MXPA01007332A - Zsig67:a member of the human secretin-glucagon-vip hormone family - Google Patents

Abstract

The secretin-glucagon-vasoactive intestinal peptide (VIP) family includes polypeptidic hormones that are crucial regulators of pancreatic, biliary, and gastrointestinal physiology. Byvirtue of their important biological functions, these polypeptides have been developed as therapeutics and as diagnostic tools. Zsig67 is a new member of the human secretin-glucagon-VIP family.

Description

ZSIG67 MEMBER OF THE HUMAN HORMONE FAMILY OF SECRETINA-GLUCAGON-VASOACTIVE INTESTINAL PEPTIDE TECHNICAL FIELD The present invention relates generally to a new polypeptide having diagnostic and therapeutic uses. In particular, the present invention relates to a new polypeptide, designated as "Zsig67", and to nucleic acid molecules encoding Zsig67.

BACKGROUND OF THE INVENTION Cellular differentiation of multicellular organisms is controlled by hormones and polypeptide growth factors. These diffusible molecules allow cells to communicate with each other and act in concert to form tissues and organs and repair and regenerate damaged tissue. Examples of hormones and growth factors include the steroid hormones, the hormone for t-irides, the follicle-stimulating hormone, the interferons, the Ref: 131873 in erleucinas, the growth factor derived from platelets, the epidermal growth factor, and the stimulating factor of the colonies anuí oci to-ma eró phage, among others. Hormones and growth factors influence cellular metabolism by agglutinating receptor proteins. Certain receptors are integral membrane proteins that are linked to the hormone or growth factor, outside the cell, and that bind to the signaling pathways within the cell, such as the second messenger systems. Other classes of receptors are soluble intracellular molecules. Of special interest are the pancreatic hormones, which are elements of the vasoactive intestinal glutagon-secrete (VIP) secretin family, which are used for therapy and diagnosis (Geoghegan and Pappas, Ann.Surgery 225: 145 (1997). ), Ulrich et al., Gastroenterol 114: 382 (1998), Walsh, "Hormones of Therapeutic Interest", in Biopharmaceuticals: Biochemistry and Biotechnology, pages 256-292 (John Wiley &Sons 1998)). Secretin, for example, is used as an adjunct to the radiological assessment of inflammatory conditions of the pancreas, to diagnose Zollinger-Ellison syndrome, and to treat intrahepatic cholestasis. Glucagon is used in the emergency treatment of insulin-induced hypoglycemia in diabetic patients, and to reduce duodenal contractility and pilrospasms during upper gastrointestinal endoscopy. Although new uses of known members of this family may be discovered, there is a need for the provision of new polypeptide hormones for biopharmaceuticals.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a new polypeptide, designated "Zsig67". The present invention also provides Zsig67 variant polypeptides and Zsig67 fusion proteins, as well as nucleic acid molecules that encode such polypeptides and proteins, and methods for using these nucleic acid molecules and amino acid sequences.

DESCRIPTION OF THE INVENTION 1. The present invention provides nucleic acid molecules that encode a new polypeptide that is a family member of the secretin-glucagon-VIP hormone. An exemplary nucleic acid molecule containing a sequence encoding the polypeptide designated "Zsig67" has the nucleotide sequence of SEQ ID NO: 1. The encoded polypeptide has the following amino acid sequence: MRTPNTSFLV LASQPLLVLI SLSALILASY SSPLLTRVSL ETVRTKEDGR HNDFNKIKDK DASRAGRERG YRNFLFHFHL SLFPHNSPNS ISKGFKFHVS YRKK (SEQ ID NO: 2). Thus, the Zsi g67 gene described herein encodes a 104 amino acid polypeptide. The Zsig67 protein has a number of structural features, including a signal sequence located at amino acid residues 1 to 28 of SEQ ID NO: 2. The protein also contains the following N-terminal cleavage site which is a portion of a element of the hormone secretin-glucagon-VIP: RH [SAN] D, wherein the amino acids acceptable for a given position are indicated within the square parentheses (amino acid residues 50 to 53 of SEQ ID NO: 2). Cleavage at this site could produce a polypeptide having Asn52 at the N-terminus. There is also a potential di-basic peptide cleavage site in Lysl04. Cleavage at this site could remove amino acid residues 102-104, and Tyrl04 could be converted to an amide. Consequently, Zsig67 can occur with a C-term subjected to amidation. Compositions with other elements of the secretin-glucagon-VIP peptide hormone family indicate that a polypeptide consisting of amino acid residues 52 to 85 of SEQ ID NO: 2 can effectively bind to the Zsig67 congnato receptor. Additional Zsig67 peptides can be generated by cleavage from a variety of monobasic sites or by cleavage similar to furin. The cleaved products include the following amino acid sequences of SEQ ID NO: 2: residues 29 to 45 amino acids, residues 29 to 48 amino acid residues, 29 to 48 amino acid residues subjected to amidation, amino acid residues 29 to 50, residues from amino acids 29 to 55, amino acid residues 29 to 63, amino acid residues 29 to 68, amino acid residues 51 to 63, amino acid residues 51 to 65 subjected to amidation, amino acid residues 51 to 67, amino acid residues 51 to 92 , amino acid residues 51 to 104, amino acid residues 68 to 92, amino acid residues 68 to 101, amino acid residues 68 to 104, amino acid residues 70 to 101, amino acid residues 73 to 92, amino acid residues 73 to 101, and amino acid residues 73 to 104. The Zsig67 gene resides on chromosome 8q24. As discussed below, this region is associated with numerous conditions and alterations. Studies were performed in which Zsig67 nucleic acid molecules were amplified from various human tissues using the polymerase chain reaction. The results indicate that the Zsig67 mRNA is present at high levels in the following tissues: pituitary, prostate, adrenal gland, uterus, liver, thyroid and lymphoid knot. In addition, the Zsig67 mRNA appears to be present, albeit at a lower level, in the testes, skeletal muscle and spleens, while in the kidney, placenta, pancreas and spinal cord they still seem to contain lower levels of Zsig67 mRNA. Accordingly, the nucleic acid molecules encoding Zsig67 can be used to differentiate between various tissues. In addition, the observation that Zsig67 is produced in numerous tissues including the pituitary, liver, prostate and thyroid, indicate that the protein can be used as a release hormone to regulate metabolic homeostasis or contractility in a variety of tissues. As described herein, the present invention provides isolated polypeptides having an amino acid sequence that is at least 70%, at least 80% or at least 90% identical to the amino acid sequence of SEQ ID NO: 2, wherein such isolated polypeptides can specifically bind to an antibody that specifically binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. An exemplary polypeptide is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 Additional exemplary polypeptides include the following: (a) a polypeptide comprising the amino acid sequence of amino acid residues 29 to 45 of SEQ ID NO: 2, (b) a polypeptide comprising the sequence of amino acid residues 51 to 63 of SEQ ID NO: 2, (c) a polypeptide comprising the amino acid sequence of residues 52 to 101 of SEQ ID NO: 2, '(d) a polypeptide comprising the sequence amino acid residue of amino acid residues 68 to 92 of SEQ ID NO: 2, (e) a polypeptide comprising the amino acid sequence of amino acid residues 70 to 101 of SEQ ID NO: 2, and (f) a polypeptide comprising the amino acid sequence of amino acid residues 73 to 92 of SEQ ID NO: 2. Illustrative polypeptides also include: (a) a polypeptide comprising the amino acid sequence of amino acid residues 29 to 48 of SEQ ID NO: 2, (b) a polypeptide comprising the amino acid sequence of amino acid residues 29 to 48 of SEQ ID NO: 2, wherein the last amino acid residue of the aforementioned sequence is subjected to amidation, (c) a polypeptide comprising the amino acid sequence of amino acid residues 29 to 50 of SEQ ID NO. : 2, (d) a polypeptide comprising the amino acid sequence of amino acid residues 29 to 55 of SEQ ID NO: 2, (e) a polypeptide comprising the amino acid sequence of amino acid residues 29 to 63 of SEQ ID NO: 2, (f) a polypeptide comprising the amino acid sequence of amino acid residues 29 to 68 of SEQ ID NO: 2, (g) a polypeptide comprising the amino acid sequence of the amino acid residues 29 to 101 of SEQ ID NO: 2, wherein the last amino acid residue of the mentioned sequence is subjected to amidation, and (h) a polypeptide comprising the amino acid sequence of amino acid residues 29 to 104 of SEQ ID NO: 2. The additional exemplary polypeptides include: (a) a polypeptide comprising the amino acid sequence of amino acid residues 51 to 65 of SEQ ID NO: 2, wherein the last amino acid residue of the mentioned sequence is subjected to amidation, (b) a polypeptide comprising the amino acid sequence of amino acid residues 51 to 67 of SEQ ID NO: 2, (c) a polypeptide comprising the amino acid sequence of amino acid residues 51 to 92 of SEQ ID NO: 2, ( d) a polypeptide comprising the amino acid sequence of amino acid residues 51 to 104 of SEQ ID NO: 2, (e) a polypeptide comprising the amino acid sequence of amino acid residues 52 to 101 of SEQ ID NO: 2, where the last The amino acid residue of the mentioned sequence is subjected to amidation, (f) a polypeptide comprising the amino acid sequence of amino acid residues 52 to 104 of SEQ ID NO: 2, (g) a polypeptide comprising the sequence of amino acid of amino acid residues 68 to 101 of SEQ ID NO: 2, (h) a polypeptide comprising the amino acid sequence of amino acid residues 68 to 104 of SEQ ID NO: 2, (i) a polypeptide that comprises the amino acid sequence of amino acid residues 73 to 101 of SEQ ID NO: 2; and (j) a polypeptide comprising the amino acid sequence of amino acid residues 73 to 104 of SEQ ID NO: 2. The present invention also provides isolated polypeptides comprising an amino acid sequence that is at least 70%, at least 80%, or at least 90% identical to the amino acid sequence of amino acid residues 52 to 85 of SEQ ID NO: 2, wherein such isolated polypeptides can specifically bind to an antibody that specifically binds to a polypeptide consisting of amino acid residues 52 to 85 of SEQ ID NO: 2. The present invention also includes isolated polypeptides comprising at least 15 , or at least 30 contiguous amino acid residues of an amino acid sequence of SEQ ID NO: 2, selected from the group consisting of: (a) amino acid residues 29 to 104, (b) amino acid residues 52 to 104, (c) amino acid residues 52 to 85, and (d) amino acid residues 68 to 104 and (e) amino acid residues 73 to 104. Illustrative polypeptides include polypeptides that either comprise or consist of e, amino acid residues (a) to (e). The present invention also provides antibodies and antibody fragments that specifically bind to such polypeptides. Exemplary antibodies include polyclonal antibodies, murine monoclonal antibodies, humanized antibodies derived from murine monoclonal antibodies, and human monoclonal antibodies. Exemplary antibody fragments include F (ab ') 2, F (ab) 2, Fab', Fab, Fv, scFv, and minimal recognition units. The present invention further includes compositions comprising a carrier and a peptide, polypeptide or antibody described herein. The present invention also provides isolated nucleic acid molecules encoding a Zsig67 polypeptide, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule having the nucleotide sequence of SEQ ID NO. : 3, (b) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 2, and (c) a nucleic acid molecule that remains hybridized after stringent washing conditions to an acid molecule nucleic acid sequence having the nucleotide sequence of nucleotides 111-422 of SEQ ID NO: 1, or the complement of nucleotides 111-422 of SEQ ID NO: 1. Exemplary nucleic acid molecules include those in which any difference between the amino acid sequence encoded by the nucleic acid molecule and the corresponding amino acid sequence of SEQ ID NO: 2 is due to a conservative substitution of amino oacid The present invention further contemplates isolated nucleic acid molecules comprising a nucleotide sequence of nucleotides 195-422 of SEQ ID NO: 1. The present invention also includes vectors and expression vectors comprising such nucleic acid molecules. Such expression vectors may comprise a transcription promoter, and a transcription terminator, wherein the promoter is operably linked to the nucleic acid molecule, and wherein the nucleic acid molecule is operably linked to the transcription terminator. . The present invention also includes recombinant host cells comprising these vectors and expression vectors. Exemplary host cells include bacterial, fungal, fungal, insect, mammalian and plant cells. Recombinant host cells comprising such expression vectors can be used to produce Zsig67 polypeptides by culturing such recombinant host cells comprising the expression vector and producing the Zsig67 protein and optionally, isolation of the Zsig67 protein from the recombinant host cells cultured. The present invention also contemplates methods for detecting the presence of Zsig? RNA in a biological sample, comprising the steps of (a) contacting a Zsig67 nucleic acid probe under hybridization conditions with either (i) molecules of test RNA isolated from the biological sample, or (ii) nucleic acid molecules synthesized from the isolated RNA molecules, wherein the probe has a nucleotide sequence comprising a portion of the nucleotide sequence of the nucleotides 111-422 of SEQ ID NO: 1, or its complement, and (b) detect the hybrid formation of the nucleic acid probe and either the test RNA molecules or the synthesized nucleic acid molecules, wherein the presence of hybrids indicates the presence of Zsig67 of RNA in the biological sample. As an illustration, the biological sample can be a human biological sample, such as a biopsy or autopsy specimen. The present invention further provides methods for detecting the presence of the Zsig67 polypeptide in a biological sample, comprising the steps of: (a) contacting the biological sample with an antibody or an antibody fragment that specifically binds to a polypeptide consisting of of the amino acid sequence of SEQ ID NO: 2, wherein the contact is made under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibodies or linked antibody fragments. Such antibodies or antibody fragments may further comprise a detectable label selected from the group consisting of radioisotope, fluorescent label, chemiluminescent label, enzyme label, bioluminescent label, and colloidal gold. An exemplary biological sample is a human biological sample, such as a biopsy or autopsy specimen. The present invention also provides equipment for performing these detection methods. For example, a kit for detecting the expression of the Zsig67 gene may comprise a container comprising a nucleic acid molecule, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of nucleotides 111-422 of SEQ ID NO: 1, (b) a nucleic acid molecule comprising the complement of nucleotides 111-422 of the nucleotide sequence of SEQ ID NO: 1, ( c) a nucleic acid molecule that is a fragment of (a) consisting of at least eight nucleotides, and (d) a nucleic acid molecule that is a fragment of (b) consisting of at least eight nucleotides. Such a device may comprise a second container comprising one or more reagents capable of indicating the presence of the nucleic acid molecule. On the other hand, a kit for the detection of the Zsig67 protein may comprise a container comprising an antibody, or an antibody fragment, that specifically binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. The present invention also contemplates anti-idiotype antibodies, or anti-idiotype antibody fragments, that specifically bind to an antibody or antibody fragment that specifically binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 In addition, the present invention provides fusion proteins comprising a portion of Zsig67 polypeptide, and an immunoglobulin portion, such as an immunoglobulin heavy chain constant region. Portions of illustrative Zsig67 polypeptides include: (a) a polypeptide consisting of the amino acid sequence of amino acid residues 1 to 101 of SEQ ID NO: 2, (b) a polypeptide consisting of the amino acid sequence of the amino acid residues 1 to 104 of SEQ ID NO: 2, (c) a polypeptide consisting of the amino acid sequence of amino acid residues 29 to 104 of SEQ ID NO: 2, (d) a polypeptide consisting of the amino acid sequence of amino acid residues 29 to 101 of SEQ ID NO: 2, (e) a polypeptide consisting of the amino acid sequence of amino acid residues 52 to 104 of SEQ ID NO: 2, (f) ) a polypeptide consisting of the amino acid sequence of amino acid residues 52 to 101 of SEQ ID NO: 2, and (g) a polypeptide consisting of the amino acid sequence of amino acid residues 52 to 85 of the SEC ID NO: 2. Suitable additional polypeptides are described herein. These and other aspects of the invention will become apparent upon reference to the following detailed description. 2 ,. Definitions In the following description, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the invention. As used herein, "nucleic acid", or "nucleic acid molecule" refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR) ), and fragments generated by any ligation, excision, endonuclease action and exonuclease action. The nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (e.g., α-enantiomeric forms of nucleotides that originate naturally), or a combination of both. The modified nucleotides may have alterations in their sugar portions and / or in the pyrimidine or purine base portions. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or the sugars can be functionalized as ethers or esters. However, the whole sugar portion can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogues. Examples of modifications in a base portion include alkylated purines and pyrimidines, purines or acylated pyrimidines, or other well-known heterocyclic substitutes. The nucleic acid monomers can be linked by phosphodiester linkages or analogues of such linkages. Link analogs. phosphodiester include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphorus anilidate, phosphoramidate and the like. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids", which comprise modified or naturally occurring nucleic acid bases bound to a polyamide structure. The nucleic acids can be either single-stranded or double-stranded. The term "complement of a nucleic acid molecule" refers to a nucleic acid molecule having a complementary nucleotide sequence and reverse orientation compared to a reference nucleotide sequence. For example, the 5 'sequence ATGCACGGG 3' is complementary to 5 ' CCCGTGCAT3 '. The term "contig" or "contiguous" denotes a nucleic acid molecule having a contiguous portion of sequence identical or complementary to another nucleic acid molecule. The contiguous sequences are said to "overlap" to a given sequence portion of the sequence of a nucleic acid molecule either in its entirety or along a partial portion of the nucleic acid molecule. The term "degenerate nucleotide sequence" denotes a nucleotide sequence that includes one or more degenerate codons when compared to a reference nucleic acid molecule encoding a polypeptide. Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residues (ie, the GAU and GAC triplets each encode Asp). The term "structural gene" refers to a nucleic acid molecule, which is transcribed into a messenger RNA (mRNA), which is then translated into an amino acid sequence characteristic of a specific polypeptide. An "isolated nucleic acid molecule" is a nucleic acid molecule that does not integrate into the genomic DNA of an organism. For example, a nucleic acid molecule encoding a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule. Another example of an "isolated nucleic acid molecule, is a chemically synthesized nucleic acid molecule that is not integrated into the genome of an organism." A nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome of that species A "nucleic acid molecule construct" is a nucleic acid molecule, either single strand or double strand, which has been modified through a human invention to contain several segments of nucleic acid combined and juxtaposed in an arrangement that does not exist in nature. "Linear DNA" denotes non-circular DNA molecules, which have free 5 'and 3' ends. Linear DNA can be prepared from closed circular DNA molecules, such as plasmids, by enzymatic digestion or physical disruption.

"Complementary DNA (cDNA)" is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a complementary primer is used for the mRNA portions, for the initiation of reverse transcription. Those skilled in the art can also use the term "cDNA" to refer to a double-stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand. The term "cDNA" also refers to a clone of a cDNA molecule, synthesized from an RNA template. A "promoter" is a sequence of nucleotides that directs the transcription of a structural gene. Typically, a promoter is located in the 5 'non-coding region of a gene, proximal to the transcriptional initiation site of a structural gene. The elements of the sequence within the promoters that function in the initiation of transcription are frequently characterized by consensual nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, CATT sequences, specific elements of differentiation (DSEs, McGehee et al., Mol.

Endocrinol, 7: 551 (1993)), cyclic AMP response elements (CREs), serum response elements (SREs, Treisman, Seminars in Cancer Biol. 1:47 (1990)), glucocorticoid response elements (GREs) ), and binding sites for other transcription factors, such as CRE / ATF (O'Relly et al., J. Biol. Chem. 257; 19938 (1992)) AP2 (Ye et al., J. Biol. Chem. 269: 25128 (1994)), SPI, protein binding element of. cAMP response (CREB; Loeken, Gene Expr., 3: 253 (1993)) and octamer factors (see in general, Watson et al., eds., - Molecular Biology of the Gene, 4th ed. (The Ben j amin / Cummings Publishing Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J. 303: 1 (1994)). If a promoter is a different inducible promoter, then, the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter. The repressible promoters are also known. A "core promoter" contains the nucleotide sequences essential for the function of the promoter, including the TATA box and the start of transcription. By this definition, a core promoter may or may not have detectable activity in the absence of specific sequences that enhance activity or confer tissue-specific activity. A "regulatory element" is a sequence of nucleotides that modulates the activity of a core promoter. For example, a regulatory element may contain a nucleotide sequence that binds to cellular factors, allowing exclusively or preferentially the transcription in particular cells, tissues or organelles. These types of regulatory elements are normally associated with genes that are expressed in a "cell-specific", "tissue-specific" or "organelle-specific" manner.

An "enhancer" is a type of regulatory element that increases the efficiency of the transcription, with respect to the distance or orientation of the improver in relation to the initial site of the transcription. "Heterologous DNA" refers to a DNA molecule or a population of DNA molecules, which np naturally exist within a given host cell. The heterologous DNA molecules for a particular host cell type contain DNA derived from the host cell species (ie, endogenous DNA) to the extent that host DNA is combined with non-host DNA (ie, exogenous DNA). . For example, a DNA molecule containing a non-host DNA segment, which encodes a polypeptide operably linked to a host DNA segment comprising a transcription promoter, is considered to be a heterologous DNA molecule. On the other hand, a heterologous DNA molecule can comprise an endogenous gene operably linked to an exogenous promoter. As another illustration, a DNA molecule comprising a gene derived from a wild type cells is considered to be heterologous DNA if that DNA molecule is introduced into a mutant cell lacking the wild-type gene. A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides". A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptide components, such as carbohydrate groups. Carbohydrates and other nonpeptide substituents can be added to a protein by the cell in which the protein is produced, and will vary with the cell type. Proteins are defined here in terms of their basic amino acid structures; Substituents such as carbohydrate groups are generally unspecified, but nevertheless may be present.

A peptide or polypeptide encoded by a non-host DNA molecule is a peptide or "heterologous" polypeptide. An "integrated genetic element" is a segment of DNA that has been incorporated into a chromosome of a host cell after the element enters the cell through human manipulation. Within the present invention, the integrated genetic elements are most commonly derived from linearized plasmids that are introduced into the cells by electroporation or other techniques. The integrated genetic elements are passed through the original host cells to their progeny. A "cloning vector" is a nucleic acid molecule, such as a plasmid, cosmid, or bacteriophage, that has the ability to replicate autonomously in a host cell. Cloning vectors typically contain one to a small number of restriction endonuclease recognition sites that allow the insertion of a nucleic acid molecule in a certain manner without loss of an essential biological function of the vector, as well as sequences of nucleotides encoding a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. The marker genes typically include genes that provide resistance to tetracycline or resistance to ampicillin. An "expression vector" is a nucleic acid molecule that encodes a gene that is expressed in a host cell. Typically, an expression vector comprises a transcription promoter, a gene, and a rascription terminator. The expression of the gene is usually placed under the control of a promoter, and the gene is said to be "operably linked" to the promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.

