0:2; (g) a polypeptide consisting of amino acid number 29 (Asp) to amino acid number 34 (Ser) of SEQ ID N0:2; and (h) a 2 0 polypeptide consisting of amino acid number 51 (Leu) to amino acid number 56 (Glu) of SEQ ID N0:2; and wherein the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal.
Within another aspect, the present invention provides an antibody produced by the method as disclosed above, which binds to a zsig66 polypeptide.
25 Within one embodiment, the antibody disclosed above is a monoclonal antibody.
Within another aspect, the present invention provides an antibody which specifically binds to a polypeptide as disclosed above.
Within another aspect, the present invention provides a method for detecting a genetic abnormality in a patient, comprising: obtaining a genetic sample 3 0 from a patient; producing a first reaction product by incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO:1 or the complement of SEQ ID NO:I, under conditions wherein said polynucleotide will hybridize to complementary polynucleotide sequence; visualizing the first reaction product; and comparing said first reaction product to a control reaction product from a wild type patient, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient.
Within another aspect, the present invention provides a pharmaceutical composition comprising an isolated polypeptide as disclosed above, wherein the polypeptide is in combination with a pharmaceutically acceptable vehicle.
These and other aspects of the invention will become evident upon l0 reference to the following detailed description of the invention and attached drawings.
BRIEF DESCRIPTION OF THE DRAWING
The figure is a hydrophobicity plot of zsig66, determined from a Hopp/Woods hydrophilicity profile based on a sliding six-residue window, with buried I5 G, S, and T residues and exposed H, Y, and W residues ignored.
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:
2 o The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purif cation or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-2 5 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, FIagTM peptide (Hopp et al., Biotechnolo~y 6:1204-10, 1988), streptavidin binding peptide, or other antigenic epitope or binding domain.
See, in 3 o general, Ford et al., Protein Expression and Purification 2: 95-107, 1991.
DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ).
The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation 5 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 tenor 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 1 o 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 1 ~~ polypeptide.
The term "complementlanti-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 complemendanti-complement pair. ether exemplary complement/anti-complement 2 0 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 < l p9 M- l .
The term "complements of a polynucleotide molecule" denotes a 2 5 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 3 0 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 "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 2 0 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 3 0 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" 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.
1 « "Paralogs" are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, a-globin, ~i-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 S' to the 3' end.
15 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 2 0 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.
25 A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. PoIypeptides 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 3 0 RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the S' 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 Iigand) and mediates the effect of the ligand on the cell.

10 Membrane-bound receptors are characterized by a mufti-peptide structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecules) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. In general, receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., 2 0 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 poIypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
The term "splice variant" is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of 3 0 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 t10%.
All references cited herein are incorporated by reference in their entirety.
1 ~~ The present invention is based in part upon the discovery of a novel DNA sequence that encodes a novel polypeptide. The polypeptide and corresponding polynucleotide were isolated from a human pituitary library. The polypeptide has been designated zsig66.
The novel zsig66 polypeptides of the present invention were initially identified by querying an EST database for proteins homologous to proteins having a secretory signal sequence. These proteins are characterized by an upstream methionine start site and a hydrophobic region of approximately 13 amino acids, followed by a peptide signal peptidase cleavage site. An EST database was queried for novel DNA
sequences whose translations would meet these search criteria. An EST was found and 2 0 its corresponding cDNA was sequenced. The zsig66 nucleotide sequence is believed to encode the entire coding sequence of the predicted protein. Zsig66 may be a novel pituitary hormone, pituitary hormone activating hormone, cell-cell signaling molecule, growth factor, cytokine, nerve cell modulating protein, secreted extracellular matrix associated protein with growth factor hormone activity, or the like, and is a member a 2 5 novel protein family.
The sequence of the zsig66 poIypeptide was obtained from a single clone believed to contain its corresponding polynucleotide sequence. The clone was obtained from a pituitary library. Other libraries that might also be searched for such sequences include fetal libraries, pancreas, liver, ovary, placenta, and the like.
3 0 The nucleotide sequence of a representative zsig66-encoding DNA is described in SEQ ID NO:I, and its deduced 84 amino acid sequence is described in SEQ ID N0:2. In its entirety, the zsig66 polypeptide (SEQ ID N0:2) represents a full-length polypeptide segment (residue 1 (Met) to residue 84 (Gly) of SEQ ID
N0:2). The domains and structural features of zsig66 are further described below.
Analysis of the zsig66 polypeptide encoded by the DNA sequence of 5 SEQ ID NO:1 revealed an open reading frame encoding 84 amino acids (SEQ ID
N0:2) comprising a predicted signal peptide of 27 amino acid residues (residue 1 (Met) to residue 27 (Ser) of SEQ ID N0:2}, and a mature polypeptide of 57 amino acids (residue 28 (Ala) to residue 84 (Gly) of SEQ ID N0:2). Further, post-translational processing may truncate zsig66, for example at a cysteine bond, and still provide a short 10 active polypeptide. As such, amino acids residues 69 (Phe) to 84 (Gly) of SEQ ID
N0:2, preferably amino acids residues 69 (Phe) to 83 (Ser) of SEQ ID N0:2, and more preferably amino acids residues 70 (Cys) to 83 (Ser) of SEQ ID N0:2, comprise an active zsig66 polypeptide. In another embodiment, the Serine residue of the short active polypeptide amino acid residues 69 (Phe) to 83 (Ser) of SEQ ID N0:2 or amino 15 acid residues 70 (Cys) to 83 (Ser) of SEQ ID N0:2 can further be amidated at the C-terminal Serine residue. The structure of zsig66 surrounding the C-C bond (see below), shows some similarity with vasopressin and oxytocin. As such, zsig66 can have cardiovascular or metabolic vasodilatation capability. For general references on vasopressin and oxytocin, see Rehbein, M. et al., Biol. Chem. 367:695-704, 1986;
20 Sausville, E. et al., J. Biol. Chem. 260:10236-10241, 1985; Ivell, R. et al., Endocrinol.
127:2990-2996, 1990; Mohr, E. et al., FEBS Lett. 193:12-16, 1985; Chauvet, M.-T. et al., Proc. Nat'l. Acad Sci USA 80:2839-2843, 1983; Schlesinger, D.H. and Audhya, T.I~., FEBS Lett. 128:325-328, 1981; and Light, A. and DuVigneaud, V., Proc.
Soc.
Exp. Biol. Med 98:692-696, 1958.
25 Moreover, the zsig66 polypeptide contains several regions of low variance or low degeneracy (see, Sheppard, P, et al., Gene 150:163-167, 1994).
These regions that exhibit low degeneracy are described in the following zsig66 motifs:
1) "motif 1" (corresponding to amino acids 36 (Trp) to 41 (Gly) of SEQ
ID N0:2);
3 a 2) "motif 2" (corresponding to amino acids 45 (Thr) to 50 (Phe) of SEQ
ID N0:2);

3) "motif 3" (corresponding to amino acids 56 (Glu) to 61 (Val) of SEQ
ID N0:2); and 4) "motif 4" (corresponding to amino acids 68 (Lys) to 73 (Asn) of SEQ
ID N0:2); and Motifs 1 through 4 are spaced apart from N-terminus to C-terminus in a configuration represented by the following:
AIa2g-{7}-M1-{3}-M2-{S}-M3-{6}-M4-{ 10}- Gly~, where Alaz$ is the starting residue of the mature polypeptide (as shown in SEQ ID N0:2), 1 « Glyg4 is the ending residue of the mature polypeptide(as shown in SEQ
ID N0:2), M# denotes the specific motif disclosed above (e.g., M1 is motif 1, etc.), and {#} denotes the approximate number of amino acid residues between the 1 ~ motifs, up to plus or minus 2 residues.
The presence of canserved and low variance motifs generally correlates with or defines important structural regions in proteins. Regions of low variance (e.g., hydrophobic clusters) are generally present in regions of structural importance (Sheppard, P. et al.,Gene 150:163-167, 1994). Such regions of low variance often 2 0 contain rare or infrequent amino acids, such as Tryptophan. The regions flanking and between such conserved and low variance motifs may be more variable, but are often functionally significant because they may relate to or define important structures and activities such as binding domains, biological and enzymatic activity, signal transduction, tissue localization domains and the like.
2 5 In addition, there are two Cysteine residues that form a disulfide bond located in SEQ ID N0:2 at amino acid number 70 and 74 that can be important for structure or activity of zsig66 polypeptide. Moreover, there are 2 consensus phosphorylation sites in zsig66 polypeptide: a protein kinase C (PKC) site at amino acid 53 (Ser) of SEQ ID N0:2; and a casein kinase II (CK2) site at amino acid 43 (Thr) 3 0 of SEQ ID N0:2.

Moreover the genomic structure of zsig66 is readily determined by one of skill in the art by comparing the cDNA sequence of SEQ ID NO:1 and the translated amino acid of SEQ ID N0:2 with the genomic DNA in which the gene is contained (Genbank Accession No. AC015783). For example, such analysis can be readily done 5 using FASTA as described herein. As such, the intron and exon junctions in this region of genomic DNA can be determined for the zsig66 gene. The region of human genomic DNA where zsig66 is located is shown in SEQ ID N0:24. The entire coding sequence appears to be contained in a single exon, as there are no putative introns within SEQ ID
N0:24. Thus, the present invention includes the zsig66 gene as located in human genomic DNA.
'The corresponding polynucleotides encoding the zsig66 polypeptide regions, domains, motifs, residues and sequences described above are as shown in SEQ
ID NO:1.
'the low degeneracy amino acids in zsig66 can be used as a tool to identify new family members. For instance, reverse transcription-polymerase chain reaction (RT-PCR) can be used to amplify sequences, encoding the low degeneracy motifs, from RNA obtained from a variety of tissue sources or cell lines. In particular, highly degenerate primers designed from the zsig66 sequences are useful for this purpose. In particular, degenerate oligonucleotide primers designed from the following 2 0 zsig66 amino acid sequences of motifs 1 through 5 are useful for this purpose:
a) WIQGMG (motif 1; SEQ ID N0:3), corresponding to degenerate polynucleotides of SEQ ID N0:7 and their complement;
b) TDFNPF (motif 2; SEQ ID N0:4), corresponding to degenerate polynucleotides of SEQ ID N0:8 and their complement;
c) KFCVVN (motif 3; SEQ ID NO:S), corresponding to degenerate polynucleotides of SEQ ID N0:9 and their complement;
d) EIELFV (motif 4; SEQ ID N0:6), corresponding to degenerate polynucleotides of SEQ ID NO:10 and their complement.
The present invention also provides polynucleotide molecules, including 3 0 DNA and RNA molecules, that encode the zsig66 polypeptides disclosed herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. SEQ ID NO:11 is a degenerate DNA sequence that encompasses all DNAs that encode the zsig66 polypeptide of SEQ ID N0:2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO:11 also provides all RNA
5 sequences encoding SEQ ID N0:2 by substituting U for T. Thus, zsig66 polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 252 of SEQ ID
NO:11 and their RNA equivalents are contemplated by the present invention. Table 1 sets forth the one-letter codes used within SEQ ID NO:11 to denote degenerate nucleotide positions. "Resolutions" are the nucleotides denoted by a code letter.
"Complement"
10 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.

Nucleotide Nucleotide Base Code ResolutionBase Code Complement A A T T

C ~= G G

G G C C

T T A A

R AIG Y CIT

Y CIT R AIG

M AIC K GIT

K GIT M AIC

S CIG S CIG

W AIT W AIT

H AICIT D AIGIT

B CIGIT V AICIG

V AICIG B CIGIT

D AIGIT H AICIT

N AICIGIT N AICIGIT

The degenerate codons used in SEQ ID NO:11, encompassing all possible codons for a given amino acid, are set forth in Table 2.

Three One Letter Letter Degenerate Code Code Synonymous Codons 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

AIa A GCA GCC GCG GCT GCN

Gly G GGA GGC GGG GGT GGN

Asn N AAC AAT ~y Asp D GAC GAT GAY

Glu E GAA GAG GAR

Gln Q CAA CAG
CAR

His H CAC CAT CAY

Arg R AGA AGG CGA CGC CGG CGT MGN

Lys K AAA AAG AAR

Met M ATG ATG

Ile I ATA ATC ATT ATH

Leu L CTA CTC CTG CTT TTA TTG YTN

Val V GTA GTC GTG GTT G'~

Phe F TTC TT'I' TTY

Tyr Y TAC TAT TAY

Trp W TGG TGG

Ter . TAA TAG TGA T~

Asn~Asp B ~y GIu~GIn Z SAR

Any X

W'O 00/36104 PCT/US99/29669 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 N0:2. 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." In general, see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc.
Acids Res. 14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As used herein, the term "preferential codon usage" or "preferential codons" is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2). For example, the amino acid Threonine (Thr) may be encoded by 2 0 ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential. Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA
2 5 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:11 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 3 0 for expression in various species, and tested for functionality as disclosed herein.

Within preferred embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:1, or a sequence complementary thereto, under stringent conditions. In general, stringent conditions are selected to be about S°C lower than the thermal melting point (Tm) for the specific sequence at a defned ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Suitable stringent hybridization conditions are equivalent to about a 5 h to overnight incubation at about 42°C in a solution comprising: about 40-SO%
formamide, up to about SX SSC, about SX Denhardt's solution, up to about 10%
dextran sulfate, and about 10-20 pg/ml denatured commercially-available carrier DNA;
hybridization is then followed by washing filters in up to about 2X SSC. For example, a suitable wash stringency is equivalent to O.1X SSC to 2X SSC, 0.1% SDS, at 55°C to 65°C. Stringent hybridization and wash conditions depend on the length of the probe, reflected in the Tm, hybridization and wash solutions used, and are routinely determined empirically by one of skill in the art.
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 zsig66 RNA. Such tissues and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci USA 77:5201, 1980), and include pituitary, pancreas, tissues of endocrine origin, and the like. Total RNA can be prepared using guanidinium isothiocyanate extraction followed by isolation by centrifugation in a CsCI
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 zsig66 polypeptides are then identified and isolated by, for example, hybridization or PCR.
A full-length clone encoding zsig66 can be obtained by conventional 3 0 cloning procedures. Complementary DNA (cDNA) clones are preferred, although for ~JVO 00/36104 PCT/US99/29669 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 5 thereof, for probing or priming a library. Expression libraries can be probed with antibodies to zsig66, receptor fragments, or other specific binding partners.
The polynucleotides of the present invention can also be synthesized using DNA synthesis machines. Currently the method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application 10 such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short polynucleotides (60 to 80 bp) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. However, for producing longer polynucleotides (longer than about 300 bp), special strategies are usually employed, 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.
One method for building a synthetic gene requires the initial production of a set of overlapping, complementary oligonucleotides, each of which is between 20 2 0 to 60 nucleotides long. Each internal section of the gene has complementary 3' and 5' terminal extensions designed to base pair precisely with an adjacent section.
Thus, after the gene is assembled, process is completed by sealing the nicks along the backbones of the two strands with T4 DNA ligase. In addition to the protein coding sequence, synthetic genes can be designed with terminal sequences that facilitate insertion into a 2 5 restriction endonuclease site of a cloning vector. An alternative way to prepare a full-length gene is to synthesize a specified set of overlapping oligonucleotides (40 to 100 nucleotides). After the 3' and 5' short overlapping complementary regions are annealed, large gaps still remain, but the short base-paired regions are both long enough and stable enough to hold the structure together. The gaps are filled and the DNA
duplex is 3 0 completed via enzymatic DNA synthesis by E. toll DNA polymerase I. After the enzymatic synthesis is completed, the nicks are sealed. Double-stranded constructs are 'WO 00/36104 PCT/US99/29669 sequentially linked to one another to form the entire gene sequence which is verified by DNA sequence analysis. See Glick and Pasternak, Molecular Biotechnoloav 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.
Zsig66 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a zsig66 gene. In view of the tissue-specific expression observed for zsig66 by Northern blotting, this gene region is expected to provide for ovary and pancreas-specific expression. Promoter elements 1 o from a zsig66 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 S' flanking sequences also facilitates production of zsig66 proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous zsig66 gene in a cell is altered by introducing into the zsig66 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 zsig66 5' non-coding sequence that permits homologous recombination of the construct with the endogenous zsig66 locus, whereby the sequences within the construct become operably linked with the endogenous zsig66 coding sequence. In this way, an endogenous 2 0 zsig66 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.
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 zsig66 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human zsig66 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
3 0 obtained from a tissue or cell type that expresses zsig66 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 zsig66-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 (PCR) (Mullis, U.S. Patent No.
4,683,202), using primers designed from the representative human zsig66 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 zsig66 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:I represents a single allele of human zsig66 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:1, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID N0:2. cDNAs generated from alternatively spliced mRIVAs, which retain the properties of the zsig66 polypeptide are 2 0 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 zsig66 polypeptides that are 2 5 substantially similar to the polypeptides of SEQ ID N0:2 and their orthologs. The term "substantially similar" is used herein to denote polypeptides having 70%, preferably 75%, more preferably at least 80%, sequence identity to the sequences shown in SEQ
ID N0:2 or their orthologs. Such polypeptides will more preferably be at least 90%
identical, and most preferably 95% or more identical to SEQ ID N0:2 or its orthologs.) 3 o 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.

w0 00/36104 PCTNS99/29669 Natl. Acad. Sci. USA $9:10915-9, 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 100 [length of the longer sequence plus the number of gaps introduced into the longer 1 o sequence in order to align the two sequences]

'WO 00/36104 PC'T/US99/29669 ~ N M

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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 5 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 zsig66. The FASTA algorithm is described by Pearson and Lipman, Proc. Nat'i Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enz~mol. 183:63 (1990).
10 Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID N0:2) 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 15 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 2 o 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 (NeedIeman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM J.
Appl. Math 26:787 ( I 974)), which allows for amino acid insertions and deletions.
25 Preferred parameters for FASTA analysis are: ktup=1, gap opening penalty=10, gap extension penalty=I, 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 3 0 acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.
'The present invention includes nucleic acid molecules that encode a polypeptide having one or more conservative amino acid changes, compared with the amino acid sequence of SEQ ID N0:2. The BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat'1 Acad. Sci. USA 89:10915 (1992)).
Accordingly, the BLOSUM62 substitution frequencies can be used to define l0 conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. As used herein, the language "conservative amino acid substitution" refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at Ieast 2 (e.g>., 2 or 3).
Variant zsig66 polypeptides or substantially homologous zsig66 polypeptides are characterized as having one or more amino acid substitutions, 2 0 deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 2 5 residues, or an affinity tag. The present invention thus includes polypeptides of from about 35 to about 90 amino acid residues, or in some embodiments, IS to 16 amino acid residues (e.g., residues 69-83 of SEQ ID N0:2 with or without an amide group), that comprise a sequence that is at least 80%, preferably at least 90%, and more preferably 95% or more identical to the corresponding region of SEQ ID N0:2. Polypeptides 3 0 comprising affinity tags can further comprise a proteolytic cleavage site between the 'WO 00/36104 PCT/US99/29669 zsig66 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 ~iydrophobic: leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine 2 5 The present invention further provides a variety of other polypeptide fusions and related multimeric proteins comprising one or more polypeptide fusions.
For example, a zsig66 polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in this regard include immunoglobulin constant region domains.
3 0 Immunogiobulin-zsig66 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric zsig66 analogs. Auxiliary domains can be fused to zsig66 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen). For example, a zsig66 polypeptide or protein could be targeted to a predetermined cell type by fusing a zsig66 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell. In this way, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. A zsig66 polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain. Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34:1-9, 1996.
The proteins of the present invention can also comprise non-naturally occurring amino acid residues. Non-naturally occurnng amino acids include, without limitation, traps-3-methylproIine, 2,4-methanoproline, cis-4-hydroxyproline, traps-4-hydroxyproline, N methylglycine, alto-threonine, methylthreonine, hydroxyethyIcysteine, 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 tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of 2 0 piasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents.
Proteins are purified by chromatography. See, for example, Robertson et al., J. Am.
Chem. Soc.
113:2722, 1991; Ellman et al., Methods Enzymol 202:301, 1991; Chung et al., Science 259:806-9, 1993.; and Chung et al., Proc. Natl. Acad. Sci. USA 90:IOI45-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J.
Biol. Chem. 271:19991-8, 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 acids) (e.g., 2-azaphenylalanine, 3 0 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart.

See, Koide et al., Biochem. 33:7470-6, 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 zsig66 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 10 or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989;
Bass et al., Proc. Natl. Acad Sci USA 88:4498-502, I 99I ). 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, 15. Hilton et al., J. Biol. Chem. 271:4699-708, 1996. Sites of ligand-receptor or other biological interaction 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-I2, 1992;
Srnith et 20 al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et aL, FEBS Left. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related polypeptides.
Determination of amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined.
Within these 25 regions one can determine specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule. Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity and computer analysis using available software (e.g., the Insight II~ viewer and homology modeling tools; MSI, San Diego, CA), 3 0 secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, Current Upin Struct Biol 5:372-376, 1995 and Cordes et ~.~ Current Opin Struct Biol 6:3-10, 1996). In general, when designing modifications to molecules or identifying specific fragments determination of structure will be accompanied by evaluating activity of modified molecules.
Amino acid sequence changes are made in zsig66 polypeptides so as to minimize disruption of higher order structwe essential to biological activity.
For example, when the zsig66 polypeptide comprises one or more helices, changes in amino acid residues will be made so as not to disrupt the helix geometry and other components of the molecule where changes in conformation abate some critical function, for example, binding of the molecule to its binding partners. The effects of amino acid 1 o sequence changes can be predicted by, for example, computer modeling as disclosed above or determined by analysis of crystal structure (see, e.g., Lapthorn et al., Nat.
Struct. Biol. 2:266-268, 1995). Other techniques that are well known in the art compare folding of a variant protein to a standard molecule (e.g., the native protein).
For example, comparison of the cysteine pattern in a variant and standard molecules 15 can be made. Mass spectrometry and chemical modification using reduction and alkyIation provide methods for determining cysteine residues which are associated with disulfide bonds or are free of such associations (Bean et al., Anal. Biochem.
201:216-226, 1992; Gray, Prote~ 2;1732-1748, 1993; and Patterson et al., Anal. Chem.
66:3727-3732, 1994). It is generally believed that if a modified molecule does not have 2 o the same disulfide bonding pattern as the standard molecule folding would be affected.
Another well known and accepted method for measuring folding is circular dichrosism (CD). Measuring and comparing the CD spectra generated by a modified molecule and standard molecule is routine (Johnson, Proteins 7:205-214, 1990).
Crystallography is another well known method for analyzing folding and structure. Nuclear magnetic 2 5 resonance (NMR), digestive peptide mapping and epitope mapping are also known methods for analyzing folding and structural similarities between proteins and polypeptides (Schaanan et al., Science 257:961-964, 1992).
A Hopp/Woods hydrophilicity profile of the zsig66 protein sequence as shown in SEQ ID N0:2 can be generated (Hope et al., Proc. Natl. Acad.
Sci.78:3824 30 3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquier et al., Protein Enei-'neerin~ I 1:153-169, 1998). The profile is based on a sliding six-residue window.