A "recombinant host" is a cell that contains a nucleic acid molecule, such as a cloning vector or an expression vector. In the present context, an example of a recombinant host is a cell that produces Zsig67 from an expression vector. In contrast, Zsig67 can be produced by a cell that is a "natural source" of Zsig67, and that lacks a vector of express ion. "Integrative transformants" are recombinant host cells, in which the heterologous DNA has been integrated into the genomic DNA of the host cells. A "fusion protein" is a hybrid protein, expressed by a nucleic acid molecule comprising the nucleotide sequences of at least two genes. For example, a fusion protein may comprise at least part of a Zsig67 polypeptide, fused to a polypeptide that binds an affinity matrix. Such a fusion protein provides a means to isolate large amounts of Zsig67 using affinity chromatography. The term "receptor" denotes a protein associated with the cell that binds to a bioactive molecule called a "ligand". This interaction mediates the effect of the ligand on the cell. The receptors can be linked to the membrane, cytosolic or nuclear; monomeric (eg, thyroid-stimulating hormone receptor, beta-adrenergic receptor) or multimeric receptor (eg, the PDGF receptor, the growth hormone receptor, the IL-3 receptor, the GM-CSF receptor, the G-CSF receptor, the erythropoietin receptor and the IL-6 receptor). The membrane-bound receptors are characterized by a muI t i -domain structure comprising an extracellular ligand and an intracellular effector domain that is typically involved in signal transduction. In certain membrane-bound receptors, the extracellular binding domain of the ligand and the intracellular domain of the effector are located in separate polypeptides comprising the complete functional receptor. In general the binding of the ligand to a receptor, results in a conformational change in the receptor, which originates an interaction between the domain of the effector and another (s) molecule (s) in the cell, which in turn leads to an alteration in the cell's metabolism. Metabolic events that frequently link to receptor interactions include gene transcription, phosphorylation, dephosphorylation, increased cyclic AMP production, cellular calcium mobilization, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and the hydrolysis of phospholipids. The term "secretory signal sequence" denotes a DNA sequence encoding a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide directs the larger polypeptide through a secretory pathway of a cell in the which is synthesized. The larger peptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway. An "isolated polypeptide" is a polypeptide that is essentially free of contaminating cellular components, such as carbohydrates, lipids or other proteinaceous impurities associated with the polypeptide in nature. Typically, an isolated polypeptide preparation contains the polypeptide in a highly purified form, ie, at least about 80% pure, at least about 90% pure, at least about 95% pure, or more than 95% pure, or more than 99% pure. One way to show that a particular protein preparation contains an isolated polypeptide, is by the appearance of a single band, following the electrophoresis of sodium dodecyl sulfate (SDS) -polyacrylamide gel from the protein preparation and the dyeing with Brilliant Blue of Coomassie from the gel. However, the term "isolated" does not exclude the presence of some polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms. The terms "amino-terminal" and "carboxyl-terminal" are used herein to denote positions within the 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 carboxyl-terminal sequence positioned to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but not necessarily to the carboxyl terminus of the complete polypeptide. The term "expression" refers to the biosynthesis of a genetic product. For example, in the case of a structural gene, the expression involves the transcription of the structural gene in an mRNA and the translation of the mRNA into one or more polypeptides. The term "splice variant" is used herein to denote the alternative forms of RNA transcribed from a gene. Splice variation is generated naturally through the use of alternative splice sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several transcribed mRNAs of the same gene. The splice variants can encode the polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splicing variant of a mRNA transcribed from a gene. As used herein, the term "immunomodulator" includes cytosines, stem cell growth factors, 1 in f or toxins, co-e s t imulant molecules, hematopoietic factors, and synthetic analogs of these molecules. Examples of immunomodulators include tumor necrosis factor, interleukins (eg, interleukin-1, (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7. , IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, and IL-15), stimulation factors of the colony (e.g., granulocyte colony stimulating factor and stimulation factor of the macrophage colony of the annulocy to), interferons (for example, int er f eronas, -a, -ß- -?, -e, and t), the factor of growth of stem cells is designated as "Sl factor", erythropoietin and thrombopoytetine The term "complete pair / ani-complement" denotes non-identical portions that form a stable pair, associated, non-covalently, under appropriate conditions. For example, biotin and avidin (or streptavidin) are prototypical members of a complement / anti-complement pair. Other complement / anti-complement pairs include receptor / ligand pairs, antibody / antigen pairs (or incomplete antigen or epitope), pairs of antisense / antisense polynucleotides, and the like. Where the subsequent dissociation of the complement / anti-complement pair is desirable, the complement / anti-complement pair preferably has a binding affinity of less than 10 ^ M ~ l.

An "anti-idiotype antibody" is an antibody that binds to the variable region domain of an immunoglobulin. In the present context, an anti-idiotype antibody binds to the variable region of an anti-Zsig67 antibody, thus, an anti-idiotype antibody mimics an epitope of Zsig67. An "antibody fragment" is a portion of an antibody such as F (ab ') 2, F (ab) 2, Fab', Fab and the like. With respect to the structure, an antibody fragment binds to the same antigen that is recognized by the intact antibody. For example, a fragment of the monoclonal anti-Zsig67 antibody binds to an epitope of Zsig67. The term "antibody fragment" also includes a synthetic or engineered polypeptide that binds to a specific antigen, such as polypeptides consisting of the variable region of the light chain, the "Fv" fragments consisting of the variable regions. of the heavy and light chains, the single chain recombinant polypeptide molecules in which the heavy and light variable regions are connected by a peptide linker ("ScFv proteins"), and the minimum recognition units, consisting of the residues of amino acids that mimic the hypervariable region. A "chimeric antibody" is a recombinant protein that contains the variable and complementary domains that determine the regions derived from a mouse antibody, while the rest of the antibody molecule is derived from a human antibody. "Humanized antibodies" are recombinant proteins, in which murine complementarity, which determines the regions of a monoclonal antibody, has been transferred from the light and heavy variable chains of murine immunoglobulin to a variable domain of human. As used herein, a "therapeutic agent" is a molecule or atom which is conjugated to a portion of antibody to produce a conjugate which is used for therapy. Examples of therapeutic agents include drugs, toxins, iminomodulators, chelators, boron compounds, photoactive agents or dyes, and rheostats. A "detectable label" is a molecule or atom which can be conjugated with a portion of the antibody to produce a molecule useful for diagnosis. Examples of detectable labels include chelator formers, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, or other porous markers. The term "affinity tag" is used herein to denote a segment of the polypeptide that can bind to a second polypeptide to provide purification or detection of the second polypeptide or provide sites for binding the second polypeptide to a substrate. Primarily, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. The affinity tags include a tract of po 1 i -hi st idina, 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 (Grus s in eye r et al., Proc. Nati, Acad. Sci. USA 82: 7952-4, 1985), substance P, FLAG peptide (Hopp et al., Biotechnology 5 1204-10, 1988), the peptide binding strepta idina, or another antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107, 1991. Affinity tags encoding nucleic acid molecules are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ ). A "naked antibody" is a complete antibody, when it opposes an antibody fragment which is not conjugated with a therapeutic agent. Naked antibodies include both polyclonal and monoclonal antibodies, as well as certain recombinant antibodies, such as chimeric and humanized antibodies.

As used herein, the term "antibody component" includes both a whole antibody and an antibody fragment. A "immunoconjugate" is a conjugate of an antibody component with a therapeutic agent or a detectable label. As used herein, the term "antibody fusion protein" refers to a recombinant molecule that comprises an antibody component and a therapeutic agent. Examples of suitable therapeutic agents for such fusion proteins include immunomodulators ("anti- body-immunomodulatory fusion protein") and toxins ("antibody-toxin fusion protein"). An "antigen associated with the tumor" is a protein that is not normally expressed, or is expressed at lower levels by a normal counterpart cell. Examples of antigens associated with tumors include 1 f a-fe t op o t e i na, antigen cacinogenic, and Her-2 / neu. Many other illustrations of tumors associated with antigens are known to those skilled in the art). See, for example, Urban et al. , An n. R e v. Imm u n l l. 1 0: 61 1 (1992). An "objective polypeptide" or an "objective peptide" is an amino acid sequence that comprises at least one epitope, and that is expressed in a target cell, such as a tumor cell or a cell carrying an infectious agent antigen. T cells recognize the peptide epitopes presented by a major histocompatibility complex molecule for a target polypeptide or target peptide and typically cause lysis of the target cell or recruit other immune cells to the target cell site, thereby killing the cell objective . An "antigenic peptide" is a peptide, which binds a molecule of the major histocompatibility complex to form an MHC-peptide complex, which is recognized by a T cell, thereby inducing a response of the cytotoxic lymphocytes upon presentation to the T cell. In this way, antigenic peptides are able to bind to a major complex molecule of hyperthyroidism and introduce a cytotoxic response of T cells, such as lysis or release of a specific cytosine against the T cell. target cell, which binds or expresses the antigen. The antigenic peptide can be linked in the context of a molecule of the major histocompatibility complex class I or class II, on a cell that presents the antigen or on a target cell. In eukaryotes, polymerase II of RNA, catalyzes the transcription of a structural gene to produce mRNA. A nucleic acid molecule can be designed to contain an RNA polymerase II template in which the RNA transcript has a sequence that is complementary to that of a specific mRNA. The transcribed RNA is called "antisense RNA" and a nucleic acid molecule that encodes the antisense RNA is called an "antisense gene." Antisense RNA molecules are capable of binding to mRNA molecules, resulting in an inhibition of mRNA translation.

An "antisense oligonucleotide specific for Zsig67" or an "antisense oligonucleotide of Zsig67" is an oligonucleotide having a sequence (a) capable of forming a stable triplet with a portion of the gene sig 6 7, or (b) capable of forming a stable duplex with a portion of a transcript of Z-gene sig 6 7 mRNA. A "ribozyme" is a nucleic acid molecule that contains a catalytic center. The term includes RNA enzymes, Imadas autoempas RNAs, authentic cRNAs, and the nucleic acid molecules that carry out these catalytic functions. A nucleic acid molecule that codes for a ribozyme is called a "ribozyme gene." An "external guide sequence" is a nucleic acid molecule that directs the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, resulting in cleavage of the mRNA by RNase P. A nucleic acid molecule encoding a sequence External guidance is called "external guide sequence gene".

The term "variant Zsig67 gene" refers to nucleic acid molecules that encode a polypeptide having an amino acid sequence that is a modification of SEQ ID NO: 2. Such variants include polymorphisms that occur naturally in the Zsig67 genes, as well as synthetic genes that contain conservative amino acid substitutions of the amino acid sequence of SEQ ID NO: 2. The additional variant forms of the .Zs? g6'7 genes are nucleic acid molecules that contain inserts or deletions of the nucleotide sequences described here. A variant of the gene gsi Zsi, can be identified determined if the gene is hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, or its complement, under severe conditions Alternatively, the genes of Zsig67 variants They can be identified by comparing the sequences. Two amino acid sequences have "100% amino acid sequence identity" if the amino acid residues of the two amino acid sequences are the same when aligned for maximum correspondence. Similarly, two nucleotide sequences have "100% identity of the nucleotide sequence", if the nucleotide residues of the two nucleotide sequences are the same when aligned by their maximum correspondence. Sequence comparisons can be carried out using sets of standard programs, such as those included in the set of LASERGENE or biometric software programs, which are produced by DNASTAR (Madison Wisconsin). Other methods for comparing two nucleotide or amino acid sequences by determining the optimal alignment are well known to those skilled in the art (see, for example, Peruski and Peruski, The Internet and the New Biology: Tools for Genomic and Molecular Research (ASM Press Inc. 1997), Wu et al., (Eds.), "Information Superhighway and Computer Databases of Nucleic Acids and Proteins", in Methods in Gene Biotechnology, pages 123-151 (CRC Press Inc, 1997), and Bishop (ed.), Guide to Human Genome Computing 2nd Edition (Academic Press, Inc. 1998)). The particular methods for determining the identity of the sequences are described below. Regardless of the particular method used to identify a variant Zsig67 gene or a variant Zsig67 polypeptide, a variant gene or polypeptide encoded by a variant gene can be characterized by the ability to specifically bind to an anti-Zsig67 antibody. The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene that occupies the same chromosomal location. Allelic variation becomes natural through a mutation, and can result in genotypic or phenotypic polymorphism within populations. Mutations of the genes can be either silent (without change in the encoded polypeptide) or they can be encoded polypeptides having the altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.

The term "ortholog" denotes a polypeptide or protein obtained from a species that is the functional counterpart of a polypeptide or protein of a different species. The sequence differences between the orthologs are the result of the evolution of the species. "Paralogs" are distinct but structurally related proteins made by an organism. It is believed that paralogs are generated through genetic duplication. For example, α-globin, β-globin, and myoglobin are paralogs ent r e s i. The present invention includes gene fragments Zs i g 6 7. Within the context of this invention, a "functional fragment" of a Zs i g 6 7 gene refers to a nucleic acid molecule that encodes a portion of a Zsig67 polypeptide, which specifically binds to an anti-Zsig67 antibody. As used here, the term "component Zsig67"includes both a Zsig67 polypeptide and a Zsig67 functional fragment.

The term "cytotoxin" refers to a molecule or atom that is capable of causing the death of a cell, or that is capable of inhibiting the growth of a cell. Examples of cytotoxins include drugs, inactive ribosome proteins, immunomodulators, chelators, boron compounds, photoactive agents or dyes, radioisotopes, and the like. A "Zsig67 signaling composition" comprises a Zsig67 component and a cytotoxin. The association between the Zsig67 component and the cytotoxin can be covalent or non-covalent. For example, the association of the cytotoxin of the Zsig67 component in the Zsig67 conjugates and the signaling fusion proteins of Zsig67 is covalent, while the Zsig67 signaling liposomes illustrate a non-covalent lens association. A "Zsig67 conjugate" is a type of signaling composition of Zsig67, which is produced by covalently linking a Zsig67 component and a cytotoxin either directly or via the binding agent.

As used herein, the term "Zsig67 signaling fusion protein" refers to a recombinant molecule comprising a Zsig67 polypeptide component and a cytotoxin. Due to the imprecision of the standard analytical methods, it is understood that the molecular weights of the polymers are approximate values. When such value is expressed as "around" X or "approximately" X, the set value of X will be understood to be exact to ± 10%. 3 . Production of a Gen Zs ± gß l Human Nucleic acid molecules encoding a human Zs ig 67 gene can be obtained by selecting a human cDNA or a genomic library, using polynucleotide probes based on the nucleotide sequence of SEQ ID NO: 1. These techniques are standards and well established. As an illustration, a nucleic acid molecule encoding a human Zs i g 67 gene can be isolated from a human cDNA library. In this case, the first step should be to prepare the cDNA library by isolating RNA from the tissue, such as pancreatic islet cells, using methods well known to those skilled in the art. In general, RNA isolation techniques should provide a method for breaking cells, a means to inhibit RNase-directed degradation of RNA and a method for separating RNA from DNA, protein and polysaccharide contaminants. For example, total RNA can be isolated by freezing the tissue in nitrogen, grinding the frozen tissue with a mortar and pestle to cause lysis of the cells, removing the embossed or milled tissue with a solution of f ool 1 / c 1 form gold, to remove the proteins, and separate the RNA from the remaining impurities by selective precipitation with lithium chloride (see for example, Ausubel et al., (eds), Short Protocols in Molecular Biology, 3rd Edition, pages 4-1 to 4-6 (John Wiley &Sons 1995) ["Ausubel (1995)"]; Wu et al., Methods in Gene Biotechnology, pages 33-41 (CRC Press, Inc., 1997) ["Wu (1997)"]). Alternatively, the total RNA can be isolated from the pancreatic tissue, by extraction of the tissue impregnated with a guanidinium isothiocyanate, extraction with organic solvents, and separation of the RNA from the contaminants using differential centrifugation (see, for example, Chirgwin et al., Biochemistry 18; 52 (1979), Ausubel (1995) on pages 4-1 to 4-6, Wu (1997) on pages 33-41). To construct the cDNA library, the poly (A) + RNA must be isolated from a total RNA preparation. Poly (A) + RNA can be isolated from total RNA, using the standard technique of. Chromatography in 1 i-go (dT) -cellosis (see for example, Aviv and Lender, Proc. Nat'l Acad. Sci. USA 55: 1408 (1972); Ausubel (1995) on pages 4-11. to 4-12). The double-stranded cDNA molecules are synthesized from poly (A) + RNA using techniques well known to those skilled in the art (see, for example, Wu (1997), on pages 41-46). In addition, commercially available equipment can be used to synthesize double-stranded cDNA molecules. For example, such equipment is available from Life Technologies, Inc. (Gaithersburg, MD), CLONTECH Laboratoires, Inc. (Palo Alto, CA), Promega Corporation (Madison, Wl) and STRATAGENE (La Jolla, CA). Several cloning vectors are suitable for the construction of a cDNA library. For example, a cDNA library can be prepared in a vector derived from a bacteriophage, such as a vector. See, for example, Huynh et al., "Constructing and Screening cDNA Librarles in? GtlO and? Gtll," in DNA Cl on i n g: A Pra ct i ca l Appro a ch Vo l. 1, Glover (ed), page 49 (IRL Press 1985); Wu (1997) on pages 47-52. Alternatively, the double-stranded cDNA molecules can be inserted into a plasmid vector, such as the PBLUESCRIPT vector.

(STRATAGENE; La Jolla, CA), a LAMDAGEM-4 (Promega Corp.) or other commercially available vectors. Suitable cloning vectors can be obtained from the American Type Culture Collection (Manassass, VA). To amplify the cloned cDNA molecules, the cDNA library is inserted into a prokaryotic host, using standard techniques. For example, a cDNA library can be introduced into E cells. c or l DH5 competent, which can be obtained, by examples of Life Technologies, Inc. (Gaithherburg, MD). A human genomic library can be prepared by means well known in the art (see, for example, Ausubel (1995) on pages 5-1 to 5-6; Wu (1997) on pages 307-327). Genomic DNA can be isolated by lysing the tissue with the Sarkosyl detergent, digesting the lysate with proteinace K, removing the insoluble waste from the lysate by centrifugation, precipitating the nucleic acid from the lysate using isopropanol, and purifying the resuspended DNA in a gradient of densities of cesium chloride. DNA fragments that are suitable for the production of a genomic library can be obtained by randomly dividing genomic DNA or by partial digestion of genomic DNA with restriction endonucleases. Fragments of genomic DNA can be inserted into a vector, such as a bacteriophage or cosmid vector, in accordance with conventional techniques, such as the use of restriction enzymes to provide the proper term, the use of alkaline phosphatase treatment to avoid unwanted binding of DNA molecules, and ligation with appropriate ligases. The techniques for such manipulation are well known in the art (see, for example, Aus ubel (2995) on pages 5-1 to 5-.6; Wu (1997) on pages 307-327). Nucleic acid molecules encoding a human Zs ig 67 gene can also be obtained, using the polymerase chain reaction (PCR) with oligonucleotide primers having the nucleotide sequences that are based on the nucleotide sequences of the Zsig67 gene human, as described here. General methods for selecting libraries with PCR are provided, for example, by Yu et al., "Use of The Polymerase Chain to Screen Phage Libraries," in Methods i Molecular Biology, Vol. 15: PCR Protocols: Current M thods and Appl ica tions, White (ed.), pages 211-215 (Humana Press, Inc. 1993). In addition, techniques for using PCR to isolate related genes as described, for example, by Preston, "Use of Degenerate Oligonucleotide Primers and the Polymerase Chain Reaction to Clone Gene Family Members," in Methods in Molecular Biology, Vol. 15 : PCR Protocols: Current Methods and Applications, White (ed.), Pages 317-337 (Humana Press, Inc. 1993). Alternatively, the. Human genomic libraries can be obtained from commercial sources, such as Research Genetics (Huntsville, AL) and the American Type Culture Collection (Manassas, VA).

A library containing the cDNA or genomic clones can be selected with one or more polynucleotide probes based on SEQ ID NO.

NO: 1, using standard methods (see, for example, Ausubel (1995) on pages 6-1 to 6-11). The anti-Zsig67 antibodies, produced as described below, can also be used to isolate the DNA sequences encoding the human Z s i g 67 genes from the cDNA libraries. For example, antibodies can be used to select the? Gtll expression libraries, or the antibodies can be used for the immuno selection that follows hybrid selection and translation (see, for example, Ausubel (1995) in the pages). 6-12 to 6-16, Margolis et al., "Screening? Expression libraries with antibody and protein probes," in DNA Cl on ing 2: Expré si on Sys t em s, 2nd Edi on, Glover et al., (eds.), pages 1-14 (Oxford University Press (1995)). As an alternative, a Z s γ 67 gene can be obtained by synthesizing the nucleic acid molecules using mutually primed long oligonucleotides and the nucleotide sequences described herein (see, for example, Ausubel (1995), on pages 8). -8 to 8-9). The techniques established using the polymerase chain reaction provide the ability to synthesize DNA molecules at least two kilobases in length (Adang et al., Plant Molec, Biol. 21: 1131 (1993).) Bambot et al. , PCR Methods and Applications 2: 266 (1993), Dillon et al., "Use of the Polymerase Chain Reaction for the Rapid Construction of Synthetic Genes" in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), Pages 263-268, (Humana Press, Inc. 1993), and Holowachuk et al., PCR Methods Appl. 4: 299 (1995)). The nucleic acid molecules of the present invention can also be synthesized with the "gene machines" using protocols such as the phosphoramidite method. If chemically synthesized double-stranded DNA is required, for an application such as the synthesis of a gene fragment, then, each complementary strand is made separately. The production of short genes (60 to 80 base pairs) is technically simple and can be achieved through the synthesis of the complementary strands and then their alignment. For the production of longer genes (>300 base pairs), however, special strategies may be required, since the coupling efficiency of each cycle during chemical synthesis of DNA is seldom 100%. To overcome this problem, synthetic (double-stranded) genes are assembled in modular form from single-stranded fragments that are 20 to 100 nucleotides in length. For the review on polynucleotide synthesis, see for example, Glick and Pasternak, Molecule r B io technology, P incipient and Appl i cations of Recombinant DNA (ASM Press 1994), Itakura et al., Annu. Rev. Biochem. 53: 323 (1984), and Climie et al., Proc. Nat'l Acad. Sci, USA 87: 633 (1990). The sequence of the Zsig67 cDNA or genomic fragment of the Zsig67 can be determined using the standard methods. The sequences of the Zsig67 polynucleotide described herein can also be used as probes or primers to clone the 5 'non-coding regions of a Z s ig 67 gene. The promoter elements of a Zsig67 gene can be used to direct the expression of heterologous genes in, for example, the pituitary tissue of transgenic animals or patients undergoing gene therapy. The identification of genomic fragments containing a Zsig67 promoter or a regulatory element can be achieved using well-established techniques, such as clearance analysis (see, generally, Ausubel (1995)). The cloning of flanking sequences ', also facilitate the production of Zsig67 proteins, by "genetic activation", in accordance with the general methods described in US Patent No. 5,641,670. Briefly, the expression of an endogenous Zsig67 gene in a cell is altered by introducing at the site or locus of Zsig67 a DNA construct comprising at least one target sequence, a regulatory sequence, an exon, and a site of the unpaired spliced donor. The target sequence is a non-coding sequence 5 'to Zs ig 6 7, which allows the homologous recombination of the construct with the locus of endogenous Zs ig 67, thereby, the sequences within the construct become operably linked with the sequence that encodes endogenous Z-67 sig. Thus, an endogenous Z s i g 6 7 promoter can be replaced or supplemented with other regulatory sequences to provide improved, tissue-specific, or otherwise regulated expression. 4. Production of Variants of the Gene Zsigß l The present invention provides a variety of nucleic acid molecules, including the DNA and RNA molecules, that encode the Zsig67 polypeptides described herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation between those polynucleotide molecules is possible. SEQ ID NO: 3 is a degenerate nucleotide sequence encompassing all nucleic acid molecules encoding the Zsig67 polypeptides of SEQ ID NO: 2. Those skilled in the art will recognize that the degenerate sequences of SEQ ID NO. : 3, also provide all the sequences encoding SEQ ID NO: 2, by substituting U for T. Thus, the present invention contemplates the nucleic acid molecules encoding the Zsig67 polypeptide, which comprise nucleotide 111 to nucleotide 422 of SEQ ID NO: 1, and its RNA equivalents. Table 1 sets forth the one-letter codes, used in SEQ ID NO: 3, to denote the positions of the degenerate nucleotide. The "resolutions" are then nucleotides denoted by a code of letters. The "complement" indicates the code for the complementary t (s). For example, the code Y denotes either C or T, and its complement R, denotes A or G, A is complementary to T, < and G is complementary to C.

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

Table 3 A person with ordinary skill in the art will appreciate that some ambiguity is introduced in the determination of a degenerate codon, representative of all possible codons that encode an amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (ARG), and the degenerate codon for arginine (MGN), can in some circumstances, encode serine (AGY). There is a similar relationship between the codons that encode phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence can encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO: 2 The variant sequences can be easily tested for their functionality as described here. Different species may exhibit "use of preferential codons". In general, see Grantham et al., Nuc. Acids Res. 8: 1893 (1980), Haas et al., Curr. Biol. 5: 315 (1996), Wain-Hobson et al., Gene 13: 355 (1981), Grosjean and Fiers, Gene 18: 199 (1982), Holm, Nuc. Acids Res. 14: 3015 (1986), Ikemura, J. Mol Biol. 158: 513 (1982), Sharp and Matasi, Curr. Op in. Genet Dev. 4: 851 (1994), Kane, Curr. Opin. Biotechnol. 5: 494 (1995), and Makrides, Microbiol. Rev. 60: 512 (1996). As used herein, the term "use of preferential codons", or "preferential codons", is a term of the art that refers to the translation codons of the protein that are most frequently used in the cells of certain species, thus favoring one or a few representatives of the possible codons that encode each amino acid (see Table 2). For example, the amino acid Threonine (Thr) can be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells, the ACC is the most frequently used codon.; in other species, for example, in the cells of insects, yeasts, viruses or bacteria, the Thr codons may be preferential. Preferred codons for a particular species can be introduced into the polynucleotides of the present invention, by a variety of methods known in the art. The introduction of the sequences of the preferential codons in the recombinant DNA can, for example, improve the production of the protein by making the translation of the protein more efficient within a particular type or type of cells. Therefore, the sequences of the degenerate codons, described in SEQ ID NO: 3, serve as templates for optimizing the expression of the polynucleotides in various types and species, commonly used in the art and described herein. The sequences containing the preferential codons can be tested and optimized for expression in several species, and tested for their functionality as described herein. The present invention also provides polypeptides and variant nucleic acid molecules, which represent the counterparts of other species (orthologs). These species include, but are not limited to, species of mammals, birds, amphibians, reptiles, fish, insects or other vertebrate and invertebrate species. Of particular interest are the Zsig67 polypeptides from other mammalian species, including porcine, murine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human Zs i g 6 7 can be cloned using the information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cells expressing Zsig67, as described herein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences described herein. A library is then prepared from the mRNA of a positive tissue or cell line. A cDNA encoding Zsig67 can then be isolated by a variety of methods, such as by probing with whole or partial human cDNA or with one or more sets of degenerate probes based on the described sequences. A cDNA can also be cloned using the polymerase chain reaction, with the primers designed from the human-representative Zsig67 sequences described herein. Within a further method, the cDNA library can be used to transform or transfect the host cells, and expression of the cDNA of interest can be detected with an antibody to the Zsig67 polypeptide. Similar techniques can also be applied for the isolation of genomic clones. Those of skill in the art will recognize that the sequence described in SEQ ID NO: 1 represents a single allele of human Zsig67, and that allelic and alternately splicing variation is expected to occur. Allelic variants of this sequence can be cloned by probing or genomic cDNA libraries from different individuals, according to standard procedures. Allelic variants of the nucleotide sequence shown in SEQ ID NO: 1, including those containing silent mutations and those in which mutations result in changes in amino acid sequences, are within the scope of the present invention, since they are proteins which are allelic variants of SEQ ID NO: 2. The cDNA molecules, generated from alternately spliced mRNAs, which retain the properties of the Zsig67 polypeptide, are included within the scope of the present invention, as they are polypeptides encoded by such cDNAs and mRNAs. The allelic variants and the splice variants of these sequences can be cloned by probing the cDNA or genomic libraries of different individuals or tissues, according to standard procedures known in the art. Within certain embodiments of the present invention, isolated nucleic acid molecules can hybridize under severe conditions to nucleic acid molecules comprising the nucleotide sequence described herein. For example, such nucleic acid molecules can be hybridized under severe conditions to the nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO: 1, to nucleic acid molecules having the nucleotide sequence of nucleotides 111 to 422 of SEQ ID NO: 1, or to nucleic acid molecules having a nucleotide sequence complementary to SEQ ID NO: 1, or to nucleotides 111 to 422 of SEQ ID NO: 1. In general, severe conditions are selected to be about 5 ° C or lower than the thermal melting point (Tm) for the specific sequence, at a defined ionic concentration and pH. The Tm is the temperature (under the defined ionic concentration and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. A pair of nucleic acid molecules, such as DNA-DNA, RNA-NRA and DNA-RNA, can be hybridized if the nucleotide sequences have some degree of complementarity. Hybrids can tolerate the base pairs paired in the double helix, but the stability of the hybrid is influenced by the degree of mating. The Tra of the matched hybrid decreases by 1 ° C for each 1-1.5% of base paired bases. The variation in the severity of the hybridization conditions allows control over the degree of mating that will be present in the hybrid. The degree of severity increases as the hybridization temperature increases and the ionic strength of the hybridization buffer decreases. Severe hybridization conditions encompass temperatures of about 5-25 ° C below the Tm of the hybrid and a hybridization buffer of up to 1 M Na +. Higher degrees of severity at low temperatures can be achieved with the addition of formamide, which reduces the Tm of the hybrid to approximately 1 ° C per 1% formamide in the buffer solution. In general, such severe conditions include temperatures of 20-70 ° C and a hybridization buffer containing up to 6x SSC and 0-50% formamide. A higher degree of severity can be achieved at temperatures from 40-70 ° C with a hybridization buffer having up to 4x SSC and from 0-50% formamide. Highly severe conditions typically encompass temperatures of 42-70 ° C with a hybridization buffer of up to Ix SSC and 0-50% formamide. Different degrees of severity can be used during hybridization and washing to achieve maximum specific binding to the target sequence. Typically, washings after hybridization are performed at increased degrees of severity to remove probes from unhybridized polynucleotides from the complexes subjected to hybridization. The above conditions are significant for serving as a guide and are also within the abilities of a person skilled in the art to adapt these conditions for use with a particular polypeptide hybrid. The Tm for a specific target sequence is the temperature (under defined conditions) at which 50% of the target sequence will hybridize to a perfectly paired probe sequence. These conditions which influence the Tm include, the size and content of base pairs of the polynucleotide probe, the ionic strength of the hybridization solution, and the presence of destabilizing agents in the hybridization solution. Numerous equations for calculating Tm are known in the art, and are specific for DNA-RNA, and hybrid DNA-RNA and variable length polynucleotide probe sequences (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Ppres 1989); Ausubel et al., (Eds.), Current Protocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds). Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26: 221 (199)). The sequence analysis software is OLIGO 6.0 (LSR; Long Lake, MN), and Prime Premier 4. 0 (Premier Biosoft International, Palo Alto, CA), as well as sites on the Internet, are tools available to analyze a given sequence and calculate the Tm based on the user's defined criteria. Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences. Typically, hybridization of longer polynucleotide sequences, > 50 base pairs, it is carried out at temperatures of approximately 20-25 ° C below the calculated Tm. For smaller probes, < 50 base pairs, hybridization is typically carried out at Tm of or below 5-10 ° C. This allows the maximum hybridization rate for the DNA-DNA and DNA-RNA hybrids. The length of the polynucleotide sequence influences the speed and stability of hybrid formation. The sequences of smaller probes, < 50 base pairs, reaches equilibrium with complementary sequences quickly, but can form less stable hybrids. The incubation times of anyone from minutes to hours can be used to achieve the formation of hybrids. Longer probe sequences reach equilibrium more slowly, but form more stable complexes even at lower temperatures. Incubations are allowed to proceed overnight or longer. In general, the incubations are carried out for a period equal to three times the calculated Cot time. The time Cot, the time taken by the polynucleotide sequences to reassociate, can be calculated by a particular sequence by methods known in the art. The base pair composition of the polynucleotide sequence will effect the thermal stability of the hybrid complex, thereby influencing the selection of the hybridization temperature and the ionic strength of the hybridization buffer. A pair A-T is less stable than the G-C pairs in aqueous solutions containing sodium chloride. Therefore, if the G-C content is higher, the hybrid is more stable. Even the distribution of the G and C residues within the sequence can contribute positively to the stability of the hybrid. In addition, the composition of base pairs can be manipulated to alter the Tm of a given sequence. For example, 5-methyldeoxy cytine can be replaced by deoxycytidine and 5-methylorine can be replaced by thymidine to increase the Tm, while 7-diaz-2'-deoxyguans can be replaced by guanosine to reduce the dependence on Tm. The ionic concentration of the hybridization buffer also affects the stability of the hybrid. Hybridization buffers generally contain blocking agents such as the Denhart solution (Sigma Chemical Co., St. Louis, Mo.), Denatured salmon sperm DNA, tRNA, milk powder (BLOTTO), heparin or SDS, and a source Na +, such as SSC (Ix SSC: 0.15 M sodium chloride, 15 mM sodium citrate) or SSPE (lx SSPE: 1.8 M NaCl, 10 mM NaH2P0, 1 mM EDTA, pH 7.7). By decreasing the ionic concentration of the buffer, the stability of the hybrid increases. Typically, hybridization buffers contain between 10 mM - 1 M Na +. The addition of the surfactant or denaturing agents such as formamide, salts of t-ra 1, or the guanidinium cations, or thiocyanate cations to the hybridization solution, will alter the Tm of a hybrid. . Typically, formamide is used at a concentration of up to 50% to allow incubations to be carried out at lower and lower temperatures. Formamide also acts to reduce non-specific background when using RNA probes. As an illustration, a nucleic acid molecule encoding a variant Zsig67 polypeptide can be hybridized to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or su-complement) at 42 ° C, overnight in a solution comprising 50% formamide, 5xSSC (lxS SC: c 1 or 0.15M sodium rurium and 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x solution of Denhardt (100 x Denhardt's solution; Ficoll 400 at 2% (w / v), 2% polyvinylpyrrolidone (w / v), and 2% bovine serum albumin (w / v), 10% dextran sulfate and 20 μg / ml denatured DNA of salmon sperm, divided. One skilled in the art may recommend projecting variations of these hybridization conditions. For example, the hybridization mixture can be incubated at a higher temperature, such as about 65 ° C, in a solution that does not contain formamide. However, premixed hybridization solutions are available (eg, the EXPRESSHYB Hybridization Solution from CLONTECH Laboratories, Inc.), and hybridization can be carried out according to the manufacturer's instructions. Following hybridization, the nucleic acid molecules can be washed to remove the nucleic acid molecules not subjected to hybridization under severe conditions, or under very severe conditions. Typical wash severity conditions include washing in a 0.5X -2X SSC solution with 0.1% sodium dodecyl sulfate (SDS) at 55-65 ° C. That is, the nucleic acid molecules encoding a variant of the Zsig67 polypeptide, remain hybridized, after severe washing conditions with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement). , in which the wash severity is equivalent to 0.5X-2X SSC with 0.1% SDS at 55-65 ° C, including 0.5 X SSC with 0.1% SDS at 55 ° C, or 2XSSC with 0.1% SDS at 65 ° C. A person skilled in the art can easily project equivalent conditions, for example, by replacing the SSPE with SSC in the wash solution. Typical severe wash conditions include washing in a 0. lx-0.2x solution of SSC with 0.1% sodium dodecyl sulfate (SDS) at 50-65 ° C. In other words, the nucleic acid molecules encoding a variant Zsig67 polypeptide, remain hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement), in which, the severity of the wash is equivalent to 0.1x-02 SSC with 0.1% SDS at 50-65 ° C, including O.lx SSC with 0.1% SDS at 50 ° C, or 0.2x SSC with 0.1% SDS at 65 ° C. The present invention also provides isolated Zsig67 polypeptides, which have a sequence identity substantially similar to the polypeptide of SEQ ID NO: 2, or their orthologs. The term "substantially similar sequence identity" is used herein to denote polypeptides having a sequence identity of 70%, 80%, 90%, 95%, or greater than 95%, with the sequence shown in SEQ ID NO: 2, or its orthologs. The present invention also contemplates nucleic acid molecules variant of Zsig67, which can be identified using two criteria: a determination of the similarity between the polypeptide encoded with the amino acid sequence of SEQ ID NO: 2, and a hybridization assay, such as is described above. Such variants of Zsig67, include the nucleic acid molecules (1) that remain hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement) under severe wash conditions, in which the severity of the wash is equivalent to 0.5x-2x SSC with 0.1% SDS at 55-65 ° C, and (2) which encodes a polypeptide having a sequence identity of 70%, 80%, 90%, 95 %, or greater than 95%, with the amino acid sequence of SEQ ID NO: 2. Alternatively, the variants of Zsig67 can be characterized as nucleic acid molecules (1) that remain hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement), under very severe washing conditions, in which the wash severity, is equivalent to O.lx- 0.2x SSC with 0.1% SDS at 50- 65 ° C and (2) which encode a polypeptide having a sequence identity of 70%, 8 0%, 90%, 95%, or greater than 95%, with the amino acid sequence of SEQ ID NO: 2. The percentage of the identity of the sequence is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 630 (1986), and Henikoff and Henikoff, Proc. Nat'l. Acad. Sci. USA 89: 10915 (1992). Briefly, two amino acid sequences are aligned to optimize alignment records using an open space penalty of 10, an opening extension penalty of 1, and the "BLOSUM62" score matrix of Henikoff and Henikoff (ibid.) As is shown in Table 3 (amino acids are indicated by the standard one-letter codes). The identity percentage is then calculated as: ([Total number of identical pairs] / [length of the longest sequence plus the number of openings entered in the longest sequence to align the two sequences]) (100).

Tab l a 3 > * ri I je ri M rt ri 1 H? n N O • 1 ca * ri n N Ct 1 1 1 Di t ri ri ct 1 1 I 1 1 b? u > N ri rt ri 1 1 «1 s tn r i ri? soir ri ri 1 1 t 1 B 10 V) H n ri O n N Ct 1 1 1 1 1 • * o rt ti ri ri ri t 1 1 1 1 H * «rt ri on ri rt ri n 1 1 1 1 1 1 a OD rt rt H ri rl n »< t n 1 1 1 1 1 1 1 1 1 0 u > «1" N rt rt O M n n n 1 t 1 1 1 i 1 1 i i t m ti O rt rt H (1 n ri Ort rt ti ti 1 1 1 1 1 1 1 1 1 s m o n H o rt O ri ri n 1 1 1 1 1 1 i i? ? rt rt rt rt r rt r rt ri ri 1 1 1 1 1 1 1 1 1 1 1 1 1 1 a "0 rt o MHH rt ri rt n ri o ri" 1 1 • 1 i 1 1 t 1 1 1 1 Z to rl mo OOH rt rt O ti rt ri O ti «1 1 1 1 1 1 1 t i i? O rt H O O n N ri n ri ri rt ti rt 1 1 1 1 1 1 1 1 1 1 1 1 1 < f H < 1 -O H i-f O rl ri H ri ri rt ct 1 1 1 1 1 1 1 1 i 1 1 1 < Oi% 0 ü 0 H 9 8 H J ki X your & to fr S i "> Those with experience in the art will appreciate that there are many established algorithms for aligning two amino acid sequences. The "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method to examine the level of identity shared by an amino acid sequence described herein and the amino acid sequence of a putative Zsig67 variant. The FASTA algorithm is described by Pearson and Lipman, Pro c. Na t 'l A ca d. S c i. E UA 85: 2444 (1988), and by Pearson, Me t h. In zym or l. 1 83: 63 (1990). Briefly, FASTA, first characterizes the similarity of the sequence by identifying the regions shared by the sequence in question (eg, SEQ ID NO: 2) and the test sequence that has either the highest identity density (if the variable ktup is 1) or the pairs of identities (if ktup = 2) without considering the conservative substi tutions, insertions or eliminations of amino acids. The ten regions with the highest density of identities are then recorded again, comparing the similarity of all paired amino acids, using an amino acid substitution matrix, and the ends of the regions are "paired" to include only those residues that contribute to the highest record. If there are several regions with records larger than the "cutoff" value (calculated by a predetermined formula, based on the length of the sequence and the value of ktup), then the initial paired regions are examined to determine if the regions can be linked to form an approximate alignment with the separations or openings. Finally, the regions with the highest recordings of the two amino acid sequences are aligned using a modification of the Needleman-Wuns ch-Se 1 ler s algorithm (Needleman and Wunsch, J. Mo l. B i o l. 4 8: 4 4 4 (1970); Sellers, S IAM J. App l. Ma th. 2 6: 1 8 1 (1974), which allows the insertions and eliminations of amino acids. The illustrative parameters for the FASTA analysis are: ktup = l, penalty for opening the spar ation = 10, penalty for separation, extension of the separation = l, and substring matrix = BLOSUM 62.

This parameter can be entered into a FASTA program, by modifying the file matrix score ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. In zym or l. 1 83: 6 3 (1990). The FASTA program can also be used to determine the identity of the sequence of nucleic acid molecules, using a relationship as described above. For comparisons of nucleotide sequences, the value of ktup can range from one to six, preferably from three to six, more preferably three, with the other parameters set as described above. The present invention includes nucleic acid molecules that encode a polypeptide having a conservative amino acid change, when compared to the amino acid sequence of SEQ ID NO: 2. That is, variants containing one or more can be obtained. amino acid substitutions of SEQ ID NO: 2, in which an alkyl amino acid is substituted for an amino acid of alkyl in the amino acid sequence of Zsig67, an aromatic amino acid is substituted by an aromatic amino acid in an amino acid sequence of Zsig67 , an amino acid that contains sulfur, is replaced by an amino acid that contains sulfur in a sequence of amino acids, an amino acid that contains hydroxy, is replaced by an amino acid that contains hydroxy in a sequence of amino acids of Zsig67, an amino acid, is substituted by an amino acid acid in an amino acid sequence of Zsig67, a basic amino acid, is replaced by an amino Basic acid in an amino acid sequence of Zsig67, or a bibic monocarboxylic amino acid is substituted by a bibasic monocarboxylic amino acid in an amino acid sequence of Zsig67. Among the common amino acids, for example, a "conservative amino acid substitution" is illustrated by a substitution between the amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine , tyrosine and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine. Table BLOSUM62 is an amino acid substitution matrix derived from approximately 2,000 local alignments of segments of protein sequences, representing highly conserved regions of more than 500 related protein groups (Henikoff and Henikoff, Pro c. Na t 'l A ca D. S ci. E UA 89: 10915 (1992)). Accordingly, substitution frequencies of BLOSUM62 can be used to define conservative amino acid substitutions, which can be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely on chemical properties (as discussed above), the language "conservative amino acid substitutions" preferably refers to a substitution represented by a BLOSUM62 value, greater than -1. For example, a substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2 or 3. Accordingly, for this system, the preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (eg, 1, 2 or 3), while most preferred amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (eg, 2 or 3). Particular variants of Zsig67 are characterized by having at least an identity of the upper sequence of 90%, at least 97%, at least 98% or at least 99% of the corresponding amino acid sequence (for example, SEQ ID NO. NO: 2), wherein the variation in the amino acid sequence is due to one or more conservative amino acid substitutions. Conservative amino acid changes in a Zsig67 gene can be introduced by substitution nucleotides for the nucleotides mentioned in SEQ ID NO: 1. Such "conservative amino acid" variants can be obtained, for example, by oligonucleotide-directed mutagenesis, mutagenesis by linker scan, mutagenesis using the polymerase chain reaction, and the like (see, Ausubel (1995) on pages 8-10 to 8-22; and McPherson (ed.), Directed Mutagenesis: A _ Practical Approach (IRL Press 1991). A variant Zsig67 polypeptide can be identified by the ability to specifically bind to the anti i-Zsi 7 antibodies. The proteins of the present invention can also comprise amino acid residues that do not occur naturally. Amino acids that do not occur naturally include, without limitation, t rans-3-me ti lpr or 1 ina, 2, 4 -met anoproline, ci s- 4 -hydroxyprol ina, trans-4-hydroxyproline, N-me T ilglycine, 1-otonine, methyl threonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, of shidroproline, 3- and 4-methyl-3-dimethyltin, proline, -leucine, norvaline, 2-azaf eni lalanine, 3 -a za f eni 1 alanin, 4-azaf enylalanine, and 4-f luorof enylalanine. Various methods are known in the art for incorporating residues that do not occur naturally in proteins. For example, an in vitro system may be employed, where nonsense mutations are suppressed using the chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of the plasmids containing the sense mutations are typically carried out in a cell-free system, comprising an extract of E. coli S30 and commercially available enzymes and other reagents. The proteins are purified by chromatography. See, for example, Robertson et al. , J. Am. Chem. Soc 113: 2122 (1991), Eliman et al. , Methods Enzymol. 202: 301 (1991), Chung et al. , Science 259: 806 (1993), and Chung et al. , proc. Nati 'Acad. Sci. USA 90: 10145 (1993). In a second method, the 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 (1996).) Within a third method , E. coli cells are grown in the absence of a natural amino acid that must be replaced (eg, phenylalanine) and in the presence of the amino acid (s) that do not occur naturally (eg, 2-azaphenylalanine, 3 -a za feni lalanina, 4-a za feni lalanina or 4-fluoroureni 1 alanine) The amino acid that does not occur naturally, is incorporated in the protein instead of its natural counterpart, see, Koide et al., Biochem 33: 1410 (1994) .An amino acid residues that occur naturally, can be converted to species that do not occur naturally, by in vitro modification.The chemical modification can be combined with site-directed mutagenesis. site to expand also tell the range of substitutions (Wynn and Richards, Protein Sci. 2: 395 (1993)). A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, amino acids that do not occur naturally, and unnatural amino acids, can be substituted for the amino acid residues of Zsig67. The essential amino acids in the polypeptides of the present invention can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081 (1989)). , Ba.ss et al., Proc. Nat'l Acad. Sci. USA 88: 4498 (1991), Coombs and Corey "Site-Directed Mutagenesis and Protein Enginering" in Proteins: Analysis and Design, Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In the last technique. Mutations of alanine alone are introduced into each residue in the molecule, and the resulting mutant molecules are tested for their biological activity as described below to identify the amino acid residues that are critical for the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271: 4699 (1996). The location of the Zsig67 receptor that links the domains can be determined by a physical analysis of the structure, when determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffective labeling, in conjunction with the amino mutation. Acids of putative contact site. See, for example, de Vos et al., Science, 255: 306 (1992), Smith et al., J. Mol. Biol. 224: 899 (1992), and Wlodaver et al., FEBS Lett 309: 59 (1992). However, Zsig67 labeled with biotin or FITC can be used for the expression of the cloning of Zsig 67 receptors. Multiple substitutions of amino acids can be made and tested using the known methods of mutagenesis and selection, such as those described by Reidhaar -Olson and Sauer. { Science 241: 53 (1988) or Bowie and Sauer (Proc. Nat'l. Acad. Sci. USA 86: 2152 (1989)). Briefly, these authors describe methods for randomly choosing two or more positions in a polypeptide, selecting a functional polypeptide, and then sequencing the muti-polypept polypeptides to determine the spectrum of allowable substitutions in each position. Other methods that can be used include phage display (eg, Lowman et al., Biochem 30: 10832 (1991), Ladner et al., US Patent No. 5,223,409, Huse, international publication No. WO 92/06204 , and site-directed mutagenesis (Derbyshire et al., Gene 45: 145 (1986), and Net et al., DNA 7: 127, (1988)). The nucleotide variants and Zsig67 polypeptide sequences described, they can also be generated by mixing the DNA as described by Stemmer, Natura 370: 389 (1994), Stemmer Proc. Nat'l Acad. Sci. USA 91: 10741, and International Publication No. WO 97/20078. Briefly, variant DNAs are generated by homologous recombination in vitro, by randomly fragmenting an original DNA, followed by reassembly using PCR, resulting in randomly introduced point mutations.This technique can be modified using a family of original DNAs, such as the allelic variants or DNAs of different species, to introduce additional variability in the process. The selection or separation for the desired activity, followed by the additional iterations of the mutagenesis and the assay, provide the rapid "evolution" of the sequences by selecting the desirable mutations while simultaneously being selected against the deleterious changes. Mutagenesis methods as described herein can be combined with high throughput automatic screening methods to detect the activity of the mutagenized cloned polypeptides in the host cells. The mutagenized DNA molecules encoding the biologically active polypeptides, or the polypeptides of are linked to the anti-Zsig67 antibodies, can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow rapid determination of the importance of amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

The present invention also includes "functional fragments" of Zsig67 polypeptides and nucleic acid molecules that encode such functional fragments. The routine elimination assays of the nucleic acid molecules can be carried out to obtain functional fragments of a molecule encoding a Zsig67 polypeptide. As an illustration, the DNA molecule having the nucleotide sequence of SEQ ID NO: 1, can be digested with Bal 31 nuclease to obtain a series of nested deletions. The fragments are inserted into the expression vectors in appropriate reading structures, and the expressed polypeptides are isolated and tested for their ability to bind to anti-Zs i g 67 antibodies. An alternative to endonucleases digestion, is to use oligonucleotide-directed mutagenesis to introduce the deletions or stop the codons for the specific production of a desired fragment. Alternatively, particular fragments of a Zsig67 gene can be synthesized using the polymerase chain reaction. Methods for identifying functional domains are well known to those skilled in the art. For example, studies on truncation in either or both terminals of interferons have been summarized by Horisberger and Di Marco, Pharmac. Ther. 55: 507 (1995). In addition, standard techniques for the functional analysis of proteins are described, for example, by Treuter et al., Gen Genet. 240: 113 (1993), Content et al., "Expression and Preliminary Deletion Analysis of the 42 kDa 2-5A synthetase induced by human interferon", in Biological Interferon Systems, Proceeding of ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72 (Nijhoff 1978), Herschman, "The EGF Receptor," in Control of Animal Cell Proliferation, Vol. 1, Boynton et al., (eds.) pages 169-199 (Academic Press 1985 ), Coumailleau et al., J. Biol. Chem. 270: 25291 (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). In addition, sequence analysis can also identify functional fragments of Zsig67. For example, comparison with other members of the secretin-glucagon-VIP hormone family indicate that a polypeptide consisting of amino acid residues 52 to 85 of SEQ ID NO: 2, can effectively bind to the cognate Zsig67 receptor. The present invention also contemplates the functional fragments of a Zsig67 gene having amino acid changes, compared to the amino acid sequence of SEQ ID NO: 2. A variant Zsig67 gene, can be identified based on the structure, determining the level of identity with the nucleotide and amino acid sequences of SEQ ID NOs: 1 and 2, as discussed above. An alternative approach for identifying a variant gene on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant Zsig67 gene can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO. : 1, as described above. The present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of a Zsig67 polypeptide described herein. Such fragments or peptides may comprise an "immunogenic epitope", which is part of a protein that elicits an antibody response when the entire protein is used as an immunogen. The peptides of the portion that transports the epitope can be identified using the standard methods (see, for example, Geysen et al., Pro c. Na t '1. Acad. S ci. E UA 81: 3998 (1983) ). In contrast, polypeptide fragments or peptides can comprise an "antigenic epitope," which is a region of a protein molecule to which an antibody can specifically bind. Certain epitopes consist of a linear or contiguous portion of amino acids, and the antigenicity of such an epitope is not interrupted by the denaturing agents.