Buried G, S, and T residues and exposed H, Y, and W residues were ignored (See, Figure). For example, in zsig66, hydrophilic regions include: (1) amino acid number 1 (Met) to amino acid number 6 (Glu) of SEQ ID N0:2; (2) amino acid number 7 (Val) to amino acid number 12 (Ile) of SEQ ID N0:2; (3) amino acid number 26 (Ser) to amino acid number 32 (Ser) of SEQ ID N0:2; (4) amino acid number 29 (Asp) to amino acid number 34 (Ser) of SEQ ID N0:2; and (5) amino acid number 51 (Leu) to amino acid number 56 (Glu) of SEQ ID N0:2.
Those skilled in the art will recognize that hydrophilicity or hydrophobicity will be taken into account when designing modifications in the amino 1 C~ acid sequence of a zsig66 polypeptide, so as not to disrupt the overall structural and biological profile. Of particular interest for replacement are hydrophobic residues selected from the group consisting of Val, Leu and Iie or the group consisting of Met, Gly, Ser, Ala, Tyr and T'rp. For example, hydrophobic residues tolerant of substitution could include such residues as shown in SEQ ID NO: 2. Cysteine residues are I5 relatively intolerant of substitution.
'The identities of essential amino acids can also be inferred from analysis of sequence similarity between other proteins with zsig66. Using methods such as "FASTA" analysis described previously, regions of high similarity are identified within a family of proteins and used to analyze amino acid sequence for conserved regions. An 2 o alternative approach to identifying a variant zsig66 polynucleotide on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant zsig66 polynucleotide can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1, as discussed above.
Other methads of identifying essential amino acids in the polypeptides 25 of the present invention are procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081 ( 1989), Bass et- al., Proc. Natl Acad. Sci. USA 88:4498 ( 1991 ), Coombs and Corey, "Site-Directed Mutagenesis and Protein Engineering," in Proteins: Anal sis and Design, Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In the latter 3 0 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 (1996).
The present invention also includes functional fragments of zsig66 polypeptides and nucleic acid molecules encoding such functional fragments. A
"functional" zsig66 or fragment thereof defined herein is characterized by its proliferative or differentiating activity, by its ability to induce or inhibit specialized cell functions, or by its ability to bind specifically to an anti-zsig66 antibody or zsig66 receptor (either soluble or immobilized). The present invention further provides fusion proteins encompassing: (a) polypeptide molecules comprising one or more of the motifs described above; and (b) functional fragments comprising one or more of these motifs.
The other polypeptide portion of the fusion protein may be contributed by a related, non-native and/or an unrelated secretory signal peptide that facilitates secretion of the fusion protein.
Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a zsig66 polypeptide. As an illustration, DNA molecules having the nucleotide sequence of SEQ ID NO:1 or fragments thereof, can be digested with Ba131 nuclease to obtain a series of nested deletions. These DNA fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested 2 0 for zsig66 activity, or for the ability to bind anti-zsig66 antibodies or zsig66 receptor.
One alternative to exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired zsig66 fragment. Alternatively, particular fragments of a zsig66 polynucleotide can be synthesized using the polymerase chain reaction.
Standard methods for identifying functional domains are well-known to those of skill in the art. For example, studies on the truncation at either or both termini of interferons have been summarized by Horisberger and Di Marco, Pharmac.
Ther.
66:507 (1995). Moreover, standard techniques for functional analysis of proteins are described by, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993);
Content 3 0 et al., "Expression and preliminary deletion analysis of the 42 kDa 2-SA
synthetase induced by human interferon," in Bioloeical Interferon Systems Proceedin~ES of ISIR-TNO Meetine on Interferon Systems, Cantell (ed.), pages 65-72 (Nijhoff 1987);
Herschman, "The EGF Receptor," in Control of Animal Cell Proliferation 1 Boynton et al., (eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J.
Biol. Chem.
270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995); and Meisel et- al., Plant Molec. Biol.
_30:1 ( 1996).
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 rnutagenized 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 zsig66 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 2 o 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 2 5 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 3 0 polypeptides (e.g., secreted and detected by antibodies; or measured by a signal transduction type assay) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid deten~nination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
In addition, the proteins of the present invention (or polypeptide 5 fragments thereof] can be joined to other bioactive malecules, particularly other cytokines, to provide mufti-functional molecules. For example, one or more domains, hydrophilic regions, or regions containing motifs 1-4 from zsig66 can be joined to other proteins to enhance their biological properties or efficiency of production.
The present invention thus provides a series of novel, hybrid molecules 10 in which a segment comprising one or more of the domains, hydrophilic regions, or regions containing motifs 1-4 of zsig66 is fused to another polypeptide.
Fusion is preferably done by splicing at the DNA level to allow expression of chimeric molecules in recombinant production systems. The resultant molecules are then assayed for such properties as improved solubility, improved stability, prolonged clearance half life, 15 improved expression and secretion levels, and pharmacodynamics. Such hybrid molecules may further comprise additional amino acid residues (e.g. a polypeptide linker) between the component proteins or polypeptides.
Using the methods discussed herein, one of ordinary skill in the art can identify and/or prepare a variety of polypeptides that are substantially similar to SEQ
2 o ID N0:2 or allelic variants thereof and retain the properties of the wild-type protein.
For example, using the methods described above, one could identify a receptor binding domain on zsig66; an extracellular ligand-binding domain of a receptor for zsig66;
heterodimeric and homodimeric binding domains; other functional or structural domains; affinity tags; or other domains important for protein-protein interactions or 25 signal transduction. Such polypeptides may also include additional polypeptide segments as generally disclosed above.
For any zsig66 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 zsig66 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 Laboratorv Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., eds., Current Protocols in Molecular BioloQV, John Wiley and Sons, Inc., NY, 1987.
In general, a DNA sequence encoding a zsig66 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 zsig66 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 zsig66, 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 zsig66 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 3 0 encoding the palypeptide 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).
Alternatively, the secretory signal sequence contained in 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 residue 1 (Met) to residue 27 (Ser) of SEQ ID N0:2 is 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 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, Viroloey_ 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-S, 1982), DEAE-2 0 dextran mediated transfection (Ausubel et al., ibid.), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90, 19$9; 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,$21; 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 S70 (ATCC No. CRL 10314), 293 (ATCC No. CRL
1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g.
CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, VA. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No.
4,956,288. Other suitable promoters include those from metallothionein genes (U.S.
Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured, for example 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, mufti-drug resistance, puromycin acetyltransferase) can also be used. In addition to selection of transfectants by agents, alternative markers that introduce an altered phenotype, such as 2 0 green fluorescent protein, or cell surface proteins such as CD4, CDB, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci.
(Bangalore) 11:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis 3 0 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., WO 00/36104 PCT/US99l29669 Baculovirus Expression Vectors' A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, C. D., Ed., Baculovirus Expression Protocols Methods in Molecular Biolo~v, Totowa, NJ, Humana Press, 1995. The second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, V.A, et al., J Virol 67:4566-79, 1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBac 1 T"" (Life Technologies) containing a Tn7 transposon to move the DNA
encoding the zsig66 polypeptide into a baculovirus genome maintained in E.
coli as a large plasmid called a "bacmid." 'fhe pFastBac 1 T"" transfer vector utilizes the AcNPV
polyhedrin promoter to drive the expression of the gene of interest, in this case zsig66.
However, pFastBac 1 T"" 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;
Bopping, 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 zsig66 secretory signal sequences 2 0 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 zsig66 secretory signal sequence. In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at 2 5 the C- or N-terminus of the expressed zsig66 polypeptide, for example, a GIu-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 zsig66 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 3 o genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus that expresses zsig66 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 5 Pasternak, Molecular Biotec-; principles and Annlications o f Recombinant DNA, ASM Press, Washington, D.C., 1994. Another suitable cell line is the High FiveOT"" cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No.
5,300,435). Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 IIT"" (Life Technologies) or ESF 921 T""
(Expression 10 Systems) for the Sf9 cells; and Ex-ce11O405T"" (JRH Biosciences, Lenexa, KS) or Express FiveOT"" (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 15 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 zsig66 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 20 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.
25 Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POTI vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
3 0 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., IO 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 P.
methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. For polypeptide production in P.
methanolica, it is preferred that the promoter and terminator in the plasmid be that of a P.
methanolica gene, such as a P. methanolica alcohol utilization gene (AUGI or AUG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate 2 0 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 P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21 ), which allows 2 5 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 UGI and A UG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRBl ) are preferred. Electroporation is used to facilitate the introduction of a plasmid 30 containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. 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 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 zsig66 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 2 0 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 2 5 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. P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a 30 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 P. methanolica is YEPD (2% D-glucose, 2% BaetoTM Peptone (Difco Laboratories, Detroit, MI), 1 % BactoTM 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 zsig66 polypeptides (or chimeric zsig66 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, polyacryIamide, specialty siIicas, and the like. PEI, DEAF, QAE
and Q derivatives are preferred. Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF
2 0 (Pharmacia), Toyopearl butyl 650 (Toro Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or poIyacrylic 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 sunnorts r"a~ hP mn~ifP,~ ,.,:.~, ..o,.".:..,. ~______ ~,__.
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 3 o 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 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 Chromatoeranhw Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and .'~ Scopes, R. (ed.) Protein Purification: Principles and practice, Springer-Verlag, New York, 1987.
The polypeptides of the present invention can be isolated by exploitation of their biological and structural properties. 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 E mol., 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 2 0 constructed to facilitate purification.
Moreover, using methods described in the art, polypeptide fusions, or hybrid zsig66 proteins, are constructed using regions or domains of zsig66 in combination with those of paralogs, orthologs, or heterologous proteins (Sambrook et al., ibid., AltschuI et al., ibid., Picard. D., Cur. Opin. Bioioav 5:511-515, 1994, and 2 5 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.
3 0 Fusion polypeptides 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 one or more 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 domains) conferring a biological function may be swapped between zsig66 of the 5 present invention with the functionally equivalent domains) from another protein.
Such domains include, but are not limited to, the secretory signal sequence, and motifs 1 through 4. 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 similar proteins (e.g. paralogs, or orthologs) or heterologous proteins, depending on the l0 fusion constructed. Moreover, such fusion proteins rnay exhibit other properties as disclosed herein.
Standard molecular biological and cloning techniques can be used to swap the equivalent domains between the zsig66 polypeptide and those polypeptides to which they are fused. Generally, a DNA segment that encodes a domain of interest, 15 e.g., a zsig66 domain described herein, is operably linked in frame to at least one other DNA segment encoding an additional polypeptide (for instance an analogous domain or region in a similar protein), and inserted into an appropriate expression vector, as described herein. Generally DNA constructs are made such that the several DNA
segments that encode the corresponding regions of a polypeptide are operably linked in 2 0 frame to make a single construct that encodes the entire fusion protein, or a functional portion thereof. For example, a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by a mature polypeptide; or a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by a mature protein. Such fusion 25 proteins can be expressed, isolated, and assayed for activity as described herein.
zsig66 polypeptides or fragments thereof may also be prepared through chemical synthesis. zsig66 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.
3 0 Polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. Methods for synthesizing polypeptides are well known in the art. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Kaiser et al., Anal. Biochem. 34:595, 1970. After the entire synthesis of the desired peptide on a solid support, the peptide-resin is with a reagent which cleaves the polypeptide from the resin and removes most of the side-chain protecting groups. Such methods are well established in the art.
The activity of molecules of the present invention can be measured using a variety of assays that measure for example, signal transduction, cell motility, steroidogenesis, mitogenesis or binding. Such assays are well known in the art.
The zsig66 polypeptides of the present invention can be used to study pancreatic cell proliferation or differentiation. Such methods of the present invention generally comprise incubating a cells, (3 cells, 8 cells, F cells and acinar cells in the presence and absence of zsig66 polypeptide, monoclonal antibody, agonist or antagonist thereof and observing changes in islet cell proliferation or differentiation.
Similarly, the zsig66 polypeptides of the present invention can be used to study pituitary cell proliferation or differentiation.
A further aspect of the invention provides a method for studying insulin.
Such methods of the present invention comprise incubating adipocytes in a culture medium comprising zsig66 polypeptide, monoclonal antibody, agonist or antagonist 2 0 thereof ~ insulin and observing changes in adipocyte protein secretion or differentiation.
The present invention also provides methods of studying mammalian cellular metabolism. Such methods of the present invention comprise incubating cells to be studied, for example, an appropriate human cell line, ~ zsig66 polypeptide, 2 5 monoclonal antibody, agonist or antagonist thereof, and observing changes in adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, or the like.
Also, zsig66 polypeptides, agonists or antagonists thereof may be therapeutically useful for promoting wound healing, for example, in the pancreas. To verify the presence of this capability in zsig66 polypeptides, agonists or antagonists of 3 0 the present invention, such zsig66 polypeptides, agonists or antagonists are evaluated with respect to their ability to facilitate wound healing according to procedures known in the art. If desired, zsig66 polypeptide performance in this regard can be compared to growth factors, such as EGF, NGF, TGF-a, TGF-(3, insulin, IGF-I, IGF-II, fibroblast growth factor (FGF) and the like. In addition, zsig66 polypeptides or agonists or antagonists thereof may be evaluated in combination with one or more growth factors to identify synergistic effects.
In addition, zsig66 polypeptides, agonists or antagonists thereof may be therapeutically useful for anti-microbial applications. To verify the presence of this capability in zsig66 polypeptides, agonists or antagonists of the present invention, such zsig66 polypeptides, agonists or antagonists are evaluated with respect to their l0 antimicrobial properties according to procedures known in the art. See, for example, Barsum et al., Eur. Resnir J 8~5~: 709-14, 1995; Sandovsky-Losica et al., J.
Med. Vet.
Mycol fEn_ghand) 28~4~: 279-87, 1990; Mehentee et al., J. Gen. Microbiol (Englandl 135 (Pt. 8): 2181-8, 1989; Segal and Savage, Journal of Medical and Veterina_tv Mycolo~w 24: 477-479, 1986 and the like. If desired, zsig66 polypeptide performance in this regard can be compared to proteins known to be functional in this regard, such as proline-rich proteins, lysozyme, histatins, lactoperoxidase or the like. In addition, zsig66 polypeptides or agonists or antagonists thereof may be evaluated in combination with one or more antimicrobial agents to identify synergistic effects.
Anti-microbial protective agents may be directly acting or indirectly 2 0 acting. Such agents operating via membrane association or pore forming mechanisms of action directly attach to the offending microbe. Anti-microbial agents can also act via an enzymatic mechanism, breaking down microbial protective substances or the cell wall/membrane thereof. Anti-microbial agents, capable of inhibiting microorganism proliferation or action or of disrupting microorganism integrity by either mechanism set 2 5 forth above, are useful in methods for preventing contamination in cell culture by microbes susceptible to that anti-microbial activity. Such techniques involve culturing cells in the presence of an effective amount of said zsig66 polypeptide, or an agonist or antagonist thereof.
Also, zsig66 polypeptides or agonists thereof may be used as cell culture 3 0 reagents in in vitro studies of exogenous microorganism infection, such as bacterial, viral or fungal infection. Such moieties may also be used in in vivo animal models of infection. Also, the microorganism-adherence properties of zsig66 polypeptides or agonists thereof can be studied under a variety of conditions in binding assays and the like.
Proteins of the present invention are useful for example, in treating ovarian, pancreatic, ocular, blood or bone disorders, can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to the appropriate animal model. For instance, host cells expressing a secreted form of zsig66 polypeptide may be embedded in an alginate environment and injected (implanted) into recipient animals. Alginate-poly-L-lysine microencapsulation, permselective 1 n membrane encapsulation and diffusion chambers are a means to entrap transfected mammalian cells or primary mammalian cells to permit the diffusion of proteins and other macromolecules secreted or released by the captured cells to the recipient animal.
Most importantly, the capsules mask and shield the foreign, embedded cells from the recipient animal's immune response. Such encapsulations can extend the life of the 1 ~> injected cells fiom a few hours or days (naked cells) to several weeks (embedded cells).
Alginate threads provide a simple and quick means for generating embedded cells and testing, in vivo, the proteins secreted therefrom.
The materials needed to generate the alginate threads are known in the art. In an exemplary procedure, 3% alginate is prepared in sterile H20, and sterile 20 filtered. Just prior to preparation of alginate threads, the alginate solution is again filtered. An approximately 50% cell suspension (containing about 5 x I05 to about 5 x 10~ cells/ml) is mixed with the 3% alginate solution. One ml of the alginate/cell suspension is extruded into a 100 mM sterile filtered CaCl2 solution over a time period of ~IS min, forming a "thread". The extruded thread is then transferred into a solution 25 of 50 mM CaCl2, and then into a solution of 25 mM CaCl2. The thread is then rinsed with deionized water before coating the thread by incubating in a 0.01 %
solution of poly-L-lysine. Finally, the thread is rinsed with Lactated Ringer's Solution and drawn from solution into a syringe barrel (without needle). A large bore needle is then attached to the syringe, and the thread is intraperitonealIy injected into a recipient in a 3 0 minimal volume of the Lactated Ringer's Solution.

An in vivo approach for assaying proteins of the present invention involves viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV).
Adenovirus, a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for 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: (i) adenovirus can accommodate relatively large DNA inserts; (ii) can be grown to high titer; (iii) infect a broad range of mammalian cell types; and (iv) can be used with many 1 t) different promoters including ubiquitous, tissue specific, and regulatable promoters.
Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
Using adenovirus vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous 1 ~~ recombination with a ca-transfected plasmid. In an exemplary system, the essential E 1 gene has been deleted from the viral vector, and the virus will not replicate unless the E1 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 EI gene deletion. the virus cannot replicate in the 2 0 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.
Moreover, adenoviral vectors containing various deletions of viral genes 2 5 can be used in an attempt to reduce or eliminate immune responses to the vector. Such adenoviruses are EI deleted, and in addition contain deletions of E2A or E4 (Lusky, M.
et ai., J. Virol. 72:2022-2032, 1998; Raper, S.E. et al., Human Gene Therany 9:671 679, 1998). In addition, deletion of E2b is reported to reduce immune responses (Amalfitano, A. et ai., J. Virol. 72:926-933, 1998). Moreover, by deleting the entire 3 0 adenovirus genome, very large inserts of heterologous DNA can be accommodated.
Generation of so called "gutless" adenoviruses where all viral genes are deleted are r ,m~~~e..~..:. ..~ -" "CA 02355039 2001-06-13 ~ US 009929669 23-12-:2000 R.fiI'r.AC»NT SHEET
WO pQl35104 ~'CT/US99129b69 particularly advantageous for insortioa of large inscns of hetemlagous DNA.
For review, see Yeh, P. and Pcaricaudct, M., FJ, ,x,:61 S-623,199?.
The adenovirus system can also be used for proteici production in vitro.
By culturing adonovints-infected non-293 cells under conditions where the cells ere not rapidly dividing, the cells can produce proteins fot extended periods of ti=ne, For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenovirai vector encoding the scented protein of interest. The cells an then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division. Alternatively, adenovirus vector infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protean (See Gamier et al., C~totechnol, 15:145-SS, 1994). With either protocol, art e~tpressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, ar membrane fractions depending oa the disposition of the expressed protein in the cell. Within the infected 293 cell production protocol, non-secreted proteins may also be effectively obtained.
In view of the tissue distribution observed for zsig66, agonists (i;ncluding jhe natural ligand! substrate/ cofactor/ etc,) and antagonists have enormous potential in both in. vitro and in vivo applications. Compounds identified as zsigfib agonists are useful fox stimulating cell growth or signal transduction in vitro and in 24 .viva, For exa~tnplo, zsig66 and agonist compounds are useful as components of deFmed cell culture media, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in ceh culhu~e. Agonists are thus useful in specifically protuoting the growth and/or development of cells in culture.
Considering the expression of zsig6b in pituitary, zsigb6 polypeptides and zsig66 agonists may be particularly useful as research reagents, particularly for the growth of piwitary and endocrine cell types, avarian cell lines, human eggs, cells front animal embryos or primary cultures derived from these tissues. As such, xsig66 polypeptide can be provided as a supplement in cell culture medimn.
Antagonists are also useful as research reagents for charaatecizing sites 34 of ligarld-receptor interaction. Inhibitors of zsig66 activity (zsig66 antagonists) include anti-zsig66 antibodies and soluble proteins which bind asigb6 polypeptide.
Inhibitors AMENDED SHEET

of zsig66 activity (zsig66 antagonists) include anti-zsig66 antibodies and soluble zsig66 receptors, as well as other peptidic and non-peptidic agents (including ribozymes).
Zsig66 polypeptide can be used to identify inhibitors (antagonists) of its activity. Test compounds are added to the biological or biochemical assays disclosed herein to identify compounds that inhibit the activity of zsig66. In addition to those assays disclosed herein, samples can be tested for inhibition of zsig66 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of zsig66-dependent cellular responses. For example, zsig66-responsive cell lines can be transfected with a reporter gene construct that is responsive to a zsig66-stimulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a zsig66-DNA response element operably linked to a gene encoding an assay detectable 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:

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 that serve as test samples including solutions, mixtures or extracts, are 2 0 tested for the level of response to the zsig66 polypeptide. The ability of a test sample to inhibit the activity of zsig66 polypeptide on the target cells as evidenced by a decrease in zsig66 stimulation of reporter gene expression in the presence of a test sample relative to a control which was cultured in the absence of a test sample.
Assays of this type will detect compounds that directly block zsig66 binding to cell-surface receptors, 2 5 e.g., dimerization, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding. Alternatively, compounds or other samples can be tested for direct blocking of zsig66 binding to receptor using zsig66 tagged with a detectable label (e.g., '25I, biotin, horseradish peroxidase, FITC, and the like). Within assays of this type, the ability of a test sample to inhibit the binding of labeled zsig66 to 3 0 the receptor is indicative of inhibitory activity, which can be confirmed through WO 00/36104 PCT/lJS99/29669 secondary assays. Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.
Alternatively, the above methodology may be used to identify agonists of zsig66 activity. Candidate compounds serving as test samples including solutions, 5 mixtures or extracts, are tested for the ability to mimic the activity of zsig6b polypeptide on the target cells as evidenced by stimulation of reporter gene expression in the presence of a test sample and the absence of zsig66, relative to a control (cultured in the absence of a test sample and the absence of zsig66 polypeptide), using assays as described above.
A zsig66 polypeptide can be expressed as a fusion with an immunoglobuhn heavy chain constant region, typically an Fc fragment, which contains two constant region domains and lacks the variable region. Methods for preparing such fusions are disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584. Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfide bonded 15 to each other and two non-Ig polypeptides are arrayed in closed proximity to each other. Fusions of this type can be used to affinity purify ligand, as an in vitro assay tool, or an antagonist of zsig66. For use in assays, the chimeras are bound to a support via the Fc region and used in an ELISA format.
A zsig66 polypeptide can also be used for purification of receptor or 2 0 polypeptides to which it binds. The zsig66 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-2 5 hydroxysuccinirnide activation, epoxide activation, sulfhydryl activation, and hydrazide activation. The resulting medium will generally be configured in the form of a column, and membrane fractions containing receptors are passed through the column one or more times to allow the receptor to bind to the ligand zsig66 polypeptide. The receptor is then eluted using changes in salt concentration, chaotropic agents (guanidine HCl), or 3 0 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 1 (? 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 S 1: 660-72, 1949) 2 0 and calorimetric assays (Cunningham et al., Science 253:545-48, 1991;
Cunningham et al., Science 245:821-25, 1991).
As a ligand, the activity of zsig66 polypeptide can be measured by a silicon-based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent 2 5 physiologic cellular responses. An exemplary device is the CytosensorT""
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 3 0 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enz mol 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 extracelIular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including zsig66 polypeptide, its agonists, or antagonists.
Preferably, the microphysiometer is used to measure responses of a zsig66-responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to zsig66 polypeptide. Zsig66-responsive eukaryotic cells comprise cells into which a receptor for zsig66 has been transfected creating a cell that is responsive to zsig66;
or cells naturally responsive to zsig66 such as cells derived from pituitary tissue.
Differences, l0 measured by a change, for example, an increase or diminution in extracellular acidification, in the response of cells exposed to zsig66 polypeptide, relative to a control not exposed to zsig66, are a direct measurement of zsig66-modulated cellular responses. Mareover, such zsig66-modulated responses can be assayed under a variety of stimuli. Using the microphysiometer, there is provided a method of identifying agonists of zsig66 polypeptide, comprising providing cells responsive to a zsig66 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 2 0 response is shown as a measurable change extracellular acidification rate.
Moreover, culturing a third portion of the cells in the presence of zsig66 polypeptide and the absence of a test compound can be used as a positive control for the zsig66-responsive cells, and as a control to compare the agonist activity of a test compound with that of the zsig66 polypeptide. Moreover, using the microphysiometer, there is provided a method of identifying antagonists of zsig66 polypeptide, comprising providing cells responsive to a zsig66 polypeptide, culturing a first portion of the cells in the presence of zsig66 and the absence of a test compound, culturing a second portion of the cells in the presence of zsig66 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 3 0 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 zsig66 polypeptide, can be rapidly identified using this method.
Moreover, zsig66 can be used to identify cells, tissues, or cell lines which respond to a zsig66-stimulated pathway. The microphysiometer, described 5 above, can be used to rapidly identify Iigand-responsive cells, such as cells responsive to zsig66 of the present invention. Cells can be cultured in the presence or absence of zsig66 polypeptide. Those cells which elicit a measurable change in extracelluIar acidification in the presence of zsig66 are responsive to zsig66. Such cell lines, can be used to identify antagonists and agonists of zsig66 polypeptide as described above.
10 The polypeptides, nucleic acid andlor antibodies of the present invention may be used in treatment of disorders associated with gonadal development, pregnancy, pubertal changes, menopause, ovarian cancer, fertility, ovarian function, polycystic ovarian syndrome, pancreas, diabetes, eye disease, pituitary function, blood pressure regulation, water balance, osteoporosis, and other bone diseases. The molecules of the 15 present invention may used to modulate or to treat or prevent development of pathological conditions in such diverse tissue as pancreas and ovary. In particular, certain syndromes or diseases may be amenable to such diagnosis, treatment or prevention. Moreover, natural functions, such as ovulation, may be suppressed or controlled for use in birth control by molecules of the present invention. For a review 2 0 on pituitary function and diseases of which zsig66 may effect or regulate, see Irnura, H.
(ed) The Pituitary Gland, Raven Press, New York, 1994.
As a pituitary-expressed polypeptide, the zsig66 polypeptide of the present invention may act in the neuroendocrine/exocrine cell fate decision pathway in organ systems in the body, and is therefore capable of regulating the expansion of 2 5 neuroendocrine and exocrine cells in the pancreas. One such regulatory use is that of islet cell regeneration. Also, it has been hypothesized that the autoimmunity that triggers IDDM starts in utero, and zsig66 polypeptide is a developmental gene involved in cell partitioning. Assays and animal models are known in the art for monitoring the exocrine/neuroendocrine cell lineage decision, for observing pancreatic cell balance and 3 0 for evaluating zsig66 polypeptide, fragment, fusion protein, antibody, agonist or antagonist in the prevention or treatment of the conditions set forth above.

The molecules of the present invention will be useful for treating growth disorders, adrenal disorders, hormone and pubertal disorders, reproductive disorders and cancers, menopause, disorders associated with production of breast milk in humans and animals, enhancing production of breast milk in humans and animals, and pituitary disorders and cancers. The polypeptides, nucleic acid and/or antibodies of the present invention can be used in treatment of disorders and complications associated with these diseases. The molecules of the present invention can be used to modulate pituitary function, pituitary hormones, reproductive organs, enhance fertility, birth control, and the like, or to treat or prevent development of pathological conditions in such diverse tissue as pancreas, other endocrine tissues and pituitary. In particular, certain syndromes and diseases may be amenable to such diagnosis, treatment or prevention.
Zsig66 polypeptide is expressed in the pituitary and may have additional biological activity as described below.
Oogenesis is the process by which a diploid stem cell proceeds through multiple stages of differentiation, culminating in the formation of a terminally differentiated cell with a unique function, an oocyte. Unlike spermatogenesis, which begins at puberty and continues on through the life of a male, oogenesis begins during fetal development and by birth, a female's entire supply of primary oocytes are stored in the ovaries in primordial follicles and await maturation and release.
2 0 In the adult ovary, folliculogenesis starts when the follicles enter the growth phase. Early growing follicles undergo a dramatic process of cellular proliferation and differentiation. The classic control of ovarian function by luteinizing hormone (LH) and follicle stimulating hormone (FSH) is now thought to include the action of a variety of molecules that act to promote cell-cell interactions between cells of the follicle. For review, see Gougeon, A., Endocrine Rev. 17:121-155, 1996.
Hence, the mechanisms for controlling ovarian folliculogenesis and dominant follicle selection are still under investigation. As zsig66 is expressed in the pituitary, it may serve a role in regulating folliculogenesis and dominant follicle selection, by affecting proliferation or differentiation of follicular cells, affecting cell-cell interactions, 3 0 modulating hormones involved in the process, and the like.

The ovarian cycle in mammals includes the growth and maturation of follicles, followed by ovulation and transformation of follicles into corpea lutea. The physiological events in the ovarian cycle are dependent on interactions between hormones and cells within the hypothalamic-pituitary-ovarian axis, including gonadotropin releasing hormone (GnRH), LH, and FSH. In addition, estradiol, synthesized in the follicle, primes the hypothalamic-pituitary axis and is required for the mid-cycle surge of gonadotropin that stimulates the resumption of oocyte meiosis and leads to ovulation and subsequent extrusion of an oocyte from the follicle. This gonadotropin surge also promotes the differentiation of the follicular cells from l0 secreting estradiol to secreting progesterone. Progesterone, secreted by the corpus luteum, is needed for uterine development required for the implantation of fertilized oocytes. The central role of hypothalamic-pituitary-gonadal hormones in the ovarian cycle and reproductive cascade, and the role of sex steroids on target tissues and organs, e.g., uterus, breast, adipose, bones and liver, has made modulators of their activity desirable for therapeutic applications. Such applications include treatments for precocious puberty, endometriosis, uterine leiomyomata, hirsutism, infertility, pre menstrual syndrome (PMS), amenorrhea, and as contraceptive agents.
Lsig66 polypeptides, agonists and antagonists which modulate the actions of such hormones can be of therapeutic value. Moreover, zsig66 may act as a 2 0 hormone itself, and as such zsig66 polypeptides, agonists and antagonists can be of therapeutic value. Such molecules can also be useful for modulating steroidogenesis, both in vivo and in vitro, and modulating aspects of the ovarian cycle such as oocyte maturation, ovarian cell-cell interactions, follicular development and rupture, luteal function, and promoting uterine implantation of fertilized oocytes. Molecules which 2 5 modulate hormone action can be beneficial therapeutics for use prior to or at onset of puberty. For example, puberty in females is marked by an establishment of feed-back loops to control hormone levels and hormone production. Abnormalities resulting from hormone imbalances during puberty have been observed and include precocious puberty, where pubertal changes occur in females prior to the age of $.
Hormone-3 0 modulating molecules, can be used, in this case, to suppress hormone secretion and delay onset of puberty.

The level and ratio of gonadotropin and steroid hormones can be used to assess the existence of hormonal imbalances associated with diseases, as well as determine whether normal hormonal balance has been restored after administration of a therapeutic agent. Similarly, the level of zsig66 can also be measured in its relationship to hormonal imbalances. Determination of estradiol, progesterone, LH, and FSH, for example, from serum is known by one of skill in the art. Such assays can be used to monitor the hormone levels after administration of zsig66 in vivo, or in a transgenic mouse model where the zsig66 gene is expressed or the marine ortholog is deleted.
Thus, as a hormone-modulating molecule, zsig66 polypeptide can have therapeutic application for treating, for example, breakthrough menopausal bleeding, as part of a therapeutic regime for pregnancy support, or for treating symptoms associated with polycystic ovarian syndrome (PCOS), PMS and menopause. In addition, other in vivo rodent models are known in the art to assay effects of zsig66 polypeptide on, for example, polycystic ovarian syndrome (PCOS).
Proteins of the present invention may also be used in applications for enhancing fertilization during assisted reproduction in humans and in animals.
Such assisted reproduction methods are known in the art and include artificial insemination, in vitro fertilization, embryo transfer, and gamete intrafallopian transfer.
Such methods are useful for assisting those who may have physiological or metabolic disorders that 2 0 prevent or impede natural conception. Such methods are also used in animal breeding programs, e.g., for livestock, racehorses, domestic and wild animals, and could be used as methods for the creation of transgenic animals. Zsig66 polypeptides could be used in the induction of ovulation, either independently or in conjunction with a regimen of gonadotropins or agents such as clomiphene citrate or bromocriptine (Speroff et al., Induction of ovulation, Clinical Gvnecolo~ic Endocrinology and Infertility, 5'" ed., Baltimore, Williams & Wilkins, 1994). As such, proteins of the present invention can be administered to the recipient prior to fertilization or combined with the sperm, an egg or an egg-sperm mixture prior to in vitro or in vivo fertilization. Such proteins can also be mixed with oocytes prior to cryopreservation to enhance viability of the 3 0 preserved oocytes for use in assisted reproduction.

The zsig66 polypeptides, agonists and antagonists of the present invention may be directly used as or incorporated into therapies for treating reproductive disorders. DlcnrrjPrc enr~h ~~ ~"te,.t _L..__ ~_r. . . _ such therapy (Soules, "Luteal phase deficiency: A subtle abnormality of ovulation" in, Infertility: Evaluation and Treatment, Keye et al., eds., Philadelphia, WB
Sounders, 1995). Moreover, administration of gonadotropin-releasing hormone is shown to stimulate reproductive behavior (Riskin and Moss, Res. Bull. 11:481-5, 1983;-Kadar et al., Physiol. Behav. 51:601-5, 1992 and Silver et al., J. Neruoendocrin. 4:207-10, 199;
King and Millar, Cell. Mol. Neurobiol., I5:5-23, 1995). Given the high prevalence of 1 ~~ sexual dysfunction and impotence in humans, molecules, such as zsig66, which may modulate or enhance gonadotropin activity can find application in developing treatments for these conditions.
The zsig66 polypeptides of the present invention can be used to study ovarian and other reproductive tissue cell proliferation, maturation, and differentiation, i.e., by acting as a luteinizing agent that converts granulosa cells from estradiol to progesterone-producing cells. For example, such methods of the present invention generally comprise incubating granulosa cells, theca cells, oocytes or a combination thereof, in the presence and absence of zsig66 polypeptide, monoclonal antibody, agonist or antagonist thereof and observing changes in cell proliferation, maturation and 2o differentiation. See for example, Basini et al.,(J. Rep. Immunol, 3~:139-53, 1998);
Duleba et al.,(Fert. Ster. (9:335-40, 1998); and Campbell, B.K. et al., J.
Reprod. and Fert. 112:69-77, 1998).
'The molecules of the present invention are useful as components of defined cell culture media, as described herein, and may be used alone or in 2 5 combination with other cytokines and hormones to replace serum that is commonly used in cell culture. Molecules of the present invention are particularly useful in specifically promoting the growth, development, differentiation, and/or maturation of ovarian cells in culture, and may also prove useful in the study of the ovarian cycle, reproductive function, ovarian cell-cell interactions, and fertilization.
3 o In addition, zsig66 may affect other hormones aside from those in reproductive tissues. In addition, the present invention also provides methods for studying steroidogenesis and steraid hormone secretion. Such methods generally comprise incubating ovarian or other hormone-producing cells in culture medium comprising zsig66 polypeptides, monoclonal antibodies, agonists or antagonists thereof with and without gonadotropins and/or steroid hormones, and subsequently observing 5 protein and steroid secretion. Exemplary gonadotropin hormones include luteinizing hormone and follicle stimulating hormone (Rouillier et al., Mol. Reprod. Dev.
50:170-7, 1998). Exemplary steroid hormones include estradiol, androstenedione, and progesterone. Effects of zsig66 on steroidogenesis or steroid secretion can be determined by methods known in the art, such as radioimmunoassay (to detect levels of 10 estradiol, androstenedione, progesterone, and the like), and immunoradiometric assay (IRMA).
Molecules expressed in the pituitary, such as zsig66 polypeptide, and which may modulate hormones, hormone receptors, growth factors, or cell-cell interactions, of the reproductive cascade, other organ systems or the cardiovascular 15 system, or are involved in oocyte or ovarian development or other development, would be useful as markers for cancer of reproductive organs and other organ systems and as therapeutic agents for hormone-dependent cancers, by inhibiting hormone-dependent growth and/or development of tumor cells. Human reproductive system cancers such as ovarian, uterine, cervical, testicular and prostate cancers are common.
Moreover, 2 0 receptors for steroid hormones involved in the reproductive cascade are found in human tumors and tumor cell lines (breast, prostate, endometrial, ovarian, kidney, and pancreatic tumors) (Kakar et al., Mol. Cell. Endocrinol., 106:145-49, 1994;
Kakar and Jennes, Cancer Letts., 98:5?-62, 1995). 'thus, expression of zsig66 in pituitary, with concomitant effects in reproductive tissues and other organ systems suggests that 25 polypeptides of the present invention would be useful in diagnostic methods for the detection and monitoring of reproductive tissue and other cancers.
Diagnostic methods of the present invention involve the detection of zsig66 polypeptides in the serum or tissue biopsy of a patient undergoing analysis of reproductive function or evaluation for possible reproductive or other cancer or disease.
3 0 Such polypeptides can be detected using immunoassay techniques and antibodies, described herein, that are capable of recognizing polypeptide epitopes. More specifically, the present invention contemplates methods for detecting zsig66 polypeptides comprising:
exposing a test sample potentially containing zsig66 polypeptides to an antibody attached to a solid support, wherein said antibody binds to a first epitope of a '~ zsig66 polypeptide;
washing the immobilized antibody-polypeptide to remove unbound contaminants;
exposing the immobilized antibody-polypeptide to a second antibody directed to a second epitope of a zsig66 polypeptide, wherein the second antibody is associated with a detectable Iabel; and detecting the detectable label. Altered levels of zsig66 polypeptides in a test sample, such as serum sweat, saliva, biopsy, and the like, can be monitored as an indication of reproductive function or of cancer or disease, when compared against a normal control.
Additional methods using probes or primers derived, for example, from the nucleotide sequences disclosed herein can also be used to detect zsig66 expression in a patient sample, such as a blood, saliva, sweat, tissue sample, or the like. For example, probes can be hybridized to tumor tissues and the hybridized complex detected by in situ hybridization. Zsig66 sequences can also be detected by PCR
2 0 amplification using cDNA generated by reverse translation of sample mRNA
as a template (PCR Primer A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Press, 1995). When compared with a normal control, both increases or decreases of zsig66 expression in a patient sample, relative to that of a control, can be monitored and used as an indicator or diagnostic for disease.
2 5 As a pituitary hormone, zsig66 may regulate blood pressure and water (osmotic) balance in the human body as discussed herein. As such, zsig66, agonists, and antagonists have great therapeutic potential in regulating blood pressure.
For example, zsig66, agonists, and antagonists have therapeutic potential in enhancing blood pressure, for example in the case of traumatic shock; or decreasing blood 3 0 pressure, for example in the case of hypertension. Moreover water balance is important in maintaining electrolytes as well as affecting overall health. As such, zsig66, agonists, and antagonists have great therapeutic potential in regulating water balance, for example after major organ surgeries, trauma nutritional or disease states that alter such balance.
Moreover, zsig66 polypeptides may modulate mammalian energy balance. The pituitary expression of zsig66 suggests that zsig66 may exhibit effects on glucose uptake, e.g. through GLUT-1, and thermogenesis (thermoregulation). For example, zsig66 polypeptides may find utility in modulating nutrient uptake, as demonstrated, for example, by 2-deoxy-glucose uptake in the brain or the like.
Among other methods known in the art or described herein, mammalian energy balance may be 1 o evaluated by monitoring one or more of the following metabolic functions:
adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, protein synthesis, thermogenesis, oxygen utilization or the like. 'These metabolic functions are monitored by techniques (assays or animal models) known to one of ordinary skill in the art, as is mare fully set forth below. For example, the glucoregulatory effects of insulin are predominantly exerted in the liver, skeletal muscle and adipose tissue.
Insulin binds to its cellular receptor in these three tissues and initiates tissue-specific actions that result in, for example, the inhibition of glucose production and the stimulation of glucose utilization. In the liver, insulin stimulates glucose uptake and inhibits gluconeogenesis and glycogenolysis. In skeletal muscle and adipose tissue, 2 0 insulin acts to stimulate the uptake, storage and utilization of glucose.
Zsig66 polypeptide is expressed in pituitary but may exhibit extra-pituitary activity in organs that affect metabolic functions. Thus, pharmaceutical compositions of the present invention may be useful in prevention or treatment of pancreatic and other endocrine, exocrine or neurocrine disorders. For example, zsig66 2 5 may be associated with pathological regulation of the expansion of neurocrine and exocrine cells in the pancreas, as evident in IDDM, pancreatic cancer or the like, or zsig66 may be associated with pathological regulation of reproductive organs such as the ovaries. Pharmaceutical compositions of the present invention may also be involved in prevention or treatment of pancreatic conditions characterized by 3 0 dysfunction associated with pathological regulation of blood glucose levels, insulin resistance or digestive function.