It is known in the art that relatively short synthetic peptides that can mimic the epitopes of a protein can be used to stimulate the production of the antibodies against the protein (see, for example, Sutcliffe et al., Science 219: 660 (1983 )). Accordingly, the epitope-bearing peptides and polypeptides of the present invention are useful for generating the antibodies that bind to the polypeptides described herein. The peptides and polypeptides carrying the antigenic epitope preferably contain at least four to ten amino acids, at least ten to fifteen amino acids, or approximately 15 to approximately 30 amino acids of SEQ ID NO: 2. such peptides and polypeptides that carry the epitope, can be produced by fragmenting a Zsig67 polypeptide, or by chemical synthesis of peptides, as described herein. In addition, epitopes can be selected by display of phage or random libraries of peptides (see, for example, Lane and Stephen, Curr Opin. Immunol., 5: 268 (1993), and Cortese et al., Curr. Opin, Bio 7: 616 (1996)). Standard methods for identifying epitopes and producing antibodies from small peptides comprising an epitope are described, for example, by Mole, "Mapping Epitope", in Methods in Molecular Biology, Vol. 10, Manson (ed.) , pages 105-116 (The Humana Press, Inc. 1992), Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies Production, Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages 60 -84 (Cambridge University Press 1995), and Coligan et al., (Eds.), Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley &Sons 1997) . Despite the particular nucleotide sequence of a variant Zsig61 gene, the gene encoding a polypeptide can be characterized by its ability to specifically bind to an anti-Zsig57 antibody. For any cop Zsig67, including variants and fusion proteins, a person of ordinary skill in the art can generate a degenerate sequence of the complete polynucleotide encoding that variant, using the information set forth in Tables 1 and 2 above. In addition, those with technical expertise can use the standard program sets to project the Zsig67 variants based on the nucleotide and amino acid sequences described herein. Accordingly, the present invention includes a computer readable medium encoded with a data structure that provides at least one of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. Forms of computer readable media suitable, include magnetic media and optically readable media. Examples of magnetic media include a fixed or hard disk drive, a random access memory (RAM) chip, a flexible disk, digital linear tape (DLT), an immediate memory disk, and a ZIP disk. Optically readable media is exemplified by compact discs (eg, read-only memory (ROM) CDs, rewritable (RW) CDs, and recordable CDs), and digital discs (videos, DVDs) (e.g. DVD-ROM, DVD-RAM, and DVD + RW).

. Production of Fusion proteins from Zsig67 The fusion proteins of Zsig67 can be used to express Zsig67 in a recombinant host, and to isolate the expressed Zsig67. As described below, the particular Zsig67 fusion proteins also have uses in diagnosis and therapy. A type of fusion protein comprises a peptide guiding a Zsig67 polypeptide from a recombinant host cell. To direct a Zsig67 polypeptide in the secretory pathway of a eukaryotic host cell, a secretory signal sequence (also known as a signaling peptide, a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. While the sequence of the secretory signal can be derived from the Zsig67, a suitable signaling sequence can also be derived from another secreted or synthesized protein of n o vo. The secretory signal sequence is operably linked to a sequence encoding Zsig67, such that the two sequences are linked in the correct reading structure and positioned to direct the newly synthesized polypeptide into the secretory path of the host cell. Secretory signal sequences are commonly positioned 5 'to the nucleotide sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the nucleotide sequence of interest (see, for example, Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). Although the secretory signal sequence of Zsig67 or another protein produced by mammalian cells (e.g., the signal sequence of the plasminogen activator of the tissue type, as described, for example, in U.S. Patent No. 5,641,655) is useful for the expression of Zsig67 in a host recombinant mammal, a sequence of the yeast signal is preferred for expression in yeast cells. Examples of suitable yeast signal sequences are those derived from pheromone factor a that matches the yeast (encoded by the MFal gene), the invertase (encoded by the S UC2 gene), or the acid phosphatase, (encoded by the PH05 gene). See for example, Romanos et al. , "Expression of Cloned Genes in Yeast," in DNA Cl on i n g 2: A Pra ct i ca l Approa ch, 2nd Edition, Glover and Hames (eds.), Pages 123-167 (Oxford University Press 1995). In bacterial cells, it is often desirable to express a heterologous protein as a fusion protein to decrease toxicity, increase stability, and improve recovery of the expressed protein. For example, Zsig67 can be expressed as a fusion protein comprising, a polypeptide of S-trans fecta a of glutathione. The Glutathione S-transferase fusion proteins are typically soluble, and easily purifiable from E lysates. c or l i on immobilized glutathione columns. In similar procedures, a Zsig67 fusion protein, comprising a protein polypeptide that binds maltose, can be isolated with a column of amylose resin, while a fusion protein comprising the end of the C-terminus, of truncated protein A, can be purified using I gG-Se lighthouses a. The techniques established to express a heterologous polypeptide as a fusion protein, in a bacterial cell, are described, for example, by Williams et al. , "Expression of Foreign Proteins in E. coli Using Plasmid Vectors and Purification of Specific Polyclonal Antibodies", in DNA Cl on in g 2: A Pra cti ca l Approa ch, 2nd Edi on, Glover and Hames (Eds.) Pages 15-58 (Oxford University Press nineteen ninety five) . In addition, expression systems are commercially available. For example, the PINPOINT Xa protein purification system (Promega Corporation; Madison, Wl), provides a method for isolating a fusion protein, comprising a polypeptide that becomes biotinylated during expression with a resin comprising avidin. Peptide ends that are useful for isolating heterologous polypeptides expressed by either prokaryotic or eukaryotic cells, include polyhistidine ends (which have an affinity for the nickel chelating resin), c-myc ends, the calmodulin binding protein ( isolated with calmodulin affinity chromatography), substance P, the RYIRS end (which binds to anti-RYIRS antibodies), the Glu-Glu endpoint, and the FLAG end (which binds to the anti-HIV antibody). FLAG). See, for example, Lou et al., Arch Biochem. Biophys. 329: 215 (1996), Morganti et al. , Biotechnol. Appl. Biochem. 23:67 (1996), and Zheng et al. , Gene 185: 55 (1997). Nucleic acid molecules encoding such polypeptide ends are available, for example, from Sigma-Aldrich Corporation (St. Louis, MO). The present invention also contemplates the use of the secretory signal sequence contained in the Zsig67 polypeptides of the present invention to direct other polypeptides in the secretion path. A signal fusion polypeptide can be made, wherein a secretory signal sequence is derived from. amino acid residues 1 through 28 of SEQ ID NO: 2, operably linked to another polypeptide, using methods known in the art, and described herein. The sequence of the secretory signal, contained in the fusion polypeptides of the present invention, is preferably amino-terminally fused to an additional peptide to direct the additional peptide to the secretory path. Such constructs have numerous applications known in the art. For example, these new constructs of the secretory signal sequence can direct the secretion of an active component of a normally non-secreted protein, such as a receptor. Such fusions may be used in a transgenic animal or in a recombinant host cultured to direct the peptides through the secretory pathway. With respect to the latter, exemplary polypeptides include pharmaceutically active molecules, such as Factor Vlla, proinsulin, insulin, follicle stimulating hormone, tissue-like activator pl of the tissue type, tumor necrosis factor, interleukins (e.g. int erleucine-1 (IL-1), IL-2, IL-3, IL-4, IL5, IL6, IL-7, IL-8, IL-9, IL-10, IL, 11, IL-12, IL-13, IL-14 and IL-15) stimulation factors of the colonies (for example the factor that stimulates the colonization of granulocytes (G-CSF) and the factor that stimulates the colonization of granulocyte macrophages (GMCSF)), Interferons (for example, int er-f-arons, ~ ßf -y, - ®, -dy-t), the growth factor of stem cells designated "SI factor", erythropoietin and t rhombopoie t ina. The sequence of the secretory signal of Zsig67, contained in the fusion polypeptides of the present invention, is preferably fused amino-tminimally to an additional peptide to direct the additional peptide in the secretion path. Fusion proteins comprising a secretory signal sequence of Zsig67 can be constructed using standard techniques.

Another form of fusion protein comprises a Zsig67 polypeptide and an immunoglobulin heavy chain constant region, typically an Fc fragment, which contains two constant region domains and a hinge region, but lacks the variable region. As an illustration, Chang et al. , Patent No. 5,723,125, describes a fusion protein comprising a human interferon and an Fc fragment of human immunoglobulin. The C-terminus of the interferon is linked to the N-terminus of the Fc fragment by a linker portion of the peptide. An example of a peptide linker is a peptide comprising mainly an inert sequence of the T cell, which is immunologically inert. An exemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGG S (SEQ ID NO: 4). In this fusion protein, a preferred Fc portion is a human α4 chain, which is stable in solution and has little or no activation complement activity. Accordingly, the present invention contemplates a Zsig67 fusion protein comprising a Zsig67 portion and a human Fc fragment, wherein the C-terminus of the Zsig67 portion is linked to the N-terminus of the Fc fragment via a peptide linker, such as a peptide consisting of the amino acid sequence of SEQ ID NO: 4. The portion Z s i 7 can be a molecule of Zs i g57 or a fragment thereof. In another variation, the Zsig67 fusion protein comprises an IgG sequence, a Zsig67 portion covalently linked to the amino terminus of the IgG sequence, and a signal peptide that is covalently linked to the amino terminus of the Zsig67 portion, wherein the IgG sequence consists of the following elements in the following order: a hinge region, a CH2 domain, and a CH3 domain. Consequently, the IgG sequence lacks a CHi domain. The Zsig67 portion has a Zsig67 activity as described herein, such as the ability to bind to a Zsig67 receptor. This general procedure for producing fusion proteins comprising portions of both antibodies and non-antibodies has been described by LaRochelle et al. , EP 742830 (WO 95/21258). The fusion proteins comprising a portion Zs i g 67 and an Fc portion can be used, for example, as a testing tool. For example, the presence of a Zsig67 receptor in a biological sample can be detected using a Zsig67 antibody fusion protein, in which the portion of Zsig67 is used to direct the cognate receptor, and a macromolecule, such as a Protein A or an anti-Fc antibody, are used to detect the binding of the ion-receptor fusion protein complex. However, such fusion proteins can be used to identify agonists and antagonists that interfere with the binding of Z s i g 6 7 to its receptor. In addition, anti- i sig 67 body fusion proteins, comprising variable domains of antibodies, can be employed as therapeutic proteins, in which, the antibody portion binds to a target antigen, such as an antigen associated with the tumor The fusion proteins can be prepared by methods known to those skilled in the art, by preparing each component of the fusion protein and chemical conjugation thereof. Alternatively, a polynucleotide that encodes both components of the fusion protein in the proper reading structure can be generated using techniques known and expressed by the methods described herein. General methods for enzymatic or chemical cleavage of fusion proteins are described, for example, by Ausubel (1995), on pages 16-19 to 16-25. 6 Production of Polypeptides from Zsi g67 in Culled Cells.

The polypeptides of the present invention, which include the full-length polypeptides, the functional fragments and the fusion proteins, can be produced in host recombinant cells, following conventional techniques. To express the gene Z s i g 67, a nucleic acid molecule encoding the polypeptide must be operably linked to the regulatory sequences that control transcriptional expression in an expression vector and, then, introduced into host cells. In addition, for transcriptional regulatory sequences, such as promoters and enhancers, expression vectors can include regulatory sequences and regulatory genes and a marker gene which is suitable for the selection of cells that carry the expression vector. Expression vectors that are suitable for the production of a foreign protein in eukaryotic cells typically contain (1) prokaryotic DNA elements, which encode a bacterial origin of replication and a marker of antibiotic resistance to provide growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control the initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of the transcripts, such as a termination / polyadention sequence. As discussed above, expression vectors can also include nucleotide sequences that encode a secretory sequence that directs the heterologous polypeptide into the secretory path of a host cell. For example, an expression vector of Zsig67 may comprise a Zsig67 gene and a secretory sequence derived from a Zsig67 gene or another secreted gene. The Zsig67 proteins of the present invention can be expressed in mammalian cells. Examples of mammalian host cells include kidney cells from the African green monkey (Vero, ATCC CRL 1587), human embryonic kidney cells (293-HEK, ATCC CRL 1573), kidney cells from baby hamsters (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovarian cells (CHO-K1; ATCC CCL61; CHO DG44 (Chasin et al., Som Cel 1. Mol et al., Gen. et 2 1: 5 5 5, (1986)), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H- 4-II-E; ATCC CRL 1548), monkey kidney cells transformed with SV40 (COS-1; ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658) .For a mammalian host, the signals Transcriptional and translational regulatory can be derived from viral sources, such as an adenovirus, bovine papilloma virus, simian virus, or the like, in which regulatory signals They are associated with a particular gene, which has a high level of expression. The appropriate regulatory and translational regulatory sequences can also be obtained from mammalian genes, such as the genes of actin, collagen, myosin, and metallothionein. The transcriptional regulatory sequences include a region of the promoter sufficient to direct the initiation of RNA synthesis. Suitable eukaryotic promoters include the gene promoter for the mouse m i t i on e i n a i (Hamer, et al., J.

Molec. Appl. Genet 1: 213 (1982)), the TK promoter of Herpes virus (McKnight, Cell 31: 355 • (1982)), the SV40 early promoter (Benoist et al., Natura 290: 304 (1981)) the promoter of the Rous sarcoma virus (Gorman et al., Proc. Nat'l Acad. Sci. USA 79: 6111 (1982)), the cytomegalovirus promoter (Foecking et al., Gene 45: 101 (1980), and the tumor virus promoter in mammals (see, generally, Etcheverry, "Expression of Engineered Proteins in Mammalians Cell Culture," in Protein Engineering: Principles and Practice, Cleland et al., ( eds), pages 163-181 (John Wiley &Sons, Inc. 1996)). Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNA polymerase promoter, can be used to control the expression of the gene for Zsig67 in mammalian cells, if the prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et al. al., al., Mol. Cell, Biol. 10: 4529 (1990), and Kaufman et al., Nucí Acids Res. 19: 4485 (1991)).

An expression vector can be introduced into host cells using a variety of standard techniques, including calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated administration, electroporation, and the like. Preferably, the transfected cells can be screened and propagated to provide host recombinant cells comprising the expression vector stably integrated into the genome of the host cells. Techniques for introducing vectors into eukaryotic cells and techniques for selecting such stable transformants using a dominant selectable marker are described, for example, by Ausubel (1995) and by Murria (ed.), Gen e Tra ns fer and Expre si ón Pro tocols (Humana Press 1991.) For example, the appropriate selectable marker is a gene that provides resistance for the antibiotic neomycin. In this case, the selection is carried out in the presence of a drug of the neomycin type, such as G-418 or the like. The selection systems can also be used to increase the level of expression of the gene of interest, a process called "amplification". The amplification is carried out by cultivating high fectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select the cells that produce high levels of the products of the introduced genes. A suitable, suitable, selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other genes for drug resistance (eg, hygromycin resistance, resistance to drugs, puromycin acetyltransferase) can also be used. Alternatively, markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, class I MHC, and alkaline phosphatase of the placenta, can be used to separate the transfected cells of those not transfected by such means as FACS separation or magnetic ray separation technology. The Zsig67 polypeptides can be produced by culturing mammalian cells using a viral delivery system. Exemplary viruses for this purpose include adenovirus, herpesvirus, vaccinia virus and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is currently the best-studied gene transfer vector for the administration of heterologous nucleic acids (for a review, see Becker et al., Me. Ce ll B iol 43: 161 (1994), and Douglas and Curil, S ci in ce &Medi cin e 4:44 (1997)). The advantages of the adenovirus system include the accommodation of relatively long DNA inserts, the ability to grow at a high titer, the ability to infect a wide range of mammalian cell types., and the flexibility that allows using them with a large number of available vectors containing different promoters. By removing portions of the adenovirus genome, large inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a transfected plasmid. One option is to eliminate the essential viral vector gene, which results in the inability to replicate, unless the El gene is provided by the host cell. Human 293 cells infected with the adenovirus vector (ATCC Nos. CRL-1573, 45504, 45505), for example, can be grown as adherent cells or in suspension cultures at a relatively high cell density, to produce significant amounts of the protein (see, Garnier et al., Cy tote chn ol., 15:45 (1994)). The genes Zs i g 67 can also be expressed in other higher eukaryotic cells, such as cells of birds, insects, yeasts, or plants. The baculovirus system provides an efficient means to introduce cloned Zsig67 genes into insect cells. Suitable expression vectors are based on the virus of multiple nuclear polyhedrosis of Au to graph a calif ornica (AcMNPV), and contain well-known promoters, such as a heat shock (70) protein promoter. Drosophila, the promoter of the immediate early gene of the nuclear polyhedra virus of Autographa cali fornica (ie-1) and the delayed early promoter 39K, the promoter of the plO virus, and the promoter of Drosophila metallothionein. A second method of preparing recombinant baculoviruses uses transposon-based systems, described by Luckow (Luckow et al., J. Virol. 67: 4566 (1993)). This system, which uses the transfer vectors, is sold in the BAC-to-BAC equipment (Life Technologies, Rockville, MD). This system uses a transfer vector, PFASTBAC (Life Technologies), which contains a Tn7 transposon to move the DNA encoding the Zsig67 polypeptide to a baculovirus genome, maintained in E. coli, as a large plasmid, called "bacmido" . See Hill-Perkins and Posee, J. Gen. Virol. 71: 971 (1990), Bronning et al. , J. Gen. Virol 75: 155 (1994), and Chazenbalk, and Rapoport, J. Biol. Chem. 270: 1545 (1995). In addition, transfer factors can include a function in the structure with the DNA encoding one end of the epitope on the C- or N-terminus of the expressed Zsig67 polypeptide, eg, one end of the Glu-Glu epitope (Gr us senmeyer et al., Pro c. Na t 'l Acad. S ci USA 82: 1952 (1985)). Using a technique known in the art, a transfer vector containing a Zsig67 gene is transformed into E. col i, and is selected by bacmidos, which contain an interrupted recombinant baculovirus indicative l a c z gene. The DNA of the bacmido, which contains the genome of the recombinant baculovirus, is then isolated, using the common techniques. The illustrative PFASTBAC vector can be modified to a considerable degree. For example, the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as the Peor promoter, p6.9 or MP), which is expressed earlier in baculovirus infection, and has been shown be advantageous for expressing the secreted proteins (see, for example, Hill-Perkins and Posee, J. Gen. Virol. 71: 971 (1990), Bonning et al., J. Gen. Virol 75: 1551 (1994), and Chazenbalk and Rapoport, J. Biol. Chem. 270: 1543 (1995) In such constructs of the transfer vector, a long or short version of the basic protein promoter can be used.In addition, the transfer vectors can be constructed, which replace the secretory signal sequences native to Zsig67, with the sequences of the secretory signal derived from insect proteins, for example, a secretory signal sequence from Ecdysteroid Gluco if 11 r as f er asa (EGT), Melitin from honey bee (Invitrogen Corporation; Carisb ad, or the gp67 baculovirus (Pharmingen: San Diego, CA), can be used in constructs, to replace the secretory signal sequence of native Zsig67. The virus or recombinant bacmido is used to transfect the host cells. Suitable host cells include cell lines derived from IPLB-S / -21, a pupal ovarian cell line from Spodoptera fru gip e rda, such as S / 9 (ATCC CRL 1711), S / 21AE, and S / 21 (Invitrogen Corporation; San diego CA), as well as the Schneider-2 cells of Dro s ophila, and the HIGH FIVEO cell line (Invitrogen), derived from Tri ch op lusiani (US Patent No. 5,300,435). Serum-free medium, commercially available, can be used to grow and maintain cells. Suitable media for Sf9 cells are the Sf900 II ™ (Life Technologies) or the ESF 921 ™ (Expression Systems); and the ExcellO405 ™ (JRH Biosciences, Lenexa, KS) or Expresss FiveO ™ (Life Technologies) for T cells. neither . When recombinant viruses are used, the cells typically grow to an inoculation density of about 2-5 x 10 5 cells at a density of 1-2 X 10 6 cells, at which time, a recombinant viral supply is added to a multiplicity of infection (MOI). ) from 0.1 to 10, more typically close to 3. Established techniques for producing recombinant proteins in baculovirus systems, are provided by Bailey et al. , "Manipulation of Baculovirus Vectors", in Methods in Molecular Biology, Volume 7: Gen Transfer and Expression Protocols, Murria (ed.), Pages 147-168 (The Humana Press, Inc. 1991), by Patel et al., "The Baculovirus Expression System", in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al., (eds.), pages 205-244 (Oxford University Press 1995), by Ausubel (1995) on pages 16-57, by Richardson (ed.), Baculovirus Expression Protocols (The Humana Press, Inc. 1995), and by Lucknow, "Insect Cell Expression Technology," in Protein Engineering: Principies and Practice, Cleland et al., (Eds.), Pages 183-218 (John Wiley &Sons, Inc., 1996). Fungal cells, including yeast cells, can also be used to express the genes described herein. Yeast species of particular interest in this regard include Saccha romyces cerevisiae, Pichia pastoris and Pichia methanol ica. Promoters suitable for expression in yeast include GALl promoters (galactose), PGK (phylogeny or kinase), ADH (alcohol dehydrogenase), AOXI (alcohol oxidase), HIS4 (Histidinol dehydrogenase), and the like. Many yeast cloning vectors have been designed and are available. These vectors include vectors based on YIp, such as vectors YIp5, YRp, such as vectors YRpl7, YEp, vectors such as YEpl3 and YCp, such as YCpl9. Methods for transforming S. cerevisiae cells with the endogenous DNA and producing the recombinant polypeptides thereof are described by, for example, Kawasaki, US Patent No. 4,599,311, Kawasaki et al., US 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. the transformed cells are selectable by their determined phenotype, by a selectable marker, commonly, drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A system for use in Saccharomyces cerevisiae is the POTl vector system, described by Kawasaki et al., (US Patent No. 4,931,373), which allows transformed cells to be selected by growing in a medium containing glucose. Additional suitable promoters and terminators, for use in yeast, include those genes for the glycolytic enzyme (see, for example, Kawasaki, US Patent No. 4,599,311, Klingsman et al., US Patent No. 4,615,974 and Bitter, Patent). North American No. 4,977,092) and the alcohol dehydrogenase genes. See also US Patents Nos. 4,990,446, 5,063,154, 5,139,936 and 4,661,454. The shaping systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces f ragilis, Ustilago maydi s, Pichia pastoris, Pichia methanolica, Pichia gu i llermondi i and Candida maltose are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132: 3459 (1986) and Cregg, U.S. Patent No. 4,882,279. The Aspergillus cells can be used according to the methods of McKnight et al., US Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are described by Sumino et al., US Patent No. 5,162,228. Methods for transforming Neurospora are described by Lambowitz et al., Patent North American No. 4,486,533. For example, the use of Pichia methanolica as a host for the production of recombinant proteins is described by Raymond, US Patent No. 5,716,808, Raymond US Patent No. 5,736,383, Raymond et al., Yeast 14: 11-23 (1998), and in international publications we. 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 the production of polypeptides 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-alcohol utilization gene. methanolica (AUG1 or AUG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitate the integration of the DNA within the host chromosomes, it is preferred that they have the complete expression segment of the plasmid flanked at both ends by the DNA sequences. A selectable marker suitable for use in Pichia methanol ica is an ADE2 gene from P. methanolica, which encodes phosphoryl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21) and which allows ade2 host cells, 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 the methanol utilization genes (AUGI and AUG2) are removed. For the production of secreted proteins, host cells deficient in vacuolar protease genes (PEP 4 and PRB1) are preferred. Electroporation is used to facilitate the production of a plasmid containing the DNA encoding a polypeptide of interest in P. methanolica cells. P. methanolica cells can be transformed by electroporation, using a pulsed electric field, which decays exponentially, having a field concentration of 2.5 to 4.5 kV / cm, preferably about 3.75 kV / cm and a constant time (t) from 1 to 40 milliseconds, more preferably approximately 20 milliseconds. Expression vectors can also be introduced into plant protoplasts, intact plant tissues or cells isolated from plants. Methods for introducing expression vectors into plant tissues include direct infection or co-culture of plant tissues with Agroba cterium tumefaciens, microprojectile-mediated distribution, DNA injection, electroporation, and the like. See, for example, Horsch et al., Science 227: 1229 (1985), Klein et al., Biotechnology. : 268 (1992), and Miki et al., "Procedures for Introducing Foreign DNA into Plants," in Methods in Plant Molecular Biology and Biotechnology Glick et al. , (eds.), pages 67-88 (CRC Press, 1993). Alternatively, the Zsig67 genes can be expressed in prokaryotic host cells. Suitable promoters, which can be used to express Zsig67 polypeptides, in host prokaryotic cells, are well known to those skilled in the art and include promoters capable of recognizing the T4, T3, Sp6 and T7 polyperases, PR promoters. and PL of the bacteriophage lambda, the trp, recA, heat shock, lacUV5, tac, Ipp-lacSpr, phoA and lacZ promoters from E. coli, the promoters of B. subtilis, the promoters of Bacillus bacteriophages, the promoters of Strep toipyces, the int promoter of bacteriophage lambda, the bla promoter of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene. Prokaryotic promoters have been reviewed by Glick, J. Ind. Microbiol. 1: 211 (1987), Watson et al., Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), and by Ausubel et al., (1995).

Preferred prokaryotic hosts include E. coli and Bacillus subtilis. Suitable E. coli strains include BL21 (DE3), BL21 (DE3) pLysS, BL21 (DE 3) pLy s E, DHl, DH4I, DH5, DH5I, DH5IF ', DH5IMCR, DH10B, DH10B / p3, DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451 and ER1647 (See, for example, Brown (ed.), Molecular Biology Labfax (Academic Press 1991)). Suitable strains of Bacillus subtilis include BR151, YB886, MI119, MI120, and B179 (see, for example, Hardy, "Bacillus Cloning Methods," in DNA Cloning: A Practical Approach, (Glover (ed.) (LRL Press 1985 )). When a Zsig67 polypeptide is expressed in a bacterium, such as E. coli, the polypeptide can be retained in the cytoplasm, typically as insoluble granules, or it can be directed to the periplasmic space by a bacterial secretion sequence. In the first case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isocyanate or urea The denatured polypeptide can then be refolded and dimerized by diluting the denatured, 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 breaking the cells (for example, by sonication or osmotic shock) to release the contents of the periplasmic space and recover the protein, thereby obviating the need for denaturation and refolding. Methods for expressing proteins in prokaryotic hosts are well known to those skilled in the art (see, for example, Williams et al., "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies, "in DNA Cloning 2: Exprés ion Systems, 2nd Edition, Glover et al., (eds.), page 15 (Oxford University Press), Ward et al.," Genetic Manipulation and Expression of Antibodies, "in Mocional Antibodies: Principies and Applications, page 137 (Wiley-Liss, Inc 1995), and Georgiou, "Expression of Proteins in Bacteria," in Protein Engineering: Principies and Practice, Cleland et al., (Eds.) Page 101 (John Wiley &Sons, Inc. 1996)). Standard methods for introducing expression vectors into cells of bacteria, yeast, insects and plants are provided, for example, by Ausubel (1995). General methods for expressing and recovering foreign proteins, produced by mammalian cell systems, are provided in, for example, Etcheverry, "Expression of Engineered Proteins in Mammalian Cell Culture," in Protein Engineering: Principles and Practice, Cleland et al., (Eds.), Pages 163 (Wiley-Liss, Inc 1996). Standard techniques for recovering the protein produced by a bacterial system are provided, for example, by Grisshammer et al. , "Purification of over-produced proteins from E. coli cells," in DNA Cloning 2: Expression Systems, 2nd Edition, Glovert et al., (Eds.), Pages 59-92 (Oxford University Press 1995). Established methods for isolating recombinant proteins from a baculovirus system are described by Richadson (ed.), Baculovirus Express Protocols (The Humana Press, Inc. 1995). As an alternative, the polypeptides of the present invention can be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. These synthesis methods are well known to those skilled in the art (see, for example, Merrifield, J. Am. Chem. Soc. 85: 2149 (1963), Stewart et al., "Solid Phase Peptide Synthesis" (2nd Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept. Prot. 3: 3 (1986), Atherton et al., Solid Phase Peptide Synthesis: A practical Approach (IRL Press 1989), Fields and Colowick, "Solid-Phase Peptide Synthesis," Methods in Enzymology volume 289 (Academic Press 1997) , and Loyd-Wi 1 liams et al., Chemical Approaches to the Synthesis of Peptides and Proteins (CRC Press, Inc. 1997). Variations in the strategies of total chemical synthesis, such as "native chemical ligation" and "ligation of expressed proteins" are also standard (see, for example, Dawson et al., Science 266: 116 (1994), Hackenberg et al. al., Proc. Nat'l. Acad. Sci. USA 94: 7845 (1997), Dawson, Methods Enzymol., 287: 34 (1997), Muir et al., Proc. Nat'l. Acad. Sci. USA ( 1998), and Severinov and Muir, J. Biol. Chem 273: 16205 (1998)). The peptides and polypeptides of the present invention comprise at least six, at least nine, or at least 15 contiguous amino acid residues of SEQ ID NO: 2. As an illustration, the polypeptides may comprise at least six, at least nine or at least 15 contiguous amino acid residues of any of the amino acid sequences of SEQ ID NO: 2, amino acid residues 29 to 104, amino acid residues 52 to 104, amino acid residues 52 to 85, amino acid residues 68 to 104 and amino acid residues 73 to 104. Within certain embodiments, of the present invention, the polypeptides comprise 20, 30, 40, 50, or more contiguous residues of these amino acid sequences. The nucleic acid molecules encoding such peptides and polypeptides are employed as primers and polymerase chain reaction probes.

Isolation of Polypeptides from Zsig67 It is preferred to purify the polypeptides of the present invention at least about 80% purity, up to at least about 90% purity, up to at least 95% purity or even, greater than 95% purity, with respect to macromolecules contaminants, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. The polypeptides of the present invention can also be purified to a pharmaceutically pure state, which is greater than 99.9% pure. Certain preparations can purify polypeptides that are substantially free of other polypeptides, particularly other polypeptides of animal origin. Fractional and / or conventional purification methods can be used to obtain preparations of purified Zsig67 from natural sources (eg, thymus tissue), and recombinant Zsig67 polypeptides and fusion Zsig67 polypeptides purified from recombinant host cells. Numerous methods for purifying proteins are known in the art. In general, precipitation with ammonium sulfate and acid extraction or chaotrope can be used for the fractionation of samples. The exemplary purification steps include hydroxyapatite chromatography, size exclusion, FPLC and reverse phase high resolution liquid chromatography. Suitable chroma tigraphic media include derivatized dextrans, agarose, cellulose, polyacrylamide, special silicas and the like. PEI, DEAE QAE and Q derivatives are preferred. Exemplary chromatographic media include those media derivatized with phenyl, butyl or octyl groups, such as Feni 1-Se lamps to FF (Pharmacia), Toyopearl butyl 650 (Toso Hass, Mo t gomeryvi 1 le, PA), Oct il-S ef arosa (pharmacia) and the like; or the poly-acrylic queens, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, crosslinked agarose beads, polystyrene beads, crosslinked polyacrylamide resins and the like which are insoluble under the conditions in which they will be used. . These supports can be modified with reactive groups that allow the binding of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and / or carbohydrate moieties. Examples of coupling chemistries include activation of cyanogen bromide, activation of N-hydroxysuccinimide, activation of epoxides, activation of sulfhydryl, activation of hydrazide, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial distributors. The selection of a particular method for the isolation of the polypeptides and their purification is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principies & Methods (Pharmacia LKB Biotechnology 1988), and Doonan, Protein Purification Protocols (The Humana Press 1996). Additional variations in the isolation and purification of Zsig67 can be projected by those skilled in the art. For example, anti-Zsig67 antibodies, obtained as is. described below, can be used to isolate large amounts of protein by immunoaffinity purification. In addition, methods for binding receptors, such as Zsig67, to receptor polypeptides linked to support media are well known in the art.

The polypeptides of the present invention can also be isolated by exploiting their particular properties. For example, chromatography by adsorption of immobilized metal ions (IMAC) can be used to purify histidine rich proteins, including those ends comprising polyhistidine ends. Briefly, a gel is charged first with divalent metal ions, to form a chelate (Sulkowski, Tren ds i n Bi o ch em 3: 1 (1985)). The proteins rich in histidine will be adsorbed by this matrix with different affinities, depending on the metal ions used, and will be eluted by competitive elution, lowering the pH, or using strong chelating agents. Other purification methods include the purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (M. Deutscher, (ed.), Me., In zym or 1. 182: 529 (1990)). Within the additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity end (e.g., the maltose binding protein, an immunoglobulin domain), can be constructed to facilitate purification. The Zsig67 polypeptides, or fragments thereof, can be prepared through chemical synthesis, as described below. The Zsig67 polypeptides can be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial amino acid residue of methionine. 8. Analogs of Zsig67 and the receiver Zsig67 A general class of Zsig67 analogs are variants that have an amino acid sequence that is a mutation of the amino acid sequence described herein.

Another general class of Zsig67 analogs is provided by anti-idiotype antibodies, and fragments thereof as described below. However, recombinant antibodies comprising variable anti-idiotype domains can be used as analogues (see for example, Monfardini et al., Proc. Assoc. Am.

Physicians 108: 420 (1996)). Since the variable domains of anti-idiotype Zsig67 antibodies mimic Z s ig67, these domains can provide either Zsig67 agonists or antagonistic activity. As an illustration, Lim and Langer, J. Interferon Res. 13: 295 (1993), describe anti-idiotypic interferon-a antibodies that have the properties of either interferon-α agonists or antagonists. Another method for identifying analogs of Zsig67 is provided by the use of combinatorial libraries. Methods for constructing and selecting for deployment of phage and other combinatorial libraries are provided, for example, by Kay et al., Phage Display of Peptides and Proteins (Academic Press 1996), Verdine, U.S. Patent No. 5,783,384, Kay et al., U.S. Patent No. 5,747,334 and Kauffman et al., U.S. Patent No. 5,723,323. Zsig67 and its analogues can be used to identify and isolate Z s ig67 receptors. For example, the proteins and peptides of the present invention can be immobilized on a column and used to bind receptor proteins of the membrane preparations that are run on the column (Hermanson et al., (Eds.), Immobilized Affinity Ligand Techniques, pages 195-202 (Academic Press 1992)). Affinity tagged or radiolabelled Zsig67 polypeptides can also be used to identify, or localize Zsig67 receptors in a biological sample (see for example, Deutscher (ed.), Methods in Enzymol., Vol.182, pages 721-37 (Academic Press 1990); Brunner et al., Ann. Rev. Biochem 62: 483 (1993); Fedan et al., Biochem. Pharmacol. 33: 1161 (1984)). Also see, Varthakavi and Minocha, < J. Gen. Virol. 77: 1875 (1996), who describe the use of anti-idiotype antibodies for the identification of receptors. As a receptor ligand, the activity of Zsig67 can be measured by a silicon-based biosensor microphysiometer, which measures the rate of extracellular acidification or proton excretion associated with receptor binding and subsequent cellular responses. An exemplary device is the CYTOSENSOR Microfiometer, manufactured by Molecular Devices Corp. (Sunnyvale, CA). A variety of cellular responses can be measured by this method, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like (see for example, McConnell et al., Science 257: 1906 (1992), Pitchford et al., Meth. Enzymol 228: 84 (1997), Arimilli et al., J. Immunol., Meth. 212: 49 (1998), and Van Liefde et al., Eur. J. Pharmacol 346: 81 (1998)). However, the microphysiometer can be used to test adherent or non-adherent eukaryotic cells. Since energy metabolism is coupled with the use of cellular ATP, any event which alters cellular ATP levels, such as receptor activation and initiation of signal transduction, will cause a change in the cellular acid section . By measuring the changes of extracellular acidification in the cell medium over time, therefore, the microphysiometer directly measures cellular responses to various stimuli, including Z s ig67, its agonists or antagonists. Preferably, the microphysiometer is used to measure responses of a eukaryotic cell responsive to Zs ig 67, compared to a control eukaryotic cell that does not respond to the Zsig67 polypeptide. Eukaryotic cells responsive to Zsig67 comprise cells in which a receptor for Zsig67 has been transfected to create a cell that is sensitive to Zs ig 67, or cells that are naturally sensitive to Z s ig67. The cellular responses modulated by Zsig67 are measured by a change (for example, an increase or decrease in extracellular acidification) in the response of cells exposed to Zsig67, compared to control cells that have not been exposed to Zs ig 7. Consequently, a microphysiometer can be used to identify cells, tissues or cell lines, which respond to a trajectory stimulated by Zsig67, and which express a functional Zsig67 receptor. As an illustration, cells expressing a functional Zsig67 receptor can be identified by (a) providing test cells, (b) incubating a first portion of the test cells in the absence of Z s ig 67, (c) incubating a second portion of the test cells in the presence of Zsig67, and (d) detecting a change (e.g., an increase or decrease in the rate of extracellular acidification, as measured by a microphysiometer) in a cellular response of the second portion of the test cells, compared to the first portion of the test cells, wherein such a change in the cellular response indicates that the test cells express a functional Zsig67 receptor. An additional negative control can be included in which, a portion of the test cells is incubated with Zsig67 and an anti-Zsi g57 antibody to inhibit the binding of Zsig67 with its congnato receptor. The microphysiometer also provides a means to identify Zsig67 agonists. For example, Zsig67 agonists can be identified by a method comprising the steps of (a) providing cells sensitive to Zsig 67, (b) incubating a first portion of the cells in the absence of a test compound, (c) incubate a second portion of the cells in the presence of a test compound, and (d) detect a change, for example, an increase or decrease in a cellular response of the second portion of the cells compared to the first portion of the cells, wherein such a change in cellular response indicates that the test compound is a Zsig67 agonist. An illustrative change in the cellular response is a measurable change in the rate of extracellular acidification, as measured by a microphysiometer. However, incubation of a third portion of the cells in the presence of a Zsig67 and in the absence of a test compound, can be used as a positive control for cells sensitive to Zsig67, and as a control to compare activity agonist of a test compound with that of Z s ig 67. An additional control may be included in which, a portion of the cells are incubated with a test compound (or Zsig67) and an anti-Zsig67 antibody to inhibit the binding of the test compound (or Zsig67) with the Zsig67 receptor. A genetic product of variant Zsig67 that lacks biological activity, can be a Zsig67 antagonist. These biologically inactive Zsig67 variants can be initially identified on the basis of hybridization assays, determination of sequence identity, or by the ability to specifically bind an anti-Z s Ig67 antibody. A Zsig67 antagonist may also be characterized by its ability to inhibit the biological response induced by Zsig67 or by a Zsig67 agonist. This inhibitory effect may result, for example, from competitive or non-competitive binding of the antagonist to the receptor Z s ig67. The microphysiometer provides a means to identify antagonists of Zs ig 67. For example, Zsig67 antagonists can be identified by a method, comprising the steps of (a) providing Zsig67 sensitive cells, (b) incubating a first portion of cells in the presence of Zsig67 and in the absence of a test compound, (c) incubate a second portion of the cells in the presence of both Zsig67 and the test compound, and (d) compare the cellular responses of the first and second cell portions, wherein a decrease in the response by the second portion, compared to the response of the first portion, indicate that the test compound is a Z67 antagonist. An illustrative change in cellular response is an extracellular acidification rate of measurable change, as measured by a microphysiometer. Zs ig 67, its agonists and antagonists, are valuable both in vivo and in vitro. For example, Zsig67 and its agonists can be used to supplement the serum-free medium, while antagonists of Zsig67 are used as search reagents for the characterization of interaction sites between Zsig67 and its receptor. In a therapeutic set, pharmaceutical compositions comprising Zsig67 antagonists can be used to inhibit the activity of Zsig67. The present invention also contemplates chemically modified Zsig67 compositions, in which a Zsig67 polypeptide is bound to a polymer. Illustrative Zsig67 polypeptides include polypeptides comprising amino acid residues 52 to 85 of SEQ ID NO: 2. Typically, the polymer is soluble in water, so that the Zsig67 conjugate is not precipitated in an aqueous environment, such as a physiological environment . An example of a suitable polymer is one that has been modified to have a single reactive group, such as an active ester for acylation, or an aldehyde for alkylation. In this way, the degree of polymerization can be controlled. An example of a reactive aldehyde is propionaldehyde of polyethylene glycol, or monoalkoxy (C _-C? O), or alkoxy derivatives thereof (see for example, Harris, et al., US Patent No. ,252,714). The polymer can be branched or unbranched. In addition, a mixture of polymers can be used to produce Z s ig 67 conjugates.