Art-recognized methods exist for monitoring all of the metabolic functions recited above. Thus, one of ordinary skill in the art is able to evaluate zsig66 polypeptides, fragments, fusion proteins, antibodies, agonists and antagonists for metabolic modulating functions. Exemplary modulating techniques are set forth below.
Adipogenesis, gluconeogenesis and glycogenolysis are interrelated components of mammalian energy balance, which may be evaluated by known techniques using, for example, ablob mice or dbldb mice. The oblob mice are inbred mice that are homozygous for an inactivating mutation at the ob (obese) locus.
Such oblob mice are hyperphagic and hypometabolic, and are believed to be deficient in 1 c) production of circulating OB protein. The dbldb mice are inbred mice that are homozygous for an inactivating mutation at the db (diabetes) locus. The dbldb mice display a phenotype similar to that of vblob mice, except dbldb mice display a more severe diabetic phenotype. Such dbldb mice are believed to be resistant to the effects of circulating OB protein. Also, various in vitro methods of assessing these parameters 1 ~; are known in the art.
Insulin-stimulated lipogenesis, for example, may be monitored by measuring the incorporation of 14C-acetate into triglyceride (Mackall et al.
J. Biol.
Chem. 251:6462-6464, 1976} or triglyceride accumulation (Kletzien et al., Mol.
Pharmacol. 41:393-398, 1992).
2 0 Glucose uptake may be evaluated, for example, in an assay for insulin-stimulated glucose transport. Non-transfected, differentiated L6 myotubes (maintained in the absence of 6418) are placed in DMEM containing 1 g/1 glucose, 0.5 or 1.0%
BSA, 20 mM Hepes, and 2 mM glutamine. After two to five hours of culture, the medium is replaced with fresh, glucose-free DMEM containing 0.5 or 1.0% BSA, 2 5 mM Hepes, I mM pyruvate, and 2 mM glutamine. Appropriate concentrations of insulin or IGF-1, or a dilution series of the test substance, are added, and the cells are incubated for 20-30 minutes. 3H or 14C-labeled deoxyglucose is added to X50 1 M
final concentration, and the cells are incubated for approximately 10-30 minutes. The cells are then quickly rinsed with cold buffer (e.g. PBS), then lysed with a suitable 3 0 lysing agent (e.g. 1 % SDS or 1 N NaOH). The cell lysate is then evaluated by counting in a scintillation counter. Cell-associated radioactivity is taken as a measure of glucose transport after subtracting non-specific binding as determined by incubating cells in the presence of cytochalasin b, an inhibitor of glucose transport. Other methods include those described by, for example, Manchester et al., Am. J. Physiol. 266 (Endocrinol.
Metab. .2~9 :E326-E333, 1994 (insulin-stimulated glucose transport).
Protein synthesis may be evaluated, for example, by comparing precipitation of 35S-methionine-labeled proteins following incubation of the test cells with 35S-methionine and ASS-methionine and a putative modulator of protein synthesis.
Thermogenesis may be evaluated as described by B. Stanley in The Biolo~v of Neuropeptide Y and Related Peptides, W. Colrners and C. Wahlestedt l0 (eds.), Humana Press, Ottawa, 1993, pp. 457-509; C. Billington et al., Am.
J. Physiol.
260:8321, 1991; N. Zarjevski et al., EndocrinoloQV 133:1753, 1993; C.
Billington et al., Am. J. Ph s~iol_ 266:81765, 1994; Heller et al., Am. J. Physiol. 252- (4 Pt 2): 8661-7, 1987; and Heller et al., Am. J. Phvsiol. 245(3): 8321-8, 1983. Also, metabolic rate, which may be measured by a variety of techniques, is an indirect measurement of thermogenesis.
Oxygen utilization may be evaluated as described by HeIler et al., Pflu ers Arch 369( 1 ): 55-9, 1977. This method also involved an analysis of hypothalmic temperature and metabolic heat production. Oxygen utilization and thenmoregulation have also been evaluated in humans as described by Haskell et al., J.
2 0 Appl. Phvsiol. 5~: 948-54, 1981.
The zsig66 polypeptides of the present invention may act in the neuroendocrine/exocrine cell fate decision pathway and is therefore capable of regulating the expansion of neuroendocrine and exocrine cells in the pancreas.
One such regulatory use is that of islet cell regeneration. Also, it has been hypothesized that 2 5 the autoimmunity that triggers IDDM starts in utero, and zsig66 polypeptide is a developmental gene involved in cell partitioning. Assays and animal models are known in the art for monitoring the exocrine/neuroendocrine cell lineage decision, for observing pancreatic cell balance and for evaluating zsig66 polypeptide, fragment, fusion protein, antibody, agonist or antagonist in the prevention or treatment of the 3 o conditions set forth above.