The Zsig67 conjugates used for therapy may comprise pharmaceutically acceptable portions of water soluble polymers. Suitable water-soluble polymers include polyethylene glycol (PEG), monomethoxy-PEG, (C1-C10) monoalkoxy-PEG, aryloxy-PEG, poly- (N-vinylpyrrolidone) PEG, tresyl monomethoxy PEG, propionaldehyde PEG, bis carbonate -succinimidyl PEG, homopolymers of propylene glycol, a copolymer of polypropylene oxide / ethylene oxide, polyoxyethylated polyols (for example, glycerol), polyvinyl alcohol, dextran, cellulose or other polymers based on carbohydrates. Suitable PEG can have a molecular weight of from about 600 to about 60,000, including, for example, 5,000, 12,000, 20,000 and 25,000. A Zsig67 conjugate can also comprise a mixture of water soluble polymers. An example of a Zsig67 conjugate comprises a Zsig67 moiety and a polyalkyl oxide moiety attached to the N-terminus of the Zs ig 6 moiety. PEG is a suitable polyalkyl oxide. As an illustration, the Zsig67 can be modified with PEG, a process known as "PEGylation". Pegylation of Zsig67 can be carried out by any of the pegylation reactions known in the art (see for example, EP 0 154 316, Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9: 249 (1992), Duncan and Spreafico, Clin. Pharmacokinet 27: 290 (1994), and Francis et al., Int J Hematol 68: 1 (1998) For example, PEGylation can be performed by an acylation reaction or by an alkylation reaction with a molecule of reactive polyethylene glycol In an alternative procedure, the Zsig67 conjugates are formed by condensation of the activated PEG, in which, a hydroxy or amino terminal group of PEG has been replaced by an activated linker (see for example, Karasiewicz et al., Patent No. 5,382,657) PEGylation by acylation typically requires reacting an active ester derived from PEG with a Zsig67 polypeptide An example of an activated PEG ester is PEG esterified to N-hydroxysuccinimide. As used herein, the term "acylation" includes the following types of bonds between Zsig67 and a water soluble polymer: amide, carbamate, urethane, and the like. Methods for preparing PEGylated Zsig67 by acylation will typically comprise the steps of (a) reacting a Zsig67 polypeptide with PEG (such as a reactive ester of an aldehyde derivative of PEG) under conditions, thereby one or more PEG groups are bound to Z sig 67, and (b) obtain the reaction products. In general, the optimal reaction conditions for the acylation reactions will be determined based on the known parameters and the desired results. For example, the largest PEG ratio: Z s i g 67, the highest percentage of the product Z s i g 67 polyPEGylated. The product of PEGylation by acylation is typically a product of poly-acylated Zs i g 67, wherein the e-amino lysine groups are PEGylated via an acyl bonding group. An example of a connection link is an amide. Typically, the resulting Zs i g 67 will be at least 95% mono, di or tri-pegylated, although some species with higher degrees of PEGylation can be formed depending on the reaction conditions. The PEGylated species can be separated from the conjugated Z s i g 67 polypeptides using standard purification methods, such as dialysis, ultrafiltration, ion exchange chromatography, affinity chromatography and the like. PEGylation by alkylation generally involves reacting a terminal aldehyde derivative of PEG with Z s i g 67 in the presence of a reducing agent. The PEG groups can be linked to the polypeptide via the -CH2-NH group. Derivatization via reductive alkylation, to produce a monoPEGylated product takes advantage of the differential reactivity of different types of primary amino groups available for derivatization. Typically, the reaction is carried out at a pH which allows one to take advantage of the pKa differences between the e-amino groups of the lysine residues and the group examined from the N-terminal residue of the protein. By such selective derivatization, the binding of a water-soluble polymer containing a reactive group such as an aldehyde to a protein is controlled. The conjugation with the polymer occurs predominantly at the N-terminus of the protein without significant modification of the other reactive groups, such as the amino groups of the side chain of lysine. The present invention provides a substantially homogeneous preparation of the monopolymer conjugates of Z s i g 67. Reductive alkylation to produce a substantially homogeneous population of a monopolymer Zs ig 67 conjugate molecule may comprise the steps of: (a) reacting a Zs ig 67 polypeptide with a reactive PEG under reductive alkylation conditions at a pH suitable for allow sel-ective modification of the a-amino group al. amino terminus of Zs i g 67, and (b) obtain the reaction products. The reducing agent used for reductive alkylation should be stable in aqueous solution and capable of reducing only the Schiff base formed in the initial process of reductive alkylation. Exemplary reducing agents include sodium borohydride, sodium cyanborohydride, dimethylamine borane, trimethylamine borane and pyridine borane. For a substantially homogeneous population of conjugates of Z s i g 67 monopolymers, the reductive alkylation reaction conditions are those which allow selective binding of the soluble polymer portion to the N-terminus of Zs i g 6 7. Such reaction conditions are generally provided for the pKa differences between the amino groups of lysine and the a-amino group at the N-terminus. The pH also affects the ratio of the polymer to the protein to be used. In general, if the pH is low, a large excess of the polymer to the protein will be desired because if the N-terminus group is less reactive, more polymer is needed to reach the optimum conditions. If the pH is higher, the polymer: Z s i g 67 does not need to be that big because. more reactive groups are available. Typically, the pH will fall within the range of 3 to 9, or 3 to 6. Other. factor to consider is the molecular weight. of the water soluble polymer. In general, the high molecular weight of the polymer, the few numbers of polymer molecules that can be bound to the protein. For PEGylation reactions, the typical molecular weight is from about 2 kDa to about 100 kDa, about 5 kDa up to about 50 kDa, or about 12 kDa up to about 25 kDa. The molar ratio of the water soluble polymer to Z s i g 67 will generally be in the range of 1: 1 to 100: 1. Typically, the molar ratio of the water soluble polymer to Zs i g 67, will be 1: 1 to 20: 1 for polyPEGylation, and 1: 1 to 5: 1 for moniPEGylation. General methods for producing conjugates comprising a polypeptide and portions of water-soluble polymers are well known in the art. See for example, Karasiewicz et al., U.S. Patent No. 5,382,657, Greenwald et al., U.S. Patent No. 5,738,846, Nieforth et al., Clin. Pharmacol. Ther. 59: 636 (1996), Monkarsh et al., Anal. Biochem. 247: 434 (1997)). The present invention contemplates compositions comprising a peptide or polypeptide described herein. Such compositions may further comprise a carrier. The carrier can be a conventional organic or inorganic carrier. Examples of carriers include water, buffer, alcohol, propylene glycol, macrogol, sesame oil, corn oil, and similar. 9. Preparation of Signaling Compositions Zsig67 A. Zsig67 Conjugates and Signaling Fusion Proteins Zsig67 A signaling composition Zsig67 comprises a Zsig67 component and a cytotoxin. Such signaling compositions can be used for the removal of tissue, a method that is employed in both experimental and therapeutic sets. Either the Zsig67 component or the cytotoxin, or both, the compound Zsig67 and the cytotoxin, can be conjugated with a soluble polymer, as discussed above. For example, polypeptide cytotoxins can be conjugated to a soluble polymer either before or after conjugation to a Zsig67 component. The soluble polymers may also be conjugated to the signaling fusion proteins Zsig67. An example of a suitable polypeptide cytotoxin is a ribosine inactivation protein. The proteins of inactivation of the ribosime Type I, are proteins of unique chains, while the proteins of inactivation of the ribosim Type II, consist of two subunits not identical (chains A and B), united by a disulfide bond (for a review, see Soria et al., Targeted Diagn. Ther.7: 193 (1992)). Type I ribosomal inactivation proteins include Saponaria officina lys polypeptides (e.g., saporin-1, saporin-2, saporin-3, saporin-6), Momordica charan tia (e.g., momordin), Byronia dioica (e.g. , bryodin, bryodin-2, Tri chosan thes kirilowii (for example, trichosantin, trichokirin), Gelonium mul tiflorum (for example, gelonin), Phytola cca americana (for example, antiviral protein of rotten weeds, proteins-II antiviral of malas rotten grasses, antiviral S-protein from rotten weeds), Phytola cca dodecandra (e.g., dodecandrin, Mirabilis antiviral protein), and the like .. Ribosim inactivating proteins are described, for example, by Walsh et al., US Patent No. 5, 635, 384. Suitable type II ribosime inactivation proteins include Ricinus communi polypeptides (eg, ricin), Abrus preca torius (eg, abrin), Adenia digi tata (eg, the modeccina), and the like. Since ribosime type II inactivation proteins include a chain that binds galactosidases and a toxic A chain that depletes adenosine, conjugates of the type II ribosomal inactivation protein should include the A chain. Additional ribosine inactivation proteins employed include bouganin, lavine, corn ribosomal inactivation proteins, vaccinia pyramidata ribosine inactivation proteins, nigrin b, basic nigrin 1, ebulin, racemosin b, lufina-a, lufina-b, lufina-S, and other ribosomal inactivation proteins known to those skilled in the art. See, for example, Bolognesi and Stirpe, International Application No. WO 98/55623, Colnaghi et al., International Application No. WO 97/49726, Hey et al., United States Patent No. 5, 635,384, Bolognesi and Stirpe, International Application. No. WO95 / 07297, Arias et al., International Publication No. WO94 / 20540, Watanabe et al., J. Biochem. 106: 6 977 (1989); Islam et al., Agrie. Biol. Chem. 55: 229 (1991), and Gao et al., FEBS Lett. 347: 257 (1994). The analogs and variants of naturally occurring ribosim inactivating proteins are also suitable for the signaling compositions described herein, and such proteins are known to those skilled in the art. Ribosome inactivation proteins can be produced using publicly available nucleotide and amino acid sequences. As an illustration, a nucleotide sequence encoding saprin-6 is described by Lorenzetti et al., U.S. Patent No. 5,529,932, while Walsh et al. , U.S. Patent No. 5,635,384, describes amino acid and nucleotide sequences of proteins inactivating the ribosim of barley and corn. Nevertheless, the proteins of inactivation of the ribosime are also commercially available. Another group of polypeptide cytotoxins includes immunomodulators. Zsig67 signaling compositions that include an immunomodulator, provide a means for delivering an immunomodulator to a target cell and are particularly employed against tumor cells. The cytotoxic effects of immunomodulators are well known to those skilled in the art. See, for example, Klegerman et al. , "Lymphokines and Monokines", in Biotechnology and Pharma cy, Pessuto et al. , (eds.), pages 53-70 (Chapman & amp;; Hall 1993). As an illustration, interferons can exhibit cell proliferation by inducing decreased expression of class I histocompatibility antigens on the surface of several cells and also increase the rate of cell destruction by cytotoxic T lymphocytes. In addition, tumor necrosis factors, such as tumor necrosis factor-a, are believed to produce cytotoxic effects by induction of DNA fragmentation. Additional polypeptide cytotoxins include ribonuclease, DNase I, Staphylococcal enterotoxin-A, diphtheria toxin, exotoxin Pseudomonas, and Pseudomonas endotoxin. See, for example, Pastan et al., Cell 47: 541 (1986), and Goldenberg, CA - A Cancer Journal for Clinicians 44:43 (1994). Other suitable toxins are known to those skilled in the art. The conjugates of the cytotoxic polypeptides and the Zsig67 components can be prepared using standard techniques for the conjugation of polypeptides. As an illustration of the general procedure, methods for the conjugation of FGF with saporin are described by Lappi et al., Biochem. Biophys. Res. Common. 160: 911 (1989), Soria et al., Targeted Diagn. Ther. 7: 193 (1992), Buechler et al., Eur. J. Biochem. 234: 106 (1995), Behar-Cohen et al., Invest. Ophthalmol. Vis. Sci. 35: 2434 (1995), Lappi and Baird, U.S. Patent No. 5,191,067, Calabresi et al., U.S. Patent No. 5,478,804, and Lappi and Baird, U.S. Patent No. ,576,288. Additional procedures for conjugation of polypeptides are known to those skilled in the art. For example Lam and Kelleher, U.S. Patent No. 5,055,291, describes the production of antibodies conjugated with either the A fragment of the diphtheria toxin or ricin toxin. The Zsig67 signaling fusion proteins can also be produced using standard techniques as discussed above. As an illustration, the recombinant saporin FGF proteins are described by Lappi et al. , J. Biol. Chem. 259: 12552 (1994), Behar-Cohen et al. , Invest. Oph thalmol. Vis. Sci. 36: 2434 (1995), McDonald et al. , Protein Expr. Purif 8: 91 (1996), and Lappi et al. , United States Patent No. 5,916,772. In a similar way, Landgraf et al. , Biochemistry 37: 3220 (1998), produces a fusion protein comprising a portion of the epidermal growth factor and the diphtheria toxin. Methods for the preparation of fusion proteins comprising a portion of cytotoxic polypeptide are well known in the art of production of antibody-toxin fusion proteins. For example, fusion proteins of Pseudomonas antibody exotoxin A have been described by Chaudhary et al., Nature 339: 394 (1989), Brinkmann et al., Proc. Nati Acad. Sci. USA 88: 8616 (1991), Batra et al., Proc. Nati Acad. Sci. USA 89: 5867 (1992), Friedman et al., J. Immunol. 150: 3054 (1993), Wels et al., Int. J. Can. 60: 131 (1995), Fominaya et al., J. Biol. Chem. 271: 10560 (1996), Kuan et al., Biochemistry 35: 2872 (1996), and Schmidt et al., Int. J. Can. 55: 538 (1996). Antibody-toxin fusion proteins containing a diphtheria toxin moiety have been described by Kreitman et al., Leukemia 7: 553 (1993), Nicholls et al., J. Biol. Chem. 268: 5302 (1993) , Thompson et al., J. Biol. Chem. 270: 28031 (1995), and Vallera et al., Blood 88: 2342 (1996). Deonarain et al., Targeting Tumor 1: 111 (1995), have described an antibody-toxin fusion protein having an RNase portion, while Linardou et al., Cell Biophys: 24-25: 243 (1994), produce a protein antibody-toxin fusion comprising a component I Dnasa. Gelonin was used as the toxin portion in the antibody-toxin fusion protein of Better et al., J. Biol. Chem. 270: 14951 (1995). As an additional example, Dohlsten et al., Proc. Nati Acad. Sci. USA 91: 8945 (1994), reported an antibody-toxin fusion protein comprising Staphylococcal enterotoxin-A. In addition, antibody fusion proteins comprising an interleukin-2 are described by Boleti et al., Ann. Oncol. 5: 945 (1995), Noicolet et al., Cancer Gener Ther. 2: 161 (1995), Becker et al., Proc. Nati Acad. Sci. USA 93: 7826 (1996), Hank et al., Clin. Cancer Res. 2: 1951 (1996), and Hu et al., Cancer Res. 55: 4998 (1996), while Yang et al., Hum. Antibodies Hybridomas 6: 129 (1995), describes a fusion protein that includes an F (ab ') 2 fragment and a fragment and a portion of tumor necrosis factor-a. These methods can be used to prepare the Zsig67 signal fusion fusion proteins described herein as an alternative to a polypeptide cytotoxin., the Zsig67 signaling compositions may comprise a radioisotope such as the cytotoxin portion. For example, a signaling composition of Zsig67 may comprise a radioisotope emitting α, a radioisotope emitting β, a radioisotope emitting α, an Auger electron emitter, an agent that captures neutrons that emit particles to, or a radioisotope that decays from the capture of the electron. Suitable radioisotopes include 198Au, 199Au, 33P, 33P, 125I, 131I, 123I, 90Y, 186Re, 188Re, 67Cu, 211At, 47Sc, 103Pb, 109Pd, 212Pb, 71Ge, 77As, 105Rh, 113Ag, 119Sb, 121Sn, 131Cs, 143Pr, 161Tb, 177Lu, 1910s, 193MPt, 197Hg, and the like. A radioisotope can be attached to a component Zsig67 directly or indirectly via a chelating agent. For example, 67Cu, is considered one of the most promising radioisotopes for radioimmunotherapy because of its 61.5 hours of half-life and abundant supply of β-particles and β-rays, can be conjugated to a Zsig67 component using the chelating agent, p-brormoacetamido-benzyl-tetraethylaminotetraacetic acid. Chase and Shapiro, "Medical Applications of Radioisotopes," in Gennaro (Eds.), Remington's Pharmaceutical Sciences, 19th Edition, pages 843-865 (Mack Publishing Company 1995). As an alternative, 90Y, which emits a β-energetic particle, may be coupled to a Zsig67 component using diethylenetriaminepentaacetic acid. However, an exemplary method suitable for the direct radiolabelling of a Zsig67 component with 131? is described by Stein et al., Antibody Immunoconj. Radiopharm. 4: 703 (1991).

Alternatively, boron sums such as carboranes can be bound to the Zsig67 components using standard techniques. Another type of cytotoxin suitable for the preparation of the Zsig67 conjugates is a chemotherapeutic drug. Chemotherapeutic drugs include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, antibiotics, epipodophyllotoxins, platinum coordination complexes and the like. Specific examples of the chemotherapeutic drugs include methotrexate, doxorubicin, daunorubicin, cytosinerabinoside, cis-platinum, vindesine, mitomycin, bleomycin, melphalan, chlorambucil, and the like. Suitable chemotherapeutic agents are described in Remington 's Pharma ceu ti cal Sciences, 19th Ed. (Mack Publishing Co., 1995), and in Goodman And Gilman' s The Pharma ceutical Ba sis Of Therapeutics, 7th Ed. (MacMillan Publishing Co. 1985). Other chemotherapeutic agents are known to those skilled in the art. In another procedure, the conjugates of Zsig67 are prepared by conjugating photoactive agents or dyes to a Zsig67 component. Fluorescents and other chromogenes, or dyes, such as forphyrins sensitive to visible light, have been used to detect and treat lesions by directing the appropriate light in the lesion. This type of "photoradiation", "phototherapy", or "photodynamic" therapy is described, for example, by Mew et al., J. Immunol. 130: 1473 (1983), Jori et al, (eds.), Photodynamic Therapy Of Tumors And Other Diseases (Librería Progetto 1985), Oseroff et al., Proc. Na ti. Acad. Sci. USA 83: 8744 (1986), van den Bergh, Chem. Bri tain 22: 430 (1986), Hasan et al., Prog. Clin. Bi ol. Res. 288: 471 (1989), Tatsuta et al., Larsers Surg. Med. 9: 422 (1989), and Pelegrin et al. Cancer 67: 2529 (1991). Another general type of cytotoxins employed is a tyrosine kinase inhibitor. Since the activation of tyrosine kinase proliferation has been suggested to play a role in the development and progression of tumors, this activation can be inhibited by the Zsig67 components that supply tyrosine kinase inhibitors. Suitable tyrosine kinase inhibitors include isoflavones, such as genisteins (5, 7, 4 '-trihydroxyisoflavone), (7,4'-dihydroxyisoflavone) daidzein and biocanin A (4-methoxygenistein), and the like. As an illustration of the general procedure, methods for the conjugation of tyrosine inhibitors to a growth factor are described by Uckun, U.S. Patent No. 5,011,995. The present invention also includes Zsig67 signaling compositions comprising a nucleic acid molecule encoding a cytotoxin. Such signaling compositions may comprise, for example, a polypeptide-polylysine Zsig67 conjugate fused to an expression vector comprising a cytotoxin gene.

B. Signaling Liposomes Zsig67 Liposomes provide a means to deliver therapeutic polypeptides to a subject intravenously, intraperitoneally, intrathecally, intramuscularly, subcutaneously, or via oral administration, inhalation or intranasal administration. Liposomes are microscopic vesicles consisting of one or more lipid bilayers surrounding the aqueous compartments (see, in general, Bakker-Woundenberg et al., Eur J. Clin. My crobiol Infect. Dis 12 (Suppl 2): S61 (1993), Kim, Drugs 46: 618 (1993), and Ranade, "Site-Specific Drug Delivery Using Liposomes as Carriers", in Drug Delivery Sys tems, Ranade and Hollinger (eds.), Pages 3-24 (CRC Press nineteen ninety five)). Liposomes are similar in composition to cell membranes and as a result, liposomes can be administered safely and are biodegradable. Depending on the method of preparation, the liposomes may be unilamellar or multilamellar, and the liposomes may vary in size with diameters ranging from 0.02 μm or greater than 10 μm. A variety of agents can be encapsulated with the liposomes: the partition of the hydrophobic agents in the bilayers and the partition of the hydrophilic agents within the internal aqueous space (s) (see, for example, Machy et al., Liposomes In Cell Biology And Pharma cology (John Libbey 1987), and Ostro et al., Ameri can J. Hosp. Pharm. 46: 1576 (1989)). However, it is possible to control the therapeutic bioavailability of the encapsulated agent by varying the size of the liposome, the number of bilayers, the lipid composition, as well as the charge and surface characteristics of the liposomes. Liposomes can absorb virtually any type of cell and then slowly release the encapsulated agent. Alternatively, an absorbed liposome can be subjected to endocytosis by cells that are phagocytic. Endocytosis is followed "by the intralysosomal degradation of the liposomal lipids and the release of the encapsulated agents (Scherphof et al., Ann. NY Acad. Csi. 446: 368 (1985).) After intravenous administration, the small liposomes (0.1 to 1.0 μm) are typically taken by the cells of the reticuloendothelial system, located mainly in the liver and spleens, while the larger liposomes of 3.0 μm are deposited in the lung.This preferential uptake of the smaller liposomes by the cells The reticuloendothelial system has been used to provide chemotherapeutic agents to macrophages and liver tumors.The reticuloendothelial system can be surrounded by several methods including saturation with large doses of liposome particles or inactivation of selective macrophages by pharmacological means (Claasen et al. ., Biochim, Biophi, Acta 802: 428 (1984)). The incorporation of phospholipids derived from glycolipids or polyethylene glycol in the membranes of liposomes has been shown to result in a significantly reduced uptake by the reticuloendothelial system (Alien et al. , Bi ochim. Biophis. Acta 1068: 133 (1991); Alien et al. , Biochim. Biophys. Acta 1150: 9 (1993)). Liposomes can also be prepared for particular target cells or organs by varying the phospholipid compositions, or by inserting receptors or ligands into the liposomes. For example, liposomes prepared with a high content of nonionic surfactant have been used to signal the liver (Hayakawa et al., Japanese Patent 4 424.018; Kato et al., Biol. Pharm. Bull. 16: 960. (1993). These formulations were prepared by mixing soybean phosphatidylcholine, α-tocopherol, and hydrogenated ethoxylated castor oil (HCO-60) in methanol, concentrating the mixture under vacuum, and then reconstituting the mixture with water. A liposomal formulation of dipalmitoylphosphatidylcholine (DPPC) with a mixture of stearylglucoside derived from soybeans (SG) and cholesterol (Ch), has also been shown to target the liver. (Shimizu et al., Biol. Pharm. Bull. 20: 881 (1997)).

Alternatively, several signaling ligands can be bound to the surface of the liposome such as antibodies, antibody fragments, carbohydrates, vitamins and transport proteins. For example, the liposomes can be modified with branched-type galactosylipid derivatives to the target asialoglycoprotein (galactose) receptors, which are exclusively expressed on the surface of liver cells (Kato and Sugiyama, Cri t Rev. Ther. Drug Carrier System 14: 287 (1997), Murahashi et al., Bi ol., Pharm. Bull. 20: 259 (1997)). Similarly, Wu et al. , Hepa tology 27: 772 (1998), has shown that liposomes labeled with asialofetuin lead to a shortened liposome plasma half-life and greatly enhance the uptake of liposomes labeled with asialofetuin by hepatocytes. On the other hand, hepatic accumulation of liposomes comprising branched-type galactosylipid derivatives can be inhibited by preinjection of asialofetuins (Murahashi et al., Biol. Pharm. Bull. 20: 259 (1997)).

Polyaconitilated human serum albumin liposomes provide another method for signaling liposomes to liver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94: 11681 (1997)). However, Gebo, et al., U.S. Patent? O. 4,603,044, discloses a liposome vesicle delivery system directed to the hepatocyte, which has specificity for the hepatobiliary receptors associated with specialized liver metabolic cells. The Zsig67 signaling compositions can be encapsulated within the liposomes using standard techniques for the microencapsulation of the proteins (see, for example, Anderson et al., Infect. Immun.31: 1099 (1981), Anderson et al., Cancer Res. 50: 1853 (1990), and Cohen et al., Biochim Biophys, Act 1063: 95 (1991), Alving et al. "Preparation and Use of Liposomes in Immunological Studies", in Liposome Technology, 2nd Edition, Vol. III, Gregoriads (ed.), Page 317 (CRC Press 1993), Wassef et al., Meth. Enzymol. 149: 124 (1987)). Suitable liposomes can contain a variety of portions, such as lipid derivatives of poly (ethylene glycol) (Alien et al., Biochim Biophis, Acta 1150: 9 (1993)). In addition, to provide a means for the delivery of Zsig67 signaling compositions, liposomes can be produced for target cells that express a Zsig67 receptor. Accordingly, the present invention includes liposomes comprising a Zsig67 component bonded to the surface to effect delivery of the encapsulated cytotoxins.

Production of Antibodies to Probes Zsig67 Antibodies to Zsig67 can be obtained, for example, by using the product of a Zsig67 or Zsig67 expression vector isolated from a natural source as an antigen. Particularly useful anti-Zsig67 antibodies are "specifically bound" with Zsig67. The antibodies are considered to be specifically bound if the antibodies have at least one of the following two properties: (1) antibodies bound to Zs ig 67 with a threshold level of binding activity, and (2) the antibodies do not cross-react significantly with polypeptides related to Zsig67. With respect to the first characteristic, the antibodies bind specifically if they bind to a peptide polypeptide or Zsig67 epitope, with a binding affinity (Ka) of 106 M_1 or greater, preferably 107 M "1 or larger, more preferably 108 M" 1 or larger, and more preferably 109 M 1 or larger. The binding affinity of an antibody can easily be determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51: 660 (1949)). With respect to the second feature, antibodies do not react significantly cross-linked with the related polypeptide molecules, for example, they detect Zsig67 but do not know related polypeptides using the Western blot analysis. Examples of known related polypeptides are orthologs and proteins of the same species that are members of a protein family. For example, specifically bound anti-Zsig67 antibodies bind to Zsig67, but not to polypeptides such as human secretin, glucagon, vasoactive intestinal peptide or human glucagon-like peptides. Anti-Zsig67 antibodies can be produced using peptides and polypeptides bearing the Zsig67 epitope. The peptides and antigenic polypeptides carrying the epitope, of the present invention, contain a sequence of at least six, or between 15 to about 30 amino acids contained in SEQ ID NO: 2. However, the peptides or polypeptides comprising a portion longer of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length above, including the entire amino acid sequence of a polypeptide of the invention, are also useful for inducing antibodies that bind to the Zsig67. It is desirable that the amino acid sequence of the peptide carrying the selected epitope provide substantial solubility in aqueous solvents (ie, the sequence includes relatively hydrophilic residues, whereas hydrophobic residues are preferably avoided). In addition, amino acid sequences containing proline residues may also be desirable for the production of antibodies. As an illustration, the potential antigenic sites in the extracellular domain of the Zsig67, were identified using the method of Jameson-Wolf, Jameson and Wolf, CABIOS 4: 181 (1988), when implemented by the PROTEAN program of LASERGENE (DNASTAR; Madison, Wl). The default parameters were used in this analysis. The Jameson-Wolf method predicts the potential antigenic determinants, combining the six main subroutines for the structural prediction of proteins. Briefly, the Hopp-Woods method, Hopp et al. , Proc Nat'l Acad. Sci. USA 78: 3824 (1981) was first used to identify the amino acid sequences that represent the largest hydrophilicity areas (parameter: seven averaged residues). In the second step, the method of Emini, Emini et al. , J. Virology 55: 836 (1985) was used to calculate the surface probabilities (parameter: surface decision threshold (0.6) = 1). Third, the Karplus-Schul t z method, Karplus and Schultz, Naturwissenschaften 72: 212 (1985), was used to predict the flexibility of the base structure chain (parameter: flexibility threshold (0.2) = 1). In the fourth and fifth steps of the analysis, secondary predictions of the structure were applied to the data, using the methods of Chou-Fasman, Chou, "Prediction of Protein Structural Classes from Amino Acid Composition", in Prediction of Protein Structure and the Principies of Protein With Form, Fasman (ed.), pages 549-586 (Plenum Press 1990), and Garnier-Robson, Garnier et al. , J. Mol. Biol. 120: 91 (1978) (Chou-Fasman parameters: conformation table = 64 proteins, region threshold a = 103, region threshold ß = 105, Garnier-Robson parameters: decision constants a and ß = 0). In the sixth subroutine, the flexibility parameters and hydropath / accessibility factors were combined to determine a surface contour value, designated as the "antigenic index". Finally, a spreading function of the peak was applied to the antigenic index, which extended the peaks of the main surface, by the addition of 20, 40, 60 or 80% of the value of the respective peak to count the free energy derived from the mobility of the superficial regions in relation to the interior regions. This calculation was not applied, however, to any main peak residing in a helical region, since the helical regions tend to be less flexible. The results of this analysis indicated that a peptide consisting of amino acids 45 to 72 of SEQ ID NO: 2 ("antigenic peptide 1"), its subfragments (amino acids 45 to 54 of SEQ ID NO: 2) ("peptide antigenic 2"), and 61 to 72 of SEQ ID NO: 2 (" antigenic peptide 3")), and its sub fragments (amino acids 45 to 50 (" antigenic 4"peptide), (amino acids 46 to 51 (" antigenic peptide 5")), (amino acids 47 to 52 (" pétido antigenic 6")), (amino acids 48 to 53 (" pétido antigenic 7")), (amino acids 49 to 54 (" pétido antigenic 8")), ( amino acids 61 to 66 antigenic peptide 9")) ', (amino acids 62 to 67' antigenic peptide 10") (amino acids 63 to 68 'antigenic peptide 11") (amino acids 64 to 69' antigenic peptide 12") (amino acids 65 to 70 antigenic peptide 13") (amino acids 66 to 71 antigenic peptide 14") (amino acids 62 to 72 antigenic peptide 15")), could provide suitable antigenic peptides The analysis also indicated that amino acid residues 87 to 92 of SEQ ID NO: 2 (16 antigenic peptide), could provide a suitable antigenic peptide. The present invention contemplates the use of any of the antigenic peptides 1 to 16 to generate antibodies to Zsig67. The present invention also contemplates polypeptides comprising at least one of the antigenic peptides 1 to 16. Polyclonal antibodies to the recombinant Zsig67 protein or to the Zsig67 protein isolated from natural sources, can be prepared, using methods well known to those skilled in the art. with experience in the technique. See, for example, Green et al. , "Production of Polyclonal Antisera", in Immunochemi ca 1 Protocols (Manson, ed.), Pages 1-5 (Humana Press 1992), and Williams et al., "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies ", in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al., (eds.), page 15 (Oxford University Press 1995). The immunogenicity of a Zsig67 polypeptide can be increased through the use of an auxiliary, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of Zsig67 or a portion thereof with an immunoglobulin polypeptide or with a maltose binding protein. The immunogenic polypeptide can be a full-length molecule or a portion thereof. If the portion of the polypeptide is "hapten-like", such a portion can be advantageously linked or bound to a macromolecular carrier (such as the tip peg hemocyanin (KLH), the bovine serum albumin (BSA), the tetanus toxoid ) for the internationalization Although polyclonal antibodies typically arise in animals such as horses, cows, dogs, chickens, rats, mice, rabbits, guinea pigs, goats or sheep, an anti-Zsig67 antibody of the present invention can also be derived from a subhuman primate antibody. . General techniques for producing diagnostically and therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., In International Patent Publication No. WO 91/11465, and Losman et al., Int. J. Cancer 45 : 310 (1990). Alternatively, monoclonal anti-Zsig67 antibodies can be generated. Mouse monoclonal antibodies to specific antigens can be obtained by methods known to those skilled in the art (see, for example, Kohier et al., Nature 255: 495 (1975), Coligan et al. (Eds.) , Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley &Sons 1991) ["Coligan"], Pickley et al., "Production of monoclonal antibodies agains expressed in E. Co li" , in DNA Cl on in g 2: Exprés si on Sys t em s, 2nd Edi on, Glover et al., (eds.), page 93 (Oxford University Press 1995)). Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising a product of the Zsig67 gene, verifying the presence of antibody production, removing a serum sample, removing the spleen to obtain B lymphocytes, fusing B lymphocytes with myeloma cells to produce the hybridomas, clone the hybridomas, select the positive clones which produce the antibodies for the antigen, grow the clones that produce the antibodies for the antigen, and isolate the antibodies from the cultures of hybridomas. In addition, an anti-Zsig67 antibody of the present invention can be derived from a human monoclonal antibody. Human morioclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic attack. In this technique, the elements of the locus or site of the human heavy and light chains are introduced into strains of mice derived from embryonic stem cell lines containing the objective interruptions of the locus or site of the endogenous heavy chain and light chain. Transgenic mice can synthesize human-specific antibodies to human antigens, and mice can be used to produce the hybridomas that secrete the antibody. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al. , Na t u re Gen e t. 7:13 (1994), Lonberg et al. , Na t u re 358: 856 (1994), and Taylor et al. , In t. Imm a. 5: 579 (1994). Monoclonal antibodies can be isolated and purified from hybridoma cultures, by a variety of well-established techniques. Such isolation techniques include, affinity chromatography with a protein A Sepharose, size exclusion chromatography, and ion exchange chromatography (see, for example, Colligan on pages 2.7.1-2.7.12 and pages 2.9 .1-2.9.3; Baines et al., "Purification of Immunolglobul in G (IgG)", in Methods in Molecular Biology or Gy, Vo l 1 0, pages 79-104 (The Humana Press, Inc 1992)) For particular uses, it may be desirable to prepare fragments of anti-Zsig67 antibodies. Such antibody fragments can be obtained, for example, by the proteolytic hydrolysis of the antibody. The antibody fragments can be obtained by digestion of pepsin or papain of the whole antibodies by conventional methods. As an illustration, fragments of antibodies can be produced by enzymatic cleavage of the antibodies with pepsin, to provide 5S fragments called F (ab ') 2. This fragment can be further excised, using a thiol reducing agent to produce the monovalent fragments of 3.5S Fab ', optionally, the cleavage reaction can be carried out using a blockforming group for the sulfhydryl groups resulting in cleavage of the disulfide bonds. As an alternative, an enzymatic cleavage using pepsin directly produces two monovalent Fab fragments and one Fc fragment. These methods are described, for example, by Goldenberg, U.S. Patent No. 4,331, 647, Nisonoff et al. , Arch Biochem. Biophys. 89: 230 (1960), Porter, Biochem. J. 73: 119 (1959), Edelman et al. , in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967), and by Coligan et al., 2.8.1-2.8.10 and 2.10.-2.10.1. Other methods of antibody cleavage, such as the separation of heavy chains to form light monovalent fragments of the chain, can be used, in addition, the excision of fragments and other enzymatic, chemical or genetic techniques can be used. also, in that the fragments are linked to the antigen that is recognized by the intact antibody.

For example, Fv fragments comprise an association of the VH and VL chains. 'This association can be monovalent, as described by Inbar et al. , Proc Nat'l Acad. Sci. USA 59: 2659 (1972). Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde (see, for example, Sandhu, Crit, Pev. Biotech, 12: 431 (1992)). The Fv fragments can comprise the VH and V chains which are connected by a peptide linker. These proteins that bind to the single chain antigen (scFv), are prepared by the construction of a structural gene comprising the DNA sequences encoding the VH and VL domains, which are connected by an oligonucleotide. The structural gene is inserted into an expression vector which is subsequently introduced into an expression vector, which is subsequently introduced into host cells, such as E. coli. Host recombinant cells synthesize a single polypeptide chain with a linker peptide, which forms a bridge between the two V domains. Methods for producing scFvs are described, for example, by Whitlow et al. , Methods: A Campanion to methods in enzymology 2:91 (1991) (see, also, Bird et al., Science 242: 423 (1989), Lander et al., US Patent No. 4,947,778, Pack et al., Bio / technology 11: 1271 81993), and Sandhu, supra). As an illustration, a scFV can be obtained by exposure lymphocytes for the Zsig67 polypeptide in vitro, and select libraries for displaying antibodies in phage vectors or similar vectors (for example, through the use of immobilized protein or Zsig67 peptide). or labeling). The genes encoding the polypeptides having the potential binding agglutination domains of the Zsig67 polypeptide can be obtained by peptide libraries randomly selected on phages (phage display) or on bacteria, such as E. coli. The nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random synthesis of polynucleotides. These libraries of randomly deployed peptides can be used to select the 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 substances or inorganic Techniques for creating and selecting such libraries from random displays of peptides are known in the art (Ladner et al., U.S. Patent No. 5,223,409, Ladner et al., U.S. Patent No. 4,946, 778, Ladner et al., U.S. Patent No. 5,430,484, Ladner et al., U.S. Patent No. 5,571,698, and Key et al., Phage Display of Peptides and Proteins (Academic Press, Inc. 1996)), and random peptide display libraries and equipment for selecting such libraries, are commercially available, for example, from CLONTECH Laboratories, Inc. (Palo Alto, CA), Invitrogen, Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA), and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Randomized peptide display libraries can be selected using the Zsig67 sequences described here to identify proteins, which are linked to the Zsig67. Another form of an antibody fragment is a peptide encoding a single region that determines complementarity (CDR). CDR peptides ("minimal recognition units") can be obtained by the construction of genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, using the polymerase chain reaction to synthesize the variable region from the RNA of the cells that produce the antibody (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2: 106 (1991), Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Appl ication, Ritter et al. (Eds.), Page 166 (Cambridge University Press 1995 ), and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principies and Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc. 1995)). Alternatively, an anti-Zsig67 antibody can be derived from a "humanized" mclnoclonal antibody. Humanized monoclonal antibodies are produced by transferring the mouse complementarity determining regions from the light and heavy variable chains of the mouse immunoglobulin to a human variable domain. The typical residues of the human antibodies are then replaced in the regions of the working structure of the murine counterparts. The use of the antibody components derived from the humanized monoclonal antibodies obviates the potential problems associated with the immunogenicity of the murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al. , Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al. , Nature 321: 522 (1986), Carter et al. , Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12: 431 (1992), Singer et al. , J. Immun. 150: 2844 (1993), Sudhir (ed.), Antibody Engineering Protocols (Humana Press, Inc. 1995), Kelley, "Engineering Therapeutic Antibodies," in Protein Engineering: Principies and Practice, Cleland et al. (eds.), pages 399-434 (John Wiley & amp; amp;; Sons, Inc. 1996), and by Queen et al. , U.S. Patent No. 5,693,762 (1997). Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-Zsig67 antibodies or antibody fragments, using standard techniques. See, for example, Green et al. , "Production of Polyclonal Antisera," in Methods in Molecule r Biology: Immunochemi ca 1 Protocols, Manson (ed.), Pages 1-12 (Humana Press 1992). Also, see Coligan on pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotype antibodies can be prepared using anti-Zsig67 antibodies or antibody fragments as immunogens with the techniques described above. As another alternative, humanized anti-idiotype antibodies or sub-human primate anti-idiotype antibodies can be prepared using the techniques described above. Methods for producing anti-idiotype antibodies are described, for example, by Irie, U.S. Patent No. 5,208,146, Greene, et al. , U.S. Patent No. 5,637,677, and Varthakavi and Minocha, J. Gen I saw l. 77: 1875 (1996). eleven . Generation of the Gene Expression of the Zsi g67 and Examination of the Si Chromos omal Zsig67 Nucleic acid molecules can be used to detect the expression of a Zsig67 gene in a biological sample. Such probe molecules include double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ. ID NO: 1, or a fragment thereof, as well as the single-stranded nucleic acid molecules that have the complement of the SEC nucleotide sequence. ID NO: 1, or a fragment thereof. The probe molecules can be DNA, RNA, oligonucleotides, and the like. Certain probes are linked to regions of the Zs i g 67 gene that have a low sequence similarity to comparable regions in other genes. As used, the term "portion" refers to at least eight nucleotides to at least 20 or more nucleotides. In a basic assay, a single-stranded probe molecule is incubated with RNA, isolated from a biological sample, under conditions of temperature and ionic concentration that promote base pairing between the probe and target Zsig67 RNA species . After separation of the unbound probe from the hybridized molecules, the number of hybrids is detected. Well-established hybridization methods of RNA detection include Northern analysis and dot / groove stain hybridization (see, for example, Ausubel (1995) on pages 4-1 to 4-27, and Wu et al. (Eds. .), "Analysis of Gene Expression at the RNA Level," in Methods in Gene Biotechnology, pages 225-239 (CRC Press, Inc. 1997). Nucleic acid probes can be detectably labeled with radioisotopes such as ccoommous. Alternatively, Zsig67 RNA can be detected with a non-radioactive hybridization method (see, for example, Isaac (ed.), Protocols for Nucleic Acid Ana lys is by Nonradioact ive Probes (Humana Press, Inc. 1993)). Typically, non-radioactive detection is achieved by the enzymatic conversion of chromogenic or chemiluminescent substrates. Illustrative non-radioactive portions include biotin, fluorescein, and digoxigenin. The oligonucleotide probes of Zsig67 are also useful for in vivo diagnosis. As an illustration, 18F-labeled oligonucleotides can be administered to a subject and visualized by positron emission tomography (Tavitian et al., Nature Medicine 4: 467 (1998)). The numerous diagnostic procedures take advantage of the polymerase chain reaction (PCR) to increase the sensitivity of the detection methods. Standard techniques for performing PCR are well known (see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methods and Applications (Humana Press, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo ( ed.), Clinical App lica t ions of PCR (Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)). Preferably, the PCR primers are designed to amplify a portion of the Zsig61 gene that has low sequence similarity to a comparable region in other genes. A variation of the PCR for diagnostic assays is reverse transcriptase-PCR (RT-PCR). In the RT-PCR technique, the RNA is isolated from a biological sample, it is reverse transcribed CDNA and the cDNA is incubated with the Zsig67 primers (see, for example, Wu et al., (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR," in Me th o ds in Gen e Bi ote chnolo g, pages 15-28 (CRC Press, 1997)). Then the PCR is carried out and the products are analyzed using standard techniques. As an illustration, the RNA is isolated from a biological sample using, for example, the cell lysis procedure of guanidini orthiocyanate described above. Alternatively, the solid phase technique can be used to isolate the mRNA from a cell lysate. A reverse transcription reaction can be primed with the isolated RNA using random oligonucleotides, short dT homopolymers, or antisense oligomers of Zsig67. The oligo-dT primers offer the advantage that several mRNA nucleotide sequences are amplified which can provide the target control sequences. The sequences of Zsig67 are amplified by the polymerase chain reaction using two flanking oligonucleotide primers are typically 20 bases in length.

The products of PCR amplification can be detected using a variety of approaches. For example, PCR products can be fractionated by gel electrophoresis, and visualized by staining with ethidium bromide. Alternatively, the fractionated PCR products can be transferred to a membrane, hybridized with a detectably labeled Zs i g 67 probe, and examined by autoradiography. Additional alternative approaches or methodologies include the use of deoxyribonucleic acid triphosphates labeled with digoxigenin to provide chemiluminescent detection, and the imaging color with C-TRAK. Another approach or methodology for the detection of Zsig67 expression is a cyclization probe (CPT) technology, in which a single-stranded target DNA is bound with an excess of chimeric DNA-RNA-DNA probe to form a complex, the RNA portion is cleaved with RNAase H, and the presence of the excised chimeric probe is detected (see, for example, Beggs et al., J. Clin Microbiol 34: 2985 (1996), Bekkaoui et al. , Biotechniques 20: 240 (1996)). Alternative methods for the detection of Zsig67 sequences can use methodologies such as amplification based on nucleic acid sequence (NASBA), cooperative amplification of templates or patterns by cross-linking (CATCH), and ligase chain reaction (LCR) (see, for example, Marshall et al., U.S. Patent No. 5,686,272 (1997), Dyer et al., J. Virol. Methods 60: 161 (1996), Ehricht et al. , Eur. J. Biochem 243: 358 (1997), and Chadwick et al., J. Virol. Methods 70:59 (1998)). Other standard methods are known to those skilled in the art. The probes and primers of Zsig67 can also be used to detect and localize the expression of the Z s ig67 gene in tissue samples. Methods for such in situ hybridization are well known to those skilled in the art (see, for example, Choo (ed.), In Situ Hybridization Protocols (Humana Press, Inc. 1994), Wu et al. (Eds.), "Analysis of Cellular DNA or Abundance of mRNA by Radioactive In Situ Hybridization (RISH)," in Methods in Gene Biotechnology, pages 259-278 (CRC Press, Inc. 1997), and Wu et al. (eds.), "Localization of DNA or Abundance of mRNA by Fluorescence in Situ Hybridization (RISH)," in Methods in Gene Biotechnology, pages 279-289 (CRC Press, Inc. 1997). Several additional diagnostic methodologies are well known to those skilled in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Coleman and Tsongalis, Molecular Diagnostics (Humana Press, Inc. 1996), and Elles, Molecule r Diagnosis of Genetic Dlsea ses (Humana Press, Inc. 1996)). The Zsig67 gene resides on chromosome 8q24. This region is associated with various conditions and alterations, including Langer-Giegiedion Syndrome, Tricor flanal filarial syndrome Type I, renal cell carcinoma, Burkitt lymphoma, idiopathic epilepsy, neonatal epilepsy, macular dystrophy, nephroblastic s, Stargardt's disease and Pendred's syndrome. Thus, the nucleotide sequences of Zsig67 can be used in lineage-based tests for various conditions and to determine whether the chromosomes of a subject contain a mutation in the Zsig67 gene. Chromosomal aberrations detectable at the site of the Zsig67 gene include, but are not limited to, aneuploidy, changes in the number of copies of genes, insertions, deletions, changes in restriction sites and rearrangements. Of particular interest are the genetic aberrations that inactivate the Zs i g 67 gene. The aberrations associated with the Zs ig 67 site can be detected using nucleic acid molecules of the present invention employing molecular genetic techniques, such as restriction fragment length polymorphism analysis, PCR techniques employing tandem repeat analysis short, amplification refractory mutation system analysis, detection of single-strand conformation polymorphism, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for example, Mathew (ed ..), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 108: 255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Humana Press , Inc. 1996), Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren (ed.), Laboratory Protoco ls for Mutation Detection (Oxford University Press 1996), Birren et al., (eds.), Genome Analysis, Vol. 2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998), 'Dracopoli et al. (eds.), Current Protocols in Human Genetics (John Wiley & amp; amp; amp;; Sons 1998), and Richards and Ward, "Molecular Diagnostic Testing," in Principies of Molecular Medicine, pages 83-88 (Humana Press, Inc. 1998)). The protein truncation test is also useful for detecting the inactivation of a gene in which translation-termination mutations produce only portions of the encoded protein (see, for example, Stoppa-Lyonnet et al., Blood 91: 3920 (1998)). According to this methodology, the RNA is isolated from a biological sample and used to synthesize the cDNA. The PCR is then used to amplify the target sequence of Zsig67 and to introduce a. RNA polymerase promoter, and a translation initiation sequence, and an ATG triplet in the structure. The PCR products are transcribed using an RNA polymerase and the transcripts are translated in vitro with a reticulocyte lysate system coupled to T7. The translation products are then fractionated by SDS-PAGE to determine the lengths of the translation products. The truncation test of the protein is described, for example, by Dracopoli et al. (eds.), Current Protocols in Human Genetics, pages 9.11.1 9.11.18 (John Wiley &Sons 1998). The present invention also contemplates the equipment to perform a diagnostic assay for the expression of the Zsig67 gene or to examine mutations in the Zsig67 gene. Such kits comprise nucleic acid probes, such as double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ. ID NO: 1, or a fragment thereof, as well as single-stranded nucleic acid molecules containing the complement of the SEC nucleotide sequence. ID NO: 1, or a fragment or portion thereof. The probe molecules can be DNA, RNA, oligonucleotides, and the like. The kits can comprise the nucleic acid primers to perform the PCR. Such equipment can contain all the necessary elements to carry out a diagnostic test as described above. A kit will comprise at least one container comprising a probe or primer of Zsig67. The equipment may also comprise a second container comprising one or more reagents capable of indicating the presence of sequences of Zs i g 67. Examples of such indicator reagents include detectable labels such as adioact labels, fluorochromes, chemiluminescent agents, and the like. A kit may also comprise a means to indicate to the user that the probes and primers of Z s i g 6 7 are used to detect the expression of the Zs i g 67 gene. For example, the written instructions may state that the attached nucleic acid molecules can be used to detect either a nucleic acid molecule encoding Zsig67, or a nucleic acid molecule having a nucleotide sequence that is complementary to a sequence. of nucleotides that encodes Zsig67. The written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert. 12. Use of Anti-Zsig67 Antibodies to Detect Zsig67 Polypeptides The present invention contemplates the use of anti-Zsig67 antibodies to select biological samples in vivo for the presence of Zsig67. In one type of viral assay, anti-Zsig67 antibodies are used in the liquid phase. For example, the presence of Zsig67 in a biological sample can be tested by mixing the biological sample with a trace amount of the labeled Zsig67 and an anti-Zsig67 antibody under conditions that promote the binding between Zsig67 and its antibody. The Zsig67 and anti-Zsig67 complexes in the sample can be separated from the reaction mixture by contacting the complex with an immobilized protein that binds to the antibody, such as an Fc antibody or Protein A from S t aphyl or c or c c u s. The concentration of Zsig67 in the biological sample will be inversely proportional to the amount of labeled Zsig67 bound to the antibody and directly related to the amount of free labeled Zsig67. Alternatively, assays can be performed in which the anti-Zsig67 antibody binds to a solid phase carrier. For example, the antibody can be attached to a polymer, such as aminodexthan, to bind the antibody to an insoluble support such as a polymer-coated bead, plate or tube. Other suitable tests will be readily apparent to those skilled in the art.

In another methodology, anti-Zsig67 antibodies can be used to detect Zsig67 in tissue sections prepared from a biopsy specimen. Such immunochemical detection can be used to determine the relative abundance of Zsig67 and to determine the distribution of Zsig67 in the tissue examined. The general techniques of immunochemistry are well established (see, for example, Ponder, "Cell Marking Techniques and Their Application," in Mammalian Development: A Practical Approach, Monk (ed.), pages 115-38 (IRL Press 1987), Coligan on pages 5.8.1-5.8.8, Ausubel (1995) on pages 14.6.1 to 14.6.13 (Wiley Interscience 1990), and Manson ( ed.), Methods In Molecular Biology, Vol. 10: Immunochemi ca 1 Protocols (The Humana Press, Inc. 1992)). Immunochemical detection can be performed by contacting a biological sample with an anti-Zsig67 antibody, and then contacting the biological sample with a detectably labeled molecule that binds to the antibody. For example, the detectably labeled molecule may comprise a portion of the antibody that binds to an anti-Zsig67 antibody. Alternatively, the anti-Zsig67 antibody can be conjugated with avidin / is trept avidin (or biotin) and the detectably labeled molecule can comprise biotin (or avidin / e st rept avidin). Numerous variations of this basic technique are well known in the art to those skilled in the art. Alternatively, an anti-Zsig67 antibody can be conjugated to a detectable label to form an anti-Zsig67 immunoconjugate. Suitable detectable labels include, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label, or colloidal gold. Methods for making and detecting such detectably labeled immunoconjugates are well known to those of ordinary skill in the art, and are described in more detail below.

The detectable label can be a radioisotope that is detected by auto-raphiography. Isotopes that are particularly useful for the purpose of the present invention are, -. Or - .n., 3 H TT, l 2 5 It, 1 3 1 tI, 3 5 Ss, y, 1 1 C. The anti immunoconjugates -Zsig67 can also be labeled with a fluorescent compound. The presence of a fluorescently labeled antibody is determined by exposing the immunoconjugate to light of a suitable wavelength and detecting the resulting fluorescence. Fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, f i coeri t er, phycocyanin, phycocyanin, o-phthaldehyde and fluorescein. Alternatively, anti-Zsig67 immunoconjugates can be detectably labeled by coupling an antibody component to a chemiluminescent compound. The presence of the immunoconjugate is determined by detecting the presence of the luminescence that arises during the course of a chemical reaction. Examples of luminescent labeling compounds include luminol, isoluminiol, an aromatic acridium ester, an imidazole, an acridium salt and an oxalate ester. Similarly, a bioluminescent compound can be used to label the anti-Zsig67 immunoconjugates of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of the luminescence. Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin. Alternatively, the anti-Zsig67 immunoconjugates can be detectably labeled by linking an anti-Zsig67 antibody component to an enzyme. When the anti-Zsig67-enzyme conjugate is incubated in the presence of an appropriate substrate, the portion of the enzyme reacts with the substrate to produce a chemical moiety that can be detected, for example, by rich, fluorometric, or visual specimens. . Examples of the enzymes that can be used to detectably label the polypeptide immunoconjugates include β-galactosidase, glucose oxidase, peroxidase and alkaline phosphatase. Those of skill in the art will know other suitable labels that may be employed in accordance with the present invention. The binding of the marker portions to the anti-Zsig67 antibodies can be complemented using standard techniques known in the art.

The typical methodology in relation to this is described by Kennedy et al, Clin. Chim. Minutes 70: 1 (1976), Schurs et al., Clin. Chim. Acta 81: 1 (1977), Shih et al. , Int'l J. Cancer 45: 1101 (1990), Stein et al. , Cancer Res. 50: 1330 (1990), and Coligan, supra. In addition, the convenience and versatility of immunochemical detection can be improved by using anti-Zsig67 antibodies that have been conjugated with avidin, streptavidin, and biotin (see, for example, Wilchek et al. (eds.), "Avidin-Biotin Technology," Methods In Enzymology, Vol. 184 (Academic Press 1990), and Bayer et al. , "Immunochemical Applications of Avidin-Bio t in Technology," in Methods In Molecular Biology, Vol 10, Manson (ed.), Pages 149-162 (The Humana Press, Inc. 1992). The methods to perform immunoassays are well established. See, for example, Cook and Self, "Monoclonal Antibodies in Diagnostic Immunoas s ays," in Monoclonal Ant ibodies: Production Engineering and Clinical Application, Ritter and Ladyman (eds.), Pages 180-208, (Cambridge University Press, 1995) , Perry, "The Role of Monoclonal Antibodies in the Advancement of Technology," in Monoclonal Antibodies Principies and Applications, Birch and Lennox (eds.), Pages 107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay ( Academic Press, Inc. 1996). In a related methodology, the Zsig67 tagged with biotin or FITC can be used to identify cells that bind to Zsig67. Such a link can be detected, for example, using flow cytometry.