zsig66 polypeptides can also be used to prepare antibodies that bind to zsig66 epitopes, peptides or polypeptides. 'The zsig66 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 5 polypeptides contain a sequence of at least 6, preferably at least 9, and more preferably at least 15 to about 30 contiguous amino acid residues of a zsig66 polypeptide (e.g., SEQ ID N0:2). Polypeptides comprising a larger portion of a zsig66 polypeptide, i.e., from 30 to 10 residues up to the entire length of the amino acid sequence are included.
Antigens or immunogenic epitopes can also include attached tags, adjuvants and l0 carriers, as described herein. Suitable antigens include the zsig66 polypeptide encoded by SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly), or a contiguous 9 to 57 amino acid fragment thereof. Other suitable antigens include motifs I through 4, as disclosed herein. Preferred peptides to use as antigens are hydrophilic peptides such as those predicted by one of skill in the art from a 15 hydrophobicity plot (See Figure). Zsig66 hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: (1) amino acid number I (Met} to amino acid number 6 (Glu) of SEQ ID N0:2; (2) amino acid number 7 (Val) to amino acid number 12 (Ile) of SEQ ID N0:2; (3) amino acid number 26 (Ser) to amino acid number 32 (Ser) of SEQ ID N0:2; (4) amino acid number 29 (Asp) to 20 amino acid number 34 (Ser) of SEQ ID N0:2; and (5) amino acid number 51 (Leu) to amino acid number 56 (Glu) of SEQ ID N0:2. Antibodies fiom an immune response generated from inoculation 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, 25 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 Hun ell, J. G. R., Ed., Monoclonal Hvbridoma Antibodies' Techniaues and Anylications, CRC Press, Inc., Boca Raton, FL, 1982.
As would be evident to one of ordinary skill in the art, polycional 3 o antibodies can be generated by inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a zsig66 polypeptide or a fragment thereof. The immunogenicity of a zsig66 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of zsig66 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
As used herein, the term "antibodies" includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(af)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 incorporating the entire non-human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered" antibody).
In some instances, humanized antibodies may retain non-human residues within the human 2 0 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. Moreover, human antibodies can be produced in transgenic, non-human animals that have been engineered to contain human immunoglobulin genes as disclosed in WIPO
Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination.
Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zsig66 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through 3 0 use of immobilized or labeled zsig66 protein or peptide). Genes encoding polypeptides having potential zsig66 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 ai., US Patent NO. 5,223,409; Ladner et al., US Patent NO.
4,946,778; Ladner et al., US Patent NO. 5,403,4$4 and Ladner et al., US Patent NO.
1 o S,S71,698) 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 sequences disclosed herein to identify proteins which bind to zsig66. These "binding polypeptides" which interact with zsig66 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 2 0 receptor, or viral binding to a receptor. The binding polypeptides can also be used for diagnostic assays for determining circulating levels of zsig66 sequences disclosed herein to identify proteins which bind to zsig66. These "binding polypeptides"
which interact with zsig66 polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to 2 5 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 zsig66 polypeptides; for detecting or quantitating 3 0 soluble zsig66 polypeptides as marker of underlying pathology or disease.
These binding polypeptides can also act as zsig66 "antagonists" to block zsig66 binding and signal transduction in vitro and in vivo. These anti-zsig66 binding polypeptides would be useful for inhibiting zsig66 activity or protein-binding.
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 known related polypeptide molecules. A threshold level of binding is determined if anti-zsig66 antibodies herein bind to a zsig66 polypeptide, peptide or epitope with an affinity at least 10-fold greater than the binding affinity to control {non-zsig66) polypeptide. It is preferred that the antibodies exhibit a binding affinity (Ka) of 106 M-1 or greater, preferably 107 M-1 or greater, more preferably 108 M 1 or greater, and most preferably 109 M 1 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-zsig66 antibodies do not significantly cross-react with known related polypeptide molecules is shown, for example, by the antibody detecting zsig66 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 zsig66, and zsig66 mutant polypeptides. Moreover, antibodies can be "screened against"
known related polypeptides, to isolate a population that specifically binds to the zsig66 polypeptides. For example, antibodies raised to zsig66 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to zsig66 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 Immunoloey, 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 Immunoloev, 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-zsig66 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 zsig66 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 zsig66 protein or polypeptide.
Antibodies to zsig66 and zsig66 binding polypeptides described herein may be used for tagging cells that express zsig66; for isolating zsig66 by affinity purification; for diagnostic assays for determining circulating levels of zsig66 polypeptides; for detecting or quantitating soluble zsig66 as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; for detecting or quantitating soluble zsig66 polypeptides as marker of underlying pathology or disease. These antibodies and binding polypeptides can also act as zsig66 "antagonists" to block zsig66 binding 2 0 and signal transduction in vitro and in vivo. These anti-zsig66 binding polypeptides would be useful for inhibiting zsig66 activity or protein-binding.
Antibodies to zsig66 may be used for tagging cells that express zsig66;
for isolating zsig66 by affinity purification; for diagnostic assays for determining circulating levels of zsig66 polypeptides; for detecting or quantitating soluble zsig66 as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zsig66 activity in vitro and in vivo.
Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the 3 0 like; indirect tags or labels may feature use of biotin-avidin or other complement/anti-complement pairs as intermediates. Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. Moreover, antibodies to zsig66 or fragments thereof may be used in vitro to detect denatured zsig66 or fragments thereof in assays, for example, Western Blots or other assays known in the art.
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 1 o instance). More specifically, zsig66 polypeptides or anti-zsig66 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 15 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 20 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 25 complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion. For these purposes, biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
In another embodiment, polypeptide-toxin fusion proteins or antibody toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for 3 0 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 celUtissue-specific delivery of generic anti-complementary-detectable/ cytotoxic molecule conjugates.
In another embodiment, zsig66-cytokine fusion proteins or antibody-cytokine fusion proteins can be used for enhancing in vivo killing of target tissues (for example, blood and bone marrow cancers), if the zsig66 polypeptide or anti-zsig66 antibody targets the hyperproliferative blood or bone marrow cell (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 zsig66 polypeptides or anti-zsig66 antibodies target an undesirable cell or tissue (i.e., a tumor or a leukemia), and the fused cytokine mediated improved target cell lysis by effector cells. Suitable cytokines for this purpose include interleukin 2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), for instance.
2 o 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 2 5 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. The stage of a cell population's differentiation is monitored by identification of markers present in the cell population. Myocytes, osteoblasts, adipocytes, 3 0 chrondrocytes, fibroblasts and reticular cells are believed to originate from a common mesenchymal stem cell (Owen et al., Ciba Fdn. Svmn. 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. The novel polypeptides of the present invention may be useful for studies to isolate mesenchymal stem cells and myocyte or other progenitor cells, both in vivo and ex vivo.
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, the present invention includes stimulating or inhibiting the proliferation of myocytes, smooth 10 muscle cells, osteoblasts, adipocytes, chrondrocytes, neuronal and endothelial cells.
Molecules of the present invention for example, may while stimulating proliferation or differentiation of cardiac myocytes, inhibit proliferation or differentiation of adipocytes, by virtue of the affect on their common precursor/stem cells. Thus molecules of the present invention may have use in inhibiting chondrosarcomas, 15 atherosclerosis, restenosis and obesity.
Assays measuring differentiation include, for example, measuring cell 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;- R.aes, Adv. Anim. Cell Biol Technol Bioprocesses, 20 161-171, 1989; all incorporated herein by reference). Alternatively, ZSIG66 polypeptide itself can serve as an additional cell-surface or secreted marker associated with stage-specific expression of a tissue. As such, direct measurement of polypeptide, or its loss of expression in a tissue as it differentiates, can serve as a marker for differentiation of tissues.
25 Similarly, direct measurement of ZSIG66 polypeptide, or its loss of expression in a tissue can be determined in a tissue or cells as they undergo tumor progression. Increases in invasiveness and motility of cells, or the gain or loss of expression of ZSIG66 in a pre-cancerous or cancerous condition, in comparison to normal tissue, can serve as a diagnostic for transformation, invasion and metastasis in 3 0 tumor progression. As such, knowledge of a tumor's stage of progression or metastasis will aid the physician in choosing the most proper therapy, or aggressiveness of treatment, for a given individual cancer patient. Methods of measuring gain and loss of expression (of either mRNA or protein) are well known in the art and described herein and can be applied to ZSIG66 expression. For example, appearance or disappearance of polypeptides that regulate cell motility can be used to aid diagnosis and prognosis of prostate cancer (Banyard, J. and Zetter, B.R., Cancer and Metast. Rev. 17:449-458, 1999). As an effector of cell motility, ZSIG66 gain or loss of expression may serve as a diagnostic for prostate and other cancers.
Moreover, the activity and effect of zsig66 on tumor progression and metastasis can be measured in vivo. Several syngeneic mouse models have been l0 developed to study the influence of polypeptides, compounds or other treatments on tumor progression. In these models, tumor cells passaged in culture are implanted into mice of the same strain as the tumor donor. The cells will develop into tumors having similar characteristics in the recipient mice, and metastasis will also occur in some of the models. Appropriate tumor models for our studies include the Lewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No. CRL-6323), amongst others. These are both commonly used tumor lines, syngeneic to the mouse, that are readily cultured and manipulated in vitro. Tumors resulting from implantation of either of these cell lines are capable of metastasis to the lung in C57BL6 mice. The Lewis lung carcinoma model has recently been used in mice to 2 0 identify an inhibitor of angiogenesis (O'Reilly MS, et al. Cell 79: 315-328,1994).
C57BL6/J mice are treated with an experimental agent either through daily injection of recombinant protein, agonist or antagonist or a one time injection of recombinant adenovirus. Three days following this treatment, 105 to 106 cells are implanted under the dorsal skin. Alternatively, the cells themselves may be infected with recombinant 2 5 adenovirus, such as one expressing zsig66, before implantation so that the protein is synthesized at the tumor site or intracellularly, rather than systemically.
The mice normally develop visible tumors within S days. The tumors are allowed to grow for a period of up to 3 weeks, during which time they may reach a size of 1 S00 - I
800 mm3 in the control treated group. Tumor size and body weight are carefully monitored 3 0 throughout the experiment. At the time of sacrifice, the tumor is removed and weighed along with the lungs and the liver. The lung weight has been shown to correlate well with metastatic tumor burden. As an additional measure, lung surface metastases are counted. The resected tumor, lungs and liver are prepared for histopathological examination, immunohistochemistry, and in situ hybridization, using methods known in the art and described herein. The influence of the expressed polypeptide in question, e.g., zsig66, on the ability of the tumor to recruit vasculature and undergo metastasis can thus be assessed. In addition, aside from using adenovirus, the implanted cells can be transiently transfected with zsig66. Use of stable zsig66 transfectants as well as use of induceable promoters to activate zsig66 expression in vivo are known in the art and can be used in this system to assess zsig66 induction of metastasis. Moreover, purified 1 o zsig66 or zsig66 conditioned media can be directly injected in to this mouse model, and hence be used in this system. For general reference see, O'Reilly MS, et al.
Cell 79:315-328, 1994; and Rusciano D, et- al. Murine Models of Liver Metastasis.
Invasion Metastasis 14:349-361, 1995.
In yet another embodiment, if the zsig66 polypeptide or anti- zsig66 antibody targets vascular cells or tissues, such polypeptide or antibody may be conjugated with a radionuclide, and particularly with a beta-emitting radionuclide, to reduce restenosis. Such therapeutic approach poses less danger to clinicians who administer the radioactive therapy. For instance, iridium-192 impregnated ribbons placed into stented vessels of patients until the required radiation dose was delivered 2 0 showed decreased tissue growth in the vessel and greater luminal diameter than the control group, which received placebo ribbons. Further, revascularisation and stent thrombosis were significantly lower in the treatment group. Similar results are predicted with targeting of a bioactive conjugate containing a radionuclide, as described herein.
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 that bind zsig66. For example, proteins and peptides of the present invention 3 0 can be immobilized on a column and membrane preparations run over the column (Immobilized Affinity Lisand Techni ues, Hermanson et al., eds., Academic Press, San Diego, CA, 1992, pp.195-202). Proteins and peptides can also be radiolabeled (Methods in Enzvmol., 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, 5 1984) and specific cell-surface proteins can be identified.
The molecules of the present invention, although expressed in pituitary, may exert their effects elsewhere and hence may be useful for treating diabetes, and pancreatic cancer. The polypeptides, nucleic acid and/or antibodies of the present invention can be used in treatment of disorders and complications associated with these 10 diseases. The molecules of the present invention can be used to modulate insulin, glucagon, and the like, or to treat or prevent development of pathological conditions in such diverse tissue as pancreas and other endocrine tissues. In particular, certain syndromes and diseases may be amenable to such diagnosis, treatment or prevention.
Since zsig66 appears to have structural similarity to vasopressin and 15 oxytocin, zsig66 could be useful as modulator blood pressure, muscle tension or and osmotic balance. For example, blood pressure modification is important in situations such as heart attack, stroke, traumatic shock, surgery, and any number of bleeding complications. As a modulator of blood pressure, muscle tension or and osmotic balance, zsig66 may modulate contractility in the organ systems and tissues that it 2 0 effects. Thus, The activity of molecules of the present invention can be measured using a variety of assays that measure cell contractility. Such assays are well known in the art.
As zsig66 is structurally related to vasopressin, it can be used to modualte tissues that contract. For example contractile tissues upon which zsig66 may act include skeletal muscle, uterus; tissues in testis, e.g., vas deferens;
prostate tissues;
25 gastrointestinal tissues, e.g., colon and small intestine; and heart. The effects of zsig66 poiypeptide, its antagonists and agonists, on tissue contractility can be measured in vitro using a tensiometer with or without electrical field stimulation. Such assays are known in the art and can be applied to tissue samples, such as aortic rings, vas deferens, ilium, uterine and other contractile tissue samples, as well as to organ systems, such as 3 o atria, and can be used to determine whether zsig66 polypeptide, its agonists or antagonists, enhance or depress contractility. Molecules of the present invention are WO 00/36104 PC'T/US99/29669 hence useful for treating dysfunction associated with contractile tissues or can be used to suppress or enhance contractility in vivo. As such, molecules of the present invention have utility in treating cardiovascular disease, infertility, in vitro fertilization, birth control, treating impotence or other male reproductive dysfunction, as well as !~ inducing birth.
The effect of the zsig66 polypeptides, antagonists and agonists of the present invention on contractility of tissues including uterus, prostate, testis, gastrointestinal tissues, and heart can be measured in a tensiometer that measures contractility and relaxation in tissues. See, Dainty et al., J. Pharmacol.
100:767, 1990;
Rhee et al., Neurotox. 16: 179, 1995; Anderson, M.B., Endo- 114:364-368, 1984;
and Downing, S.J. and Sherwood, O.D, Endocrinol. 116:1206-1214, 1985. For example, measuring vasodilatation of aortic rings is well known in the art.
Briefly, aortic rings are taken from 4 month old Sprague Dawley rats and placed in a buffer solution, such as modified Krebs solution (118.5 mM NaCI, 4.6 mM KCI, 1.2 mM
MgS0,.7Hz0, 1.2 mM KH,P04, 2.5 mM CaCI2.2H20, 24.8 mM NaHC03 and 10 mM
glucose). One of skill in the art would recognize that this method can be used with other animals, such as rabbits, other rat strains, Guinea pigs, and the like.
The rings are then attached to an isometric force transducer (Radnoti Inc., Monrovia, CA) and the data recorded with a Ponemah physiology platform (Gould Instrument systems, Inc., 2 o Valley View, OH) and placed in an oxygenated (95% OZ, 5% CO~) tissue bath containing the buffer solution. The tissues are adjusted to 1 gram resting tension and allowed to stabilize for about one hour before testing. The integrity of the rings can be tested with norepinepherin (Sigma Co., St. Louis, MO) and Carbachol, a muscarinic acetylcholine agonist (Sigma Co.). After integrity is checked, the rings are washed 2 5 three times with fresh buffer and allowed to rest for about one hour. To test a sample for vasodilatation, or relaxation of the aortic ring tissue, the rings are contracted to two grams tension and allowed to stabilize for fifteen minutes. A zsig66 polypeptide sample is then added to I , 2 or 3 of the 4 baths, without flushing, and tension on the rings recorded and compared to the control rings containing buffer only.
Enhancement 3 0 or relaxation of contractility by zsig66 polypeptides, their agonists and antagonists is directly measured by this method, and it can be applied to other contractile tissues such as uterus, prostate, and testis.
The activity of molecules of the present invention can be measured using a variety of assays that measure stimulation of gastrointestinal cell contractility, modulation of nutrient uptake and/or secretion of digestive enzymes. Of particular interest are changes in contractility of smooth muscle cells. For example, the contractile response of segments of mammalian duodenum or other gastrointestinal smooth muscles tissue (Depoortere et al., J. Gastrointestinal Motility 1:150-159, 1989, incorporated herein by reference). An exemplary in vivo assay uses an ultrasonic 1 o micrometer to measure the dimensional changes radially between commissures and longiturdinally to the plane of the valve base (Hansen et al., Society of Thoracic Surgeons 60:5384-390, 1995).
Gastric motility is generally measured in the clinical setting as the time required for gastric emptying and subsequent transit time through the gastrointestinal tract. Gastric emptying scans are well known to those skilled in the art, and briefly, comprise use of an oral contrast agent, such as barium, or a radiolabeled meal. Solids and liquids can be measured independently. A test food or liquid is radiolabeled with an isotope (e.g. 99mTc), and after ingestion or administration, transit time through the gastrointestinal tract and gastric emptying are measured by visualization using gamma 2 0 cameras (Meyer et al., Am. J. Dia. Dis 21:296, 1976; Collins et al., Gut 24:1117, 1983;
Maughan et al., Diabet. Med. 13 9 Suun. S:S6-10, 1996 and Horowitz et al., Arch.
Intern. Med. 145:1467-1472, 1985). These studies may be performed before and after the administration of a pro-motility agent to quantify the e~cacy of the drug.
T'he polypeptides, antagonists, agonists, nucleic acid and/or antibodies of 2 5 the present invention can also be used in treatment of disorders associated with gastrointestinal cell contractility, secretion of digestive enzymes and acids, gastrointestinal motility, recruitment of digestive enzymes; inflammation, particularly as it affects the gastrointestinal system; reflux disease and regulation of nutrient absorption. Specific conditions that will benefit from treatment with molecules of the 30 present invention include, but are not limited to, diabetic gastroparesis, post-surgical gastroparesis, vagotomy, chronic idiopathic intestinal pseudo-obstruction and gastroesophageai reflux disease. Additional uses include, gastric emptying for radiological studies, stimulating gallbladder contraction and antrectomy.
The motor and neurological affects of molecules of the present invention make it useful for treatment of obesity and other metabolic disorders where neurological feedback modulates nutritional absorption. The molecules of the present invention are useful for regulating satiety, glucose absorption and metabolism, and neuropathy-associated gastrointestinal disorders. Molecules of the present invention are also useful as additives to anti-hypoglycemic preparations containing glucose and as adsorption enhancers for oral drugs which require fast nutrient action.
Additionally, l0 molecules of the present invention can be used to stimulate glucose-induced insulin release.
Polynucleotides encoding zsig66 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zsig66 activity. If a mammal has a mutated or absent zsig66 gene, the zsig66 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zsig66 polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes 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
2 0 defective virus is not infectious 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 herpes simplex virus 1 (HSV 1 ) 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 zsig66 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 3 0 33:153, 1983; T'emin et al., U.S. Patent No. 4,650,?64; 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 (Felgner 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 malecules for the purpose 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, DEAF dextran, calcium phosphate precipitation, use of a gene 2 0 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.
Antisense methodology can be used to inhibit zsig66 gene transcription, such as to inhibit cell proliferation in vivo. Polynucleotides that are complementary to a segment of a zsig66-encoding polynucleotide (e.g., a polynucleotide as set froth in 2 5 SEQ ID NO:1 ) are designed to bind to zsig66-encoding mRNA and to inhibit translation of such mRNA. Such antisense polynucleotides are used to inhibit expression of zsig66 polypeptide-encoding genes in cell culture or in a subject.
The present invention also provides reagents which will find use in diagnostic applications. For example, the zsig66 gene, a probe comprising zsig66 DNA
3 0 or RNA or a subsequence thereof can be used to determine if the zsig66 gene is present on a chromosome or if a mutation has occurred. Detectable chromosomal aberrations at the zsig66 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 poIynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length 5 polymorphism (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, Chest 108:255-65, 1995).
Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes (Cox et 1 o al., Science 250:245-50, 1990). Partial or full knowledge of a gene's sequence allows one to design PCR primers suitable for use with chromosomal radiation hybrid mapping panels. Radiation hybrid mapping panels are commercially available which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL). These panels enable rapid, 15 PCR-based chromosomal localizations and ordering of genes, sequence-tagged sites (STSs), and other nonpolymorphic and polymorphic markers within a region of interest.
This includes establishing directly proportional physical distances between newly discovered genes of interest and previously mapped markers.
The precise knowledge of a gene's position can be useful for a number 2 0 of purposes, including: 1 ) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a 2 5 particular gene might have.
Sequence tagged sites (STSs) can also be used independently for chromosomal localization. An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome. An STS is defined by a pair of oligonucleotide primers that are used in a 3 o polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Since STSs are based solely on DNA sequence they can be completely described within an electronic database, for example, Database of Sequence Tagged Sites (dbSTS), GenBank, (National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), and can be searched with a gene sequence of interest for the mapping data contained within these short genomic landmark STS sequences.
The zsig66 gene can be used to detect genes of known function map to the same region of the human chromosome. In addition, zsig66 polynucleotide probes can be used to detect abnormalities or genotypes associated with human disease states as well as map to the same region of the human genome. See the Online Mendellian Inheritance of Man (OMIM) gene map, on a publicly available WWW server (http://www3.ncbi.nlm.nih.gov/htbin-post/Omim). Candidate genes for an inheritable disease which show linkage to the same chromosomal region as the zsig66 gene can hence be identified.
The zsig66 gene is located at the 4q28-q31 region of chromosome 4 (Example 3). Several known genes map to this locus and are associated with human disease states: The Fibrinogin alpha, beta, and gamma gene cluster is located at 4q28, and mutations therein are associated with diseases such as dysfibrinogenemia, hypofibrinogenemia, and fibrinogenemia which result in blood clotting malfunction and other complications (See, for example, Berg, K. and Keirulf, P. Clin. Genet.
36: 229-235, 1989). Zsig66 polynucleotide probes can be used to detect abnormalities or genotypes associated with these fibrinogen associated markers. Further, zsig66 polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 4q28-q31 deletions and translocations associated with human diseases, such as Reiger syndrome (deletion in 4q); loss of heterozygosity, or translocation 2 5 between 4q28-q31 and another chromosome; translocations involved with malignant progression of tumors; or mutations, which are expected to be involved in chromosome rearrangements in malignancy; or deletions and translocations in other cancers.
Similarly, zsig66 polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 4q28-31 trisomy and chromosome loss 3 0 associated with human diseases such as Reiger syndrome (above). Moreover, amongst other genetic loci, those for autosomal dominant pseudohypoaldosteroidism (PHA) type I (4q31. l ), anterior segment mesenchymal dysgenesis and Reiger syndrome (4q28-q31 ), Hepatitis B virus associated hepatocellular carcinoma susceptability (4q32. l ), and others, all manifest themselves in human disease states as well as map to this region of the human genome. See the Online Mendellian Inheritance of Man (OMIM) gene map, 5 and references therein, for this region of chromosome 4 on a publicly available WWW
server (http://www3.ncbi.nlm.nih.gov/htbin-postlOmim/getmap?chromosome=4q28, and surrounding regions through 4q31 ). All of these serve as possible candidate genes for an inheritable disease which show linkage to the same chromosomal region as the zsig66 gene. Thus, zsig66 polynucleotide probes can be used to detect abnormalities or genotypes associated with these defects.
Similarly, defects in the zsig66 locus itself may result in a heritable human disease state. As a pituitary hormone, defects the zsig66 molecules of the present invention can have broad reaching effects in the human body. Molecules of the present invention, such as the polypeptides, antagonists, agonists, polynucleotides and I5 antibodies of the present invention would aid in the detection, diagnosis prevention, and treatment associated with a zsig66 genetic defect. A diagnostic could assist physicians in determining the type of genetic disease and appropriate associated therapy, or assistance in genetic counseling. As such, the inventive anti-zsig66 antibodies, polynucleotides, and polypeptides can be used for the detection of zsig66 polypeptide, 2 0 mRNA or anti-zsig66 antibodies, thus serving as markers and be directly used for detecting or diagnosing known or yet to be discovered diseases, or cancers, as described herein, using methods known in the art and described herein.
In addition, zsig66 polynucleotide probes can be used to detect allelic differences between diseased or non-diseased individuals at the zsig66 chromosomal 2 5 locus. As such, the zsig66 sequences can be used as diagnostics in forensic DNA
profiling. Such profiling can be applied to commercial animals as well for use in breeding programs.
In general, the diagnostic methods used in genetic linkage analysis, to detect a genetic abnormality or aberration in a patient, are known in the art.
Most 3 0 diagnostic methods comprise the steps of (a) obtaining a genetic sample from a potentially diseased patient, diseased patient or potential non-diseased carrier of a recessive disease allele; (b} producing a first reaction product by incubating the genetic sample with a ZSMF 16 polynucleotide probe wherein the polynucieotide will hybridize to complementary polynucleotide sequence, such as in RFLP analysis or by incubating the genetic sample with sense and antisense primers in a PCR reaction under appropriate PCR reaction conditions; (iii) Visualizing the first reaction product by gel electrophoresis and/or other known method such as visualizing the first reaction product with a ZSMF 16 polynucleotide probe wherein the polynucleotide will hybridize to the complementary polynucleotide sequence of the first reaction;
and (iv) comparing the visualized first reaction product to a second control reaction product of a 1 o genetic sample from wild type patient. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the diseased or potentially diseased patient, or the presence of a heterozygous recessive carrier phenotype for a non-diseased patient, or the presence of a genetic defect in a tumor from a diseased patient, or the presence of a genetic abnormality in a fetus or pre-implantation embryo. For example, a difference in restriction fragment pattern, length of PCR products, length of repetitive sequences at the ZSIG66 genetic locus, and the like, are indicative of a genetic abnormality, genetic aberration, or allelic difference in comparison to the normal wild type control. Controls can be from unaffected family members, or unrelated individuals, depending on the test and availability of samples.
2 0 Genetic samples for use within the present invention include genomic DNA, mRNA, and cDNA isolated form any tissue or other biological sample from a patient, such as but not limited to, blood, saliva, semen, embryonic cells, amniotic fluid, and the like.
The polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO:1, the complement of SEQ ID NO:1, or an RNA equivalent thereof.
2 5 Such methods of showing genetic linkage analysis to human disease phenotypes are well known in the art. For reference to PCR based methods in diagnostics see see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humans Press, Ine.
1991), White (ed.), PCR Protocols: Current Methods and Applications (Humans Press, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humans Press, Inc.
1996), 3 0 Hanausek and Walaszek (eds.), Tumor Marker Protocols (Humans Press, Ine.
1998), Lo (ed.), Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).
Aberrations associated with the ZSIG66 locus can be detected using nucleic acid molecules of the present invention by employing standard methods for direct mutation analysis, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for l0 example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc.
1991 ), Marian, Chest 108:25 S ( 1995), Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc. 1996), Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren (ed.), Laboratory Protocols for Mutation Detection (Oxford University Press 1996), Birren et al. (eds.), Genome Analysis, Yol. 2:
Detecting Genes (Cold Spring Harbor Laboratory Press 1998), Dracopoli et al.
(eds.), Current Protocols in Human Genetics (John Wiley & Sons 1998), and Richards and Ward, "Molecular Diagnostic Testing," in Principles of Molecular Medicine, pages 83-88 (Humana Press, Inc. 1998)). Direct analysis of an ZSIG66 gene for a mutation can be performed using a subject's genomic DNA. Methods for amplifying genomic DNA, 2 0 obtained for example from peripheral blood lymphocytes, are well-known to those of skill in the art (see, for example, Dracopoli et al. (eds.), Current Protocols in Human Genetics, at pages 7.1.6 to 7.1.7 (John Wiley & Sons 1998)).
Mice engineered to express the zsig66 gene, referred to as "transgenic mice," and mice that exhibit a complete absence of zsig66 gene function, referred to as 2 5 "knockout mice," may also be generated (Snouwaert et al., Science 257:1083, 1992;
Lowell et al., Nature 366:740-42, 1993; Capecchi, M.R., Science 244: 1288-1292, 1989; Palmiter, R.D. et al. Annu Rev Genet. 20: 465-499, 1986). For example, transgenic mice that over-express zsig66, either ubiquitously or under a tissue-specific or tissue-restricted promoter can be used to ask whether over-expression causes a 3 0 phenotype. For example, over-expression of a wild-type zsig66 polypepdde, polypeptide fragment or a mutant thereof may alter normal cellular processes, resulting in a phenotype that identifies a tissue in which zsig66 expression is functionally relevant and may indicate a therapeutic target for the zsig66, its agonists or antagonists.
For example, a preferred transgenic mouse to engineer is one that over-expresses the zsig66 mature polypeptide (approximately amino acids 28 (Ala) to 84 (Gly) of SEQ ID
5 N0:2). Moreover, such over-expression may result in a phenotype that shows similarity with human diseases. Similarly, knockout zsig66 mice can be used to determine where zsig66 is absolutely required in vivo. The phenotype of knockout mice is predictive of the in vivo effects of that a zsig66 antagonist, such as those described herein, may have. The human zsig66 cDNA can be used to isolate marine l0 zsig66 mRNA, cDNA and genomic DNA, which are subsequently used to generate knockout mice. These mice may be employed to study the zsig66 gene and the protein encoded thereby in an in vivo system, and can be used as in vivo models for corresponding human diseases. Moreover, transgenic mice expression of zsig66 antisense polynucleotides or ribozymes directed against zsig66, described herein, can 15 be used analogously to transgenic mice described above.
F or pharmaceutical use, the proteins of the present invention are formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. In general, pharmaceutical 2 0 formulations will include a zsig66 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:
2 5 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 pg/kg of patient weight per day, preferably 0.5-20 _g/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 3 0 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.
The invention is further illustrated by the following non-limiting examples.
EXAMPLES
Example 1 Identification of zsig66 A. Usine an EST Sequence to Obtain Full-length zsig66 Scanning of translated cDNA library databases using a signal trap as a query resulted in identification of an expressed sequence tag (EST) sequence from a pituitary library found to be homologous to a human secretory signal sequence.
Confirmation of the EST sequence was made by sequence analyses of the cDNA from which the EST originated. This cDNA was contained in a plasmid from a human pituitary library, and was sequenced using the following primers to generate complete double stranded sequence of this clone: ZC6,768 (SEQ ID
N0:12), ZC20,137 (SEQ ID N0:13 ), ZC20,136 (SEQ ID N0:14), ZC20,132 (SEQ ID NO: i 5), 2 0 ZC20,134 (SEQ ID N0:16), ZC 19,964 (SEQ ID N0:17), ZC 19,965 (SEQ ID
N0:18), ZC694 (SEQ ID N0:19).
Example 2 Tissue Distribution Northern blot analysis was performed using Human Multiple Tissue NorthernT"" Blots (MTN I, MTN II, and MTN III) (Clontech). The cDNA described in Example 1 was used in a PCR reaction using oligos ZC21,336 (SEQ ID N0:20) and ZC21,338 (SEQ ID N0:21 ) as primers. PCR conditions were as follows:
94°C for 1.5 minutes; 35 cycles at 94°C for 15 seconds , 66°C for 30 seconds;
72°C for 30 seconds, 3 0 one cycle at 72°C for 5 minutes, followed by a 4°C hold. A
sample of the PCR
reaction product was run on a 4% agarose gel. A band of the expected size of about 220 by was seen. The 220 by PCR fragment, was gel purified using a commercially available kit (QiaexII~"'''; Qiagen) and then radioactively labeled with 32P-dCTP using Rediprime IIT"" (Amersham), a random prime labeling system, according to the manufacturer's specifications. The probe was then purified using a Nuc-TrapT''' column S (Stratagene) according to the manufacturer's instructions. ExpressHyb""
(Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at SS°C using 1-2 x 106 cpm/ml of labeled probe. The blots were then washed for 30 minutes in 2X SSC/0.1% SDS at room temperature, followed by a wash in 2X SSC/0.1% SDS at 65°C , followed by a wash in O.1X SSC/0.1% SDS at 65°C. The blots were exposed 7 days. No transcript signals were observed in the tissues represented on the blots. Moreover, an in-house produced Northern blot containing RNA from pituitary gland was also probed, following the methods and conditions described above. Again, no signals were observed, suggesting that the mRNA for zsig66 is relatively rare and/or expressed in only specific physiologic conditions.
Dot Blots were also performed using Human RNA Master Blots''' (Clontech). The methods and conditions for the Dot Blots are the same as for the Multiple Tissue Blots described above. A signal was observed in genomic DNA.
2 0 Example 3 Chromosomal Assignment and Placement of Zsi~66 Zsig66 was mapped to chromosome 4 using the commercially available version of the "Stanford G3 Radiation Hybrid Mapping Panel" (Research Genetics, Inc., Huntsville, AL). The "Stanford G3 RH Panel" contains DNAs from each of 2 5 radiation hybrid clones of the whole human genome, plus two control DNAs (the RM
donor and the A3 recipient). A publicly available WWW server (http://shgc-www.stanford.edu) allows chromosomal localization of markers.
For the mapping of Zsig66 with the "Stanford G3 RH Panel", 20 pl reactions were set up in a 96-well microtiter plate (Stratagene, La Jolla, CA) and used in 3 0 a "RoboCycler Gradient 96" thermal cycler (Stratagene). Each of the 85 PCR
reactions consisted of 2 ~xl lOX KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, CA), 1.6 ~I dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, CA), 1 ul sense primer, ZC21,450 (SEQ ID N0:22), 1 ~l antisense primer, ZC21,451 (SEQ
ID N0:23), 2 ~.1 "RediLoad" (Research Genetics, Inc., Huntsville, AL), 0.4 ul SOX
Advantage KIenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA
from 5 an individual hybrid clone or control and ddH20 for a total volume of 20 pl.
The reactions were overlaid with an equal amount of mineral oil and sealed. The PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 94oC, 35 cycles of a 45 seconds denaturation at 94oC, 45 seconds annealing at 60oC and 1 minute AND
15 seconds extension at 72oC, followed by a final 1 cycle extension of 7 minutes at 10 72oC. The reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD).
The results showed linkage of Zsig66 to the chromosome 4 marker SHGC-57096 with a LOD score of > 11 and at a distance of 0 cR_10000 from the marker.
The use of surrounding genes, which have been cytogenetically mapped, positions Zsig66 1 > in the 4q28-q31 chromosomal region.
From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

SEQUENCE LISTING
<110> ZymoGenetics. Inc.
<120> Pancreatic polypeptide zsig66 <130> 98-81 <150> US 09/212.947 <151> 1998-12-I6 <160> 24 <170> FastSEQ for Windows Version 3.0 <210>1 <211>2375 <212>DNA

<213>Homo Sapiens <220>
<221> CDS
<222> (224)...(475) <400> 1 gttgcccagt ctggtctcaa actcctaggc tcaagtgacc ctcccacttc gacctcccaa 60 agtgctggga ttacaggtgt gagctaccat gcctggctga tgttggttat ttttagcaaa 120 ataaaaactc ttagatgtct gaaactttca ctatcactct atgatctatt ataaatcaat 180 aagaatcttt aggtaggctg tccccttttt ctctctttcc tcc atg aaa gaa cac 235 Met Lys Glu His aat gaa gtg agt aaa aaa gcc att tat ttg ccc ttc ctt cta gcc cca 283 Asn Glu Val Ser Lys Lys Ala Ile Tyr Leu Pro Phe Leu Leu Ala Pro gtg ctg att cca gcc ttc agt get gat cca gtt tct gga tca ctg tgg 331 Ual Leu Ile Pro Ala Phe Ser Ala Asp Pro Val Ser Gly Ser Leu Trp ata cag ggt atg ggg gcc act cta acc gac ttt aat ccc ttc ctg gat 379 Ile Gln Gly Met Gly Ala Thr Leu Thr Asp Phe Asn Pro Phe Leu Asp i:ca get cgt gaa ata gaa ttg ttt gtt tta gtt gta ttg get ttg aag 427 Ser Ala Arg Glu Ile Glu Leu Phe Val Leu Val Val Leu Ala Leu Lys tat tgt gta gtg aat tgt cag agt cct gga ctg aac tca atc tct ggt 475 Phe Cys Val Val Asn Cys Gln Ser Pro Gly Leu Asn Ser Ile Ser Gly taattcttga tgaaatgatt tctcctttct cacagaacca aatggacatc ctcagttgtt 535 ctctgttcag gaatctccct gtggaaagtc agtggaggtt taaccaggtt gggagagagg 595 acagtcactg ctggctgctg cctactgtga attcccttca gtatccaatt tttcacttcc 655 ctgcctattc ttctaatgag aaataaccca gaagtggaac agaagaaaaa caaaacccaa 715 aaccatcaaa caagcttaag acaaattttg tcctttggct ttccagtctt ctcatctaat 775 catattattt tcagcaactt ttgataacgt gttgcttgta taaaatcaat ttgtgtgtct 835 tttttctttc attctttctt ccttttttca caataagatc ttgggagagt gaatgctaat 895 gcagttctaa agtggaagct taaaactaag aacagtttgt tctccttcca tccctttaca 955 ctgtgatctt taacatcacc gtcaaggaga gtgtgtttgg ttttgtgatc aatttagctg 1015 tcaggggttt gtgattttct gagagctgac tcttgctgct aatgaacatg atcatttata 1075 cactgtcact gtagtatagg catgttaaat aattgatcag gccctggcag ttagagaatg 1135 gcaaatgcag tccctttgtt gctatggtaa tacatttggt taggggggct gtcatttgga 1195 cc:acatggat ctggcacatg tgacataatt gcctgtggtt gaggcggttg caggagaaaa 1255 tcaacttaag aagaggtggc attttgaagt aaattaatgt atatttcagg gttttttttg 1315 ggcttaacat tgtggagtga cttattcttt tctattaaaa ggctcattct cagctaatgg 1375 taagcataga gaatgagtca agtgacagat ttgtttactt gctgacacag tggaatttgt 1435 tc:atttattg tataaaatgg ttattgtgtg aagtttacat tcctccctct attatcccct 1495 ggagggctcc ccataagtga aattgatgcc gggcttatta gggatcccga agggaaagga 1555 caacatactc tcttttgttc tctgtttttt tttctttaaa caatcataag tttttaaagg 1615 taggggctat gtttttgtat tctttcaaca aatatttatt tgttgcaagg tgctcttagg 1675 ggttgggagc acagtgtgag caaagcagat atggtccttg ccctcagagg ttagactgtg 1735 gt:ggattatg tccatttgtc tttgagtttc tcctttagcc ctatccttag ccttatcaag 1795 aaccttctca atatgtgttt ggtaagtgtt tagaatagtt tcttcctttc cagtccagtc 1855 tcataatata tgtttcttcc aatgatctat taatactatt tgtgaacaac caacacaaaa 1915 gtaaatgaaa gcttaattga cataagtgca gcccttccat tgatccatgt ccttcttttc 1975 cttcatagtt tagccaagtt ctacctctgc tgtgatgcca tccaagccaa cagcaatgtc 2035 tttctggcta tttttttact cagttgatgt gacatatttt gttctgtttt ttaaaaatgt 2095 tattattggc caggcgtggt ggctcacacc tgtaatccca gcactttggg aggccgaggc 2155 gggcagatca tgaggtcagg agatcgagac catcctggct aacacagtga aaccctgtct 2215 ctacaaaaaa ttagctgggc gtggtggcac gcgcctgtaa tcccagctac ttgggaggct 2275 gaggcaggag aatcacttga acccgggagg cggaggttgc agtgagccaa gattgtgcca 2335 ctrgctctcca gcctgggtga cagagtgaga atctgtctca 2375 <210> 2 <211> 84 <212> PRT

<213> Homo Sapiens <400> 2 Met Lys Glu His Asn Glu Ual Ser Lys Lys Ala Ile Tyr Leu Pro Phe Leu Leu Ala Pro Ual Leu Ile Pro Ala Phe Ser Ala Asp Pro Ual Ser Gly Ser Leu Trp Ile Gln Gly Met Gly Ala Thr Leu Thr Asp Phe Asn Pro Phe Leu Asp Ser Ala Arg Glu Ile Glu Leu Phe Ual Leu Ual Ual Leu Ala Leu Lys Phe Cys Ual Ual Asn Cys Gln Ser Pro Gly Leu Asn Ser Ile Ser Gly <210> 3 <211> 6 <212> PRT
<213> Artificial Sequence <220>
<223> Zsig66 Polypeptide Motifl <400> 3 Trp Ile Gln Gly Met Gly <210> 4 <211> 6 <212> PRT
<213> Artificial Sequence <220>
<223> Zsig66 Polypeptide Motif2 <400> 4 Thr Asp Phe Asn Pro Phe <210> 5 <211> 6 <212> PRT
<213> Artificial Sequence <220>

<223> Zsig66 Polypeptide Motif3 <400> 5 Lys Phe Cys Val Ilal Asn <210> 6 <211> 6 <212> PRT
<213> Artificial Sequence <220>
<223> Zsig66 Polypeptide Motif4 <400> 6 Glu Ile Glu Leu Phe Ual <210> 7 <211> 17 <212> DNA
<213> Artificial Sequence <220>
<223> Degenerate Oligonucleotide for zsig66 Motifl <221> misc_feature <222> (1). .(17) <223> n = A,T,C or G
<400> 7 tygathcarg gnatggg 17 <210> 8 <211> 17 <212> DNA
<213> Artificial Sequence <220>
<223> Degenerate Oligonucleotide for zsig66 Motif2 <221> mist feature <222> (1).x.(17) <223> n = A,T,C or G

<400> 8 acngayttya ayccntt 17 <210> 9 <211> 17 <212> DNA
<213> Artificial Sequence <220>
<223> Degenerate 0ligonucleotide for zsig66 Motif3 <221> mi sc__feature <222> (1). .(17) <223> n = A,T,C or G
<400> 9 aarttytgyg tngtnaa 1~
<210> 10 <211> 17 <212> DNA
<213> Artificial Sequence <220>
<223> Degenerate Oligonucleotide for zsig66 Motif4 <221> misc feature <222> (1).x.(17) <223> n = A,T,C or G
<400> 10 garathgary tnttygt 17 <210> 11 <211> 252 <212> DNA
<213> Artificial Sequence <220>
<223> Degenerate Nucleotide sequence for zsig66 <221> misc feature <222> (1)._..(252) <223> n = A,T,C or G

WO 00/36104 PCTNS99l29669 <400>

atgaargarcayaaygargtnwsnaaraar gcnathtayytnccnttyytnytngcnccn 60 gtnytnathccngcnttywsngcngayccn gtnwsnggnwsnytntggathcarggnatg 120 ggngcnacnytnacngayttyaayccntty ytngaywsngcnmgngarathgarytntty 180 gtnytngtngtnytngcnytnaarttytgy gtngtnaaytgycarwsnccnggnytnaay 240 wsnathwsnggn 252 <210> 12 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer ZC6768 <400> 12 gcaattaacc ctcactaaag ggaac 25 <210> 13 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer ZC20137 <400> 13 ttctcattag aagaataggc 20 <210> 14 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer ZC20136 <400> 14 gaataagtca ctccacaatg 20 <210> 15 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer ZC20132 <400> 15 tagagaatgg caaatgcagt c 21 <210> 16 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer ZC20134 <400> 16 caaggtgctc ttaggggttg 20 <210> 17 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer ZC19964 <400> 17 ggactgaact caatctctgg 20 <210> 18 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer ZC19965 <400> 18 tagtattaat agatcattgg 20 <210> 19 <211> 20 <212> DNA
<213> Artificial Sequence <220>

<223> 0liganucleotide primer ZC694 <400> 19 taatacgact cactataggg 20 <210> 20 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Oliganucleotide primer ZC21336 <400> 20 cagagattga gttcagtcca ggac 24 <210> 21 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer ZC21338 <400> 21 ctt:ccttcta gccccagtgc tga 23 <210> 22 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer ZC21450 <400> 22 ggggtttgtg attttctg 18 <210> 23 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer ZC21451 <400> 23 tttgccattc tctaactg 18 <210>24 <211>271 <212>DNA

<213>Homo sapiens <400>

tctctttcctccatgaaagaacacaatgaagtgagtaaaaaagccatttatttgcccttc 60 cttctagccccagtgctgattccagccttcagtgctgatccagtttctggatcactgtgg 120 atacagggtatgggggccactctaaccgactttaatcccttcctggattcagctcgtgaa 180 atagaattgtttgttttagttgtattggctttgaagttttgtgtagtgaattgtcagagt 240 cctggactgaactcaatctctggttaattct 271

Claims (22)

What is claimed is:

1. An isolated polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser);

(b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser);
(c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 84 (Gly);
(d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID NO:2, wherein the amino acid percent identity is determined using a FASTA
program with ktup=1, gap opening penalty=10, gap extension penalty=1, and substitution matrix=BLOSUM62, with other parameters set as default.

2. An isolated polynucleotide according to claim 1, wherein the polynucleotide is selected from the group consisting of:
(a) a polynucleotide sequence as shown in SEQ ID NO:1 from nucleotide 431 to nucleotide 472;
(b) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 425 to nucleotide 472;
(c) a polynucleotide sequence as shown in SEQ ID NO:1 from nucleotide 425 to nucleotide 475;
(d) a polynucleotide sequence as shown in SEQ ID NO:1 from nucleotide 305 to nucleotide 475;
(e) a polynucleotide sequence as shown in SEQ ID NO:1 from nucleotide 224 to nucleotide 475; and

3. An isolated polynucleotide sequence according to claim 1, wherein the polynucleotide comprises nucleotide 1 to nucleotide 252 of SEQ ID NO:11.

4. An isolated polynucleotide according to claim 1, wherein the polynucleotide encodes a polypeptide that comprises a sequence of amino acid residues selected from the group consisting of:

(a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser);
(b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser);
(c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 84 (Gly);
(d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID NO:2.

5. The isolated polynucleotide molecule of claim 1, wherein the polynucleotide encodes a polypeptide that contains motifs 1through 4 spaced apart from N-terminus to C-terminus in a configuration Ala28-[7]-M1-{3}-M2-{5}-M3-{6}-M4-{10}-Gly84, wherein M1 is "motif 1," a sequence of amino acids as shown in amino acids 36 to 41 of SEQ ID NO:2, M2 is "motif 2," a sequence of amino acids as shown in amino acids 45 to 50 of SEQ ID NO:2, M3 is "motif 3," a sequence of amino acids as shown in amino acids 56 to 61 of SEQ ID NO:2, M4 is "motif 4," a sequence of amino acids as shown in amino acids 68 to 73 of SEQ ID NO:2, and Ala28 is the Alanine residue at amino acid number 28 in SEQ ID NO:2 Gly84 is the Glycine residue at amino acid number 84 in SEQ ID NO;2, and {#} denotes the number of amino aids between the motifs;

6. An expression vector comprising the following operably linked elements;
a transcription promotor;
a DNA segment encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 8~ (Gly);
and a transcription terminator.

7. An expression vector according to claim 6, further comprising a secretory signal sequence operably linked to the DNA segment.

8. A cultured cell into which has been introduced an expression vector according to claim 6, wherein the cell expresses a polypeptide encoded by the DNA segment.

9. A DNA construct encoding a fusion protein, the DNA construct comprising:
a first DNA segment encoding a polypeptide comprising a sequence of amino acid residues selected firm the group consisting of;
(a) the amino acid sequence of SEQ ID NO: 2 from residue number 28 (Ala) to amino acid number 84 (Gly); and at least one other DNA segment encoding an additional polypeptide, wherein the first and other DNA segments are connected in-frame;
and encode the fusion protein.

10. A fusion protein produced by a method comprising:
culturing a host cell into which has been introduced a vector comprising the following operably linked elements:
(a) a transcriptional promoter;
(b) a DNA construct encoding a fusion protein according to claim 9; and (c) a transcriptional terminator; and recovering the protein encoded by the DNA segment.

11, An isolated polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser);
(b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser);
(c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number G9 (Phe) to amino acid number 84 (Gly);
(d) the amino acid sequence as shown in SEQ ID NO;2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown is SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID NO;2, wherein the amino acid percent identity is determined using a FASTA
program with ktup=1, gap opening penalty=10, gap extension penalty-1, and substitution matrix=BLOSUM62, with other parameters set as default.

12. An isolated polypeptide according to claim 11,wherein the polypeptide further comprises motifs 1 through 4 spaced apart from N-terminus to C-terminus in a configuration Ala28-(7}-M1-{3}-M2-{5}-M3-{6}-M4-{10}- Gly84, wherein M1 is "motif 2," a sequence of amino acids as shown in amino acids 36 to 41 of SEQ ID NO:2, M2 is "motif 2," a sequence of amino acids as shown in amino acids 45 to 50 of SEQ ID NO:2, M3 is "motif 3," a sequence of amino acids as shown in amino acids 56 to 61 of SEQ ID NO:2, M4 is "motif 4," a sequence of amino acids as shown in amino acids 68 to 73 of SEQ ID NO:2, and Ala28 is the Alanine residue at amino acid number 28 in SEQ ID NO:2, Gly84 is the Glycine residue at amino acid number 84 in SEQ ID NO:2, and (#y) denotes the number of amino acids between the motifs.

13, An isolated polypeptide according to claim 11, wherein the polypeptide comprises a sequence of amino acid residues selected from the group consisting of;
(a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser);
(b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser);
(c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 84 (Gly);
(d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID NO:2.

14. An isolated polypeptide according to claim 11, wherein the polypeptide further comprises an amidated C-terminal Serine residue.

15. A method of producing a polypeptide comprising:
culturing a cell according to claim 8; and isolating the polypeptide produced by the cell.

16. A method of detecting, in a test sample, the presence of a modulator of activity of a protein encoded by amino acid 28 (Ala) to amino acid number 84 (Gly) of SEQ
ID NO:2 , comprising;
transfecting a cell responsive to the protein, with a reporter gene construct that is responsive to a cellular pathway stimulated by the protein; and producing a polypeptide by the method of claim 15; and adding the polypeptide to the cell, in the presence and absence of a test sample; and comparing levels of response to the polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and determining from the comparison, the presence of the modulator of the activity of tho protein in the test sample.

17. A method of producing an antibody to a polypeptide comprising the following steps in order:
inoculating an animal with a polypeptide selected from the group consisting of:
(a) a polypeptide consisting of 9 to 57 amino acids, wherein the polypeptide has a contiguous sequence of amino acids as shown within SEQ ID
NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly);
(b) a polypeptide consisting of the amino acid sequence of SEQ ID NO:2 from residue number 28 (Ala) to amino acid number 84 (Gly);
(c) a polypeptide according to claim 11, 12 or 13;
(d) a polypeptide consisting of amino acid number 1 (Met) to amino acid number 6 (Glu) of SEQ ID NO:2;
(e) a polypeptide consisting of amino acid number 7 (Val) to amino acid number 12 (Ile) of SEQ ID NO:2;
(f) a polypeptide consisting of amino acid number 26 (Ser) to amino acid number 32 (Ser) of SEQ ID NO:2;
(g) a polypeptide consisting of amino acid number 29 (Asp) to amino acid number 34 (Ser) of SEQ ID NO;2; and (h) a polypeptide consisting of amino acid number 51 (Leu) to amino acid number 56 (Glu) of SEQ ID NO;2; and wherein the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal.

18. An antibody produced by the method of claim 17, which binds to a polypeptide of SEQ ID NO;2.

19. The antibody of claim 18, whey tho antibody is a monoclonal antibody.

20. An antibody which specifically binds to a polypeptide of claim 11, 12 or 13.

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

22. A pharmaceutical composition comprising an isolated polypeptide of claim l1, 12 or 13, wherein the polypeptide is in combination with a pharmaceutically acceptable vehicle.