The present invention also contemplates equipment for the performance of an immunological diagnostic assay for the expression of the gene Z s i g 67. Such kits comprise at least one container comprising an anti-Zsig67 antibody, or an antibody fragment. A kit may also comprise a second container comprising one or more reagents capable of indicating the presence of the anti-Zsig67 antibody or the antibody fragments. Examples of such indicator reagents include detectable labels such as a radioactive label, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label, colloidal gold, and the like. A kit may also comprise a means for transmitting to the user that Zsig67 antibodies or antibody fragments are used to detect the Zsig67 protein. For example, written instructions may state that the attached antibody or antibody fragment can be used to detect Zsig67. The written material can be applied directly to a container, or the written material can be provided in the form of an insert in the package. 13. Therapeutic Uses of Polypeptides that have Activity Zsig67 The present invention includes the use of proteins, polypeptides, and peptides described herein, which have Zsig67 activity (such as Zsig67 polypeptides, Zsig67 anti-idiotype antibodies and Zsig67 fusion proteins), to a subject who lacks an adequate amount of this polypeptide . The standard methods can be used to prepare pharmaceutically useful compositions comprising a protein, polypeptides, or peptides having Zsi g67 activity, thereby the therapeutic proteins are combined in a mixture with a pharmaceutically acceptable carrier. A composition is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a receiving patient. The buffered saline sterile phosphate solution is an example of a pharmaceutically acceptable carrier. Other suitable carriers are well known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceuticals Sciences, 19th Edition (Mack Publishing Company 1995). A pharmaceutical composition comprising molecules having Zsig67 activity may be provided in liquid, aerosol, or solid form. The liquid forms are illustrated by injectable solutions and oral suspensions. Exemplary solid forms include capsules, tablets, and controlled release forms. The last form is illustrated by miniosmotic pumps and implants (Bremer et al., Pharm.Biotechnol 10: 239 (1997); Ranade, "Implants in Drug Delivery", in Drug Deliveru Systems, Ranade and Hollinger (eds.), Pages 95-123 (CRC Press 1995); Bremer et al., "Protein Delivery with Infusion Pumps", in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), Pages 239-254 (Plenum Press 1997); Yewey et al., "Delivery of Proteins from Controlled Reléase Injectable Implant", in Protein Delivery; Physical Ssystems, Sanders and Hendren (eds.), Pages 93-117 (Plenum Press 1997)). Degradable polymer microspheres are designed to maintain high systemic levels of therapeutic proteins. The microspheres are prepared from degradable polymers such as poly (lactide-co-glycolide), polyanhydrides, poly (ortho esters), non-biodegradable ethyl vinyl acetate polymers, in which the proteins are trapped in the polymer (Gombotz and Pettit , Bioconjugate Chem. 6: 332 (1995); Ranade; "Role of Polymers in Drug Delivery", in Drug Delivery Systems, Ranade and Hollinger (eds.), Pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, " Degradable Controlled Released Systems Useful for Protein Delivery ", in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), Pages 45-92 (Plenum Pres 1997), Bartus et al., Science 281: 1161 (1988); Burke, Nature Biotechnology 16: 153 (1998), Putney, Curr Opin, Chem. Biol. 2: 548 (1998)). Polyethylene glycol (PEG) coated nanospheres can also provide carriers for intravenous administration of therapeutic proteins (see for example, Gref et al., Pharm Biotechnol 10: 167 (1997)). The administration of a molecule having Zsig67 activity for a subject can be intravenous, intraarterial, intramuscular, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, perfusion through a regional catheter, or direct intralesional injection. When therapeutic proteins are administered by injection, administration can be by continuous infusion or by single or multiple pills. Additionally, administration routes include oral, dermal, membrane-mucosal, pulmonary, and transcutaneous. Oral delivery is suitable for polyester microspheres, zein microspheres, protenoid microspheres, polycyanoacrylate microspheres, and lipid-based systems (see for example, DiBase and Morrel, "Oral Delivery of Microencapsulated Proteins", in Delivery Protein). : Physi cal Sys tems, Sanders and Hendren (eds.), Pages 255-288 (Plenum Press 1997)). The viability of an intranasal delivery is exemplified by an insulin delivery mode (see, for example, Hinchcliffe and Ilium, Adv. Drug, Deliv. Rev. 35: 199, 1999.) Dry or liquid particles comprising a Zsig67 polypeptide, a functional fragment, can be prepared and inhaled with the aid of dry powder dispersants, liquid aerosol generators, or nebulizers (for example, Pettit and Gombotz, TIBTECH 1 6: 343 (1998); Patton et al., Adv. Drug Deliv. Rev. 35: 235 (1999) This procedure is illustrated by the AERX diabetes management system, which is a hand held electronic inhaler that delivers aerosolized insulin to the lungs. proteins as large as 48,000 kDa have been delivered through the skin at therapeutic concentrations with the help of low frequency ultrasound, which illustrates the viability of subcutaneous administration (Mitragotr i et al., Science 269: 850 (1995)). Transdermal delivery using electroporation provides another means for administering the Zsig67 molecules (Potts et al., Pharm.Biotechnol 10: 213 (1997)). Generally, the dosage of the administered polypeptide, protein or peptide will vary depending upon such factors as age, weight, height, sex, general medical condition and previous medical history of the patient. Typically, it is desirable to provide the container with a dosage of a molecule having Zsi g67 activity which is in the range of about 1 pg / kg to 10 mg / kg (amount of agent / patient's body weight), although a lower or higher dosage can be administered as dictated by the circumstances. For therapy purposes, molecules having Zsig67 activity and a pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective amount. A combination of a protein, polypeptide, or peptide having Zsig67 activity and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a receiving patient. Suitable subjects include a mammalian subject, such as a human. The pharmaceutical compositions Zsig67 can be supplied as a kit comprising a container comprising the Zsig67. The Zsig67 can be provided in the form of an injectable solution for a single or multiple dose, or as a sterile powder that will be reconstituted prior to injection. Said equipment may also comprise wrn information on indications and uses of the pharmaceutical composition. However, such information may include a statement that the Zsig67 composition is contraindicated in patients with known hypersensitivity to Zsig67. 14. Therapeutic Uses of Nucleotide Sequences Zsig67 The use of the present invention includes nucleotide sequences Zsig67 to provide Zsig67 to a subject in need of such treatment. In addition, a therapeutic expression vector that inhibits the expression of the Zsig67 gene can be provided, such as an antisense molecule, a ribozyme, or an outer guide sequence molecule. There are numerous methods for introducing a Zsig67 gene into a subject, which includes the use of recombinant host cells expressing Zsig67, the delivery of pure nucleic acids encoding Zsig67, the use of a cationic lipid carrier with an acid molecule. nucleic acid encoding Zsig67, and the use of the virus expressing Zsig67, such as recombinant retrovirus, recombinant adeno-associated virus, recombinant adenovirus, and recombinant Herpes simplex virus [VSH] (see, eg, Mulligan, Science 260 : 926 (1993), Rosemberg et al., Sciences 242: 1515 (1988), LaSalle et al., Science 259: 988 (19.93), Wolff et al., Science 247: 1465 (1990), Breakfield and Deluca, The New Biologist 3: 203 (1991) In an ex vivo procedure, for example, cells are isolated from a subject, transfected with a vector expressing a Zsig67 gene, and then transplanted into the subject. Zsig67 gene, an e vector is constructed xpression in which, a nucleotide sequence encoding a Zsig67 gene is operably linked to a core promoter, and optionally a regulatory element, to control the transcription of the gene. The general requirements of an expression vector are described below. Alternatively, a Zsig67 gene can be delivered using recombinant viral vectors, including for example, adenoviral vectors (eg, Kass-Eisler et al., Proc. Nat'l. Acad. Sci. USA 90: 11498 (1993), Kolls et al. al., Proc. Nati, Acad. Sci. USA 91: 215 (1994), Li et al., Hum. Gen. Ther 4: 403 (1993), Vmcent et al., Nat. Genet. 1993), and Zabner et al., Cell 75: 207 (1993)), adenovirus-associated viral vectors (Flotte et al., Proc. Nati, Acad. Sci. USA 90: 10613 (1993)), alphaviruses such as Semliki Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66: 851 (1992), Raju and Huang, J. Vir. 65: 2501 (1991), and Xiong et al., Science 243: 1188 (1989) ), vectors of the herpes virus (Ozaki et al., Biochem. Biophys., Res. Comm. 193: 653 (1993), Panicalli and Paoletti, Proc. Nati. Acad. Sci. USA 79: 4927 (1982)), viruses pox, such as the pox virus of canaries or vaccinia virus (Fisher-Hoch et al., Proc. Nati, Acad. Sci. USA 86: 317 (1989), and Flexner et al., Ann. NY Acad. Sci. 56 9:86 (1989)), and retroviruses (eg, Baba et al., J. Neurosurg 79: 129 (1993), Ram et al., Cancer Res. 53:83 (1993), Takamiya et al., J Neurosci. Res 33: 493 (1992), Vile and Hart, Cancer Res. 53: 962 (1993), Vile and Hart, Cancer Res. 53: 3860 (1993), and Anderson et al., U.S. Patent No. 5,399,346). Within several embodiments, either the same viral vector, or a vector particle which contains the viral vector, can be used in the methods and compositions described below. As an illustration of a system, an adenovirus, a double-stranded DNA virus, of the gene transfer vector is well characterized, for the delivery of a heterologous nucleic acid molecule (for review, see Becker et al., Meth. Cell Biol. 43: 161 (1994), Douglas and Curiel, Sci ence &Medi cine 4:44 (1997)). The various adenovirus systems offer several advantages including: (i) the ability to accommodate relatively large DNA inserts, (ii) the ability to grow them to high titers, (iii) the ability to infect a wide range of cell types, and (iv) the ability to be used with many different promoters including ubiquitous, tissue-specific and regulatable promoters. In addition, adenoviruses can be administered by intravenous injection, because the viruses are stable in the bloodstream. Using adenovirus vectors where the portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA, either by direct ligation or by homologous recombination with a co-transfected plasmid. In an exemplary system, the essential gene is deleted from the viral vector, and the virus will not replicate, unless the El gene is provided by the host cell. When administered intravenously to intact animals, the adenovirus is directed primarily to the liver. Although an adenoviral system delivery with the deletion of the El gene can not replicate in the host cells, the host tissue will express and process a heterologous encoded protein. The host cells will also secrete the heterologous protein if the corresponding gene includes a signal of secretory sequence. The secreted proteins will enter the circulation of the tissue expressing the heterologous gene (eg, highly vascularized liver). However, adenoviral vectors containing several deletions of viral genes can be used to reduce or eliminate immune responses to the vector. Such adenoviruses are deleted El, and in addition, contain deletions of E2A or E4 (Lusky et al., J. Virol. 72: 2022 (1998); Raper et al. L. , Human Gene Therapy 9: 671 (1998)). The suppression of E2b has been reported to reduce immune responses (Amalfitano et al., J. Virol 72 926 (1998)). By suppressing the total adenovirus genome, very long inserts of heterologous DNA can be accommodated. The generation of so-called "pusillanimous" adenoviruses, in which viral genes are suppressed, are particularly advantageous for the insertion of large heterologous DNA inserts (for a review, see Yeh and Perricaudet, FASEB J. 11: 615 (1997)). ). High titrant bases of recombinant viruses capable of expressing a therapeutic gene can be obtained from infected mammalian cells using standard methods. For example, recombinant HSV can be prepared in Vero cells, as described by Brandt et al., J. Gen. Virol. 72: 2043 (1991), Herold et al., J. Gen. Virol 75: 1211 (1994), Visalli and Brandt, Virology 185: 419 (1991), Grau et al., Invest. Ophthalmol. Vis. Sci. 30: 2474 (1989), Brandt et al., J- Virol. Meth. 35: 209 (1992), and by Brown and MacLean (eds.), HSV Virus Protocols (Humana Press 1997). Alternatively, an expression vector comprising a Zsig67 gene can be introduced into cells of a subject, by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for the in vivo transfection of a gene encoding a marker (Felgner et al., Proc. Nati, Acad. Sci. USA 85: 8027 (1998)). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain major advantages. Liposomes can be used to direct transfection to particular cell types, which is particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney and brain. The lipids may be chemically coupled to other molecules for signaling purposes. The indicated peptides (e.g., hormones or neurotransmitters), proteins such as antibodies, or non-peptide molecules, can be chemically coupled to the liposomes. Electroporation is another alternative mode of administration of Zsig67 nucleic acid molecules. For example, Aihara and Miyazaki, Na ture Biotechnol ogy 1 6: 8 61 (1998), have demonstrated the use of in vivo electroporation for gene transfer. in the muscle. In an alternative to gene therapy, a therapeutic gene can encode an antisense Zsig67 RNA that inhibits the expression of Zsig67. Suitable sequences for anti-sense Zsig67 molecules can be derived from the Zsig67 nucleotide sequences described herein. Alternatively, an expression vector can be constructed in which, a regulatory element is operably linked to a nucleotide sequence encoding a ribozyme. Ribozymes can be designed to express the activity of the endonuclease that targets certain target sequences in an mRNA molecule (see for example, Draper and Macejak, U.S. Patent No. 5,496,698, McSwiggen, U.S. Patent No. 5,525,468, Chowrira and McSwiggen , U.S. Patent No. 5,631,359, and Robertson and Goldberg, U.S. Patent No. 5,225,337). In the context of the present invention, ribozymes include nucleotide sequences that bind to the Zsig67 mRNA. In another procedure, expression vectors can be constructed in which, a regulatory element directs the production of RNA transcripts capable of promoting RNase P-mediated cleavage of mRNA molecules encoding a Zsig67 gene. According to this method, an external leader sequence can be constructed to direct the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, which is subsequently cleaved by the cellular ribozyme (see for example, Altman et al., Patent No. 5,168,053, Yuan et al., Science 253: 1269 (1994), Pace et al., International Publication No. WO 96/18733, George et al., International Publication No. WO 96/21731, and Werner et al. ., International Publication No. WO 97/33991). Preferably, the outer leader sequence comprises a ten to fifteen nucleotide sequence complementary to the Zsig67 mRNA, and a 3'-NCCA nucleotide sequence, wherein N is preferably a purine. The external leader sequence transcripts are linked to the target mRNA species by forming base pairs between the mRNA and the complementary outer guide sequences, thus promoting the cleavage of the mRNA by RNase P to the nucleotide located on the 5 'side of the region of paired bases. In general, the dosage of a composition comprising a therapeutic vector having a nucleic acid sequence of Zsi g67, such as recombinant virus, will vary depending on such factors as age, weight, height, sex, general medical condition and the patient's previous medical history. Suitable routes of administration of therapeutic vectors include intravenous injection, intraarterial injection, intraperitoneal injection, intramuscular injection, intratumoral injection and injection into a cavity containing a tumor. A composition comprising viral vectors, non-viral vectors, or a combination of viral and non-viral vectors of the present invention, can be formulated in accordance with known methods to prepare pharmaceutically useful compositions, thereby, the vectors or viruses are combined in a mixture with a pharmaceutically acceptable carrier. As noted above, a composition, such as buffered phosphate salt is said to be "a pharmaceutically acceptable carrier" if administration can be tolerated by a recipient subject. Other suitable carriers are well known to those in the art (see, for example, Remington's Pharma ceuti ca l Sciences, 19th Ed. (Mack Publishing Co., 1995), and Gilman's Pharma cological Basis of Therapeutics, 7th. Ed.

(MacMillan Publishing Co., 1985)). For therapy purposes, an expression vector of the therapeutic gene, or a recombinant virus comprising such a vector, and a pharmaceutically acceptable carrier is administered to a subject in a therapeutically effective amount. A combination of an expression vector (or virus) and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount", if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient subject. When the subject treated with an expression vector of the therapeutic gene or a recombinant virus is a human, then the therapy is preferably somatic cell gene therapy. That is, the preferred treatment of a human with an expression vector of the therapeutic gene or a recombinant virus does not cause the introduction into the cells of a nucleic acid molecule that can be part of a human germ line and pass through the generations successive (ie, therapy of the human germline gene).

. Production of Transgenic Mice Transgenic mice can be produced by genetic engineering to overexpress the Zsig67 gene in all tissues or under the control of a tissue-specific or tissue-specific regulatory element. These overproducers of Zsig67 can be used to characterize the phenotype that results in overexpression, and transgenic animals can serve as models for human diseases caused by excess Zsig67. Transgenic mice that overexpress Zsig67 also provide bioreactor models for the production of Zsig67, such as soluble Z s i g 6 7, in the milk or blood of larger animals. Methods for producing the transgenic mice are well known to those skilled in the art (see, for example, Jabob, "Expression on Knockout of Interferons in Transgenic Mice", in Overspression and Knockout of Cytokines in Transgeni cice, Jacob (ed. ), pages 111-124 (Academic Press, Ltd. 1994), Monastersky and Robl (eds.), Transgeni c Animal Science Strategies (ASM Press 1995), and Abbud and Nilson, "Recombinant Protein Expression in Transgenic Mice," in Gene Expression Sytems: Using Na ture for the Art of Expression, Fernandez and Hoeffer (eds.), Pages 367-397 (Academic Press, Inc. 1999)). For example, a method to produce a transgenic mouse expressing a Zsig67 gene can start with fertile males, adults (stallions) (B6C3fl, 2-8 months of age (Taconic Farms, Germantown, NY)), vasectomized males (stallions) (B6D2fl, of. 2-8 months, (Taconic Farms)), fertile prepubertal females (donors) (B6C3fl, 4-5 months, (Taconic Farms)), and adult fertile females (recipients or recipients) (B6D2fl, 2-4 months, (Taconic Farms)). The donors are acclimated for a week and then injected with approximately 8 IU / gonadotropin mouse of Pregnancy Mare Serum (Sigma Chemical Company, St. Louis, MO) IP, and 46-47 hours later, 8 IU / mouse of Gonadotropin Corionica (hCG (Sigma)), IP to induce superovulation. Donors mate with stallions subsequent to hormone injections. Ovulation generally occurs within 13 hours of the hCG injection. Ovulation is confirmed by the presence of a vaginal plug the next morning after mating. The fertilized eggs are collected under a surgical microscope. The oviducts are harvested and the eggs are released into sections of uninarinalysis containing hyaluronidase (Sigma). The eggs are washed once in hyaluronidase, and twice in the W640 medium of Whitten (described, for example, by Menino and O'Claray, Biol. Reprod. 77: 159 (1986), and Dienhart and Downs, Zygote 4: 129 (1996)) that have been incubated with 5% C02, 02 to 5%, and 90% N2 at 37 ° C. The eggs are then stored in an incubator at 37 ° C / 5% C02 until microinjection. Ten to twenty micrograms of the plasmid DNA containing a sequence encoding Zsig67 are linearized, are gel purified, and resuspended in 10 M Tris-HCl (pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of 5-10 nanograms per microliter for microinjection. For example, the sequences encoding Zsig67 can encode a polypeptide comprising amino acid residues 111 to 422 of SEQ. ID NO: 2. Plasmid DNA is microinjected into harvested eggs contained in a drop of W640 medium and placed on mineral oil equilibrated with hot C02. The DNA is extracted by means of an injection needle (extracted from a borosilicate glass capillary of OD 1 mm and ID 0.75 mM), and injected into individual eggs. Each egg is penetrated with the needle of the injection, in one or both of the haploid pronuclei.

DNA picolitres are injected into the pronuclei, and the injection needle is removed without contacting the nucleoli. The procedure is repeated until all the eggs are injected. Successfully microinjected eggs are transferred to an organ tissue culture dish with a pre-gassed W640 medium for storage overnight in a 37 ° C / 5% C02 incubator. The next day, the embryos of two cells are transferred to the pregnant pessimals. The containers are identified by the presence of coupling obturators, after copulation with the vasectomized stallions. The containers are anesthetized and shaved on the left dorsal side and transferred to a surgical microscope. A small incision is made in the skin and through the muscular wall in the middle section of the abdominal area bounded by the rib cage, by the back room, and the hind leg, in the middle space between the knee and the spleen. The reproductive organs are exteriorized towards a small surgical drape. The fat layer extends towards the surgical drape, and a small forceps or forceps (Roboz, Rockville, MD) are attached to the fat layer and allowed to hang through the back or back of the mouse, preventing the organs slide backwards. With a fine transfer pipette containing mineral oil followed by the alternating W640 and air bubbles, 12 to 17 healthy embryos are transferred from two cells the day prior to injection into the container. The swollen blister is located and the oviduct is maintained between the blister and the pouch, a slit is made in the oviduct with a 28 g needle near the bag, making sure that the blister or the pouch does not break. The pipette is transferred into the crevice in the oviduct and the embryos are blown in, allowing the first air bubble to escape from the pipette. The fat layer is pushed gently into the peritoneum, and the reproductive organs are allowed to slide inward. The peritoneal wall is closed with a suture and the skin is closed with a wound clamp. The mice are recovered in a chamber heated at 37 ° C for a minimum of four hours. The containers are returned to the boxes in pairs, and left 19-21 days in gestation. After birth, leave 19-21 postpartum days before weaning. Weaned mice are classified by sex and placed in boxes separated by sex, and a 0.5 cm biopsy (used to obtain the genotype) is taken from the tail with clean scissors. Genomic DNA is prepared from the cuts in the tails using, for example, a QIAGEN DNEASY equipment following the manufacturer's instructions. Genomic DNA is analyzed by PCR using primers designed to amplify a Zsig67 gene or a selectable marker gene that is introduced into the same plasmid. After confirming that the animals are transgenic, they cross-cross in an inbred strain placing a tansgenic female with a native type male, or a transgenic male with one or two native type females. When the young are born they are weaned, separated by sex, and the cuts are made in the tail to obtain the genotype. To verify the expression of a transgene in a live animal, a hepatectomy is performed. A surgical preparation of the upper part of the abdomen is made directly below the zypuid process. Using the sterile technique, a small incision 1.5-2 cm below the sternum is made and the left lateral lobe of the liver is exteriorized. Using a 4-0 silk thread, a knot is made around the lower lobe securing it out of the body cavity. A non-traumatic clamp is used to hold the knot while a second absorbable Dexon loop (American Cyanamid; Wayne, N.J.) is placed close to the first knot. A distal cut of the Dexon node is made and approximately 100 mg of excised liver tissue is placed in a sterile petri dish. The section of the excised liver is transferred to a 14-mL round-bottom tube of polypropylene and rapidly frozen in liquid nitrogen and stored on dry ice.The surgical site is closed with suture and with wound clips, and the animal's cage on a cloth heated at 37 ° C for 24 hours postoperatively. The animal is checked daily after the operation and the wound tweezers are removed 7-10 days after surgery. The level of expression of Zsig67 mRNA for each transgenic mouse is examined using a hybridization assay of the RNA solution or the polymerase chain reaction. In addition, to produce transgenic mice that overexpress Zsig67, it is useful to genetically engineer transgenic mice that express abnormally low or do not express the gene. Such transgenic mice provide useful models for conditions associated with a deficiency of Zsig67. As discussed above, the expression of the Zsig67 gene can be inhibited using the anti-sense genes, ribozyme genes, or outer leader sequence genes. To produce the transgenic mice that sub-express the Zsig67 gene, such inhibitory sequences target the Zsig67 mRNA. Methods to produce transgenic mice that have an expression aqaémáója ± iláf i ag ae? DelaantégrHiicpar (tréculaDre £ Ej empibsid &? upaBfc al., "Gene Underexpression in Cultured Cells and Animáis by Antisense DNA and RNA Strategies," in Methods in Gene Biotechnology, pages 205-224 (CRC Press 1997). An alternative methodology for production of transgenic mice that has little or no expression of Zsig67 is to generate mice that have at least one allele of Zsig67 replaced by a non-functional Zsig67 gene. One method for designing a non-functional Zsig67 gene is to insert another gene, such as a selectable marker gene, into a nucleic acid molecule encoding Zsig67. Standard methods for producing these so-called "agénic mice" are known to those skilled in the art (see, for example, Jabob, "Expression and Knockout of Interferons in Transgenic Mice", in Overspression and Knockout of Cytokines in Transgenic Mice, Jacob ( ed.), pages 111-124 (Academic Press, Ltd. 1994), and Wu et al., "New Strategies for Gene Knockout," in Methods in Gene Biotechnology, pages 339-365 (CRC Press 1997). , thus described generally, will be more readily understood with reference to the following example, which is provided by way of illustration and is not intended to limit the present invention.

EXAMPLE 1 Construction of the Nucleic Acid Molecule that Encodes the Zsig67 RNA from the pituitary gland was purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA) and reverse transcribed in the following manner. The cDNA reaction of the first strand contained 10 μl of Poly (A) + mRNA of the human pituitary (CLONTECH), which had been selected twice with poly d (T), at a concentration of 1.0 mg / mL, and 2 μl of 20 pmol / μl of the primer of the first strand ZC6191 (GTCTG GGTTC GCTAC TCGAG GCGGC CGCTA TTTTT TTTTT TTTTT TTT; SEQ ID NO: 4) containing an XhoI restriction site. The mixture was heated at 70 ° C for 2.0 minutes and cooled by cooling on ice. The synthesis of the strand cDNA was initiated by the addition of 8 μl of the first strand buffer (SUPERSCRIPT 5x buffer, Life Technologies, Inc., Gaithersburg, MD), 4 μl of 100 mM dithiothreitol, and 2 μl of a solution of deoxynucleotide triphosphate (dNTP) each containing 10 mM of dTTP, dATP, dGTP and 5-methyl-dCTP (Pharmacia LKB Biotechnology; Piscataway, NJ) to the RNA-primer mixture. The reaction mixture was incubated at 37 ° C for 2 minutes, followed by the addition of 10 μl of the RNase H 200 U / μl reverse transcriptase (SUPERSCRIPT II, Life Technologies). The efficiency of the synthesis of the first strand was analyzed in a parallel reaction by adding 10 μCi of 32P-adCTP to a 5 μl aliquot from one of the reaction mixtures to label the reaction for analysis. The reactions were incubated at 37 ° C for 5 minutes, 45 ° C for 45 minutes, then incubated at 50 ° C for 10 minutes. The 32 P-adCTP not incorporated in the labeled reaction was removed by chromatography on a pore size 400 gel filtration column (CLONTECH). Nucleotides and primers not incorporated in the reactions of the first unlabeled strand by chromatography on a pore size 400 gel filtration column (CLONTECH). The length of the cDNA of the first strand was determined by electrophoresis in agarose gel. The reaction of the second strand contained 100 μl of the unlabeled first strand cDNA, 30 μl of the 5x polymerase buffer I (125 mM Tris-HCl, pH 7.5, 500 mM KCl, 25 mM MgCl 2, 50 mM (NH 4) 2 SO 4 ), 2.0 μl of 100 mM dithiothreitol, 3.0 μl of a solution containing 10 M of each deoxynucleotide triphosphate, 7 μl of 5 mM β-NAD, 2.0 μl of E DNA ligase. coli 10 U / μl (NEW ENGLAND BIOLABS; Beverly, MA), 5 μl of DNA polymerase I of 10 U / μl E. coli (NEW ENGLAND BIOLABS), and 1.0 μl of RNase H 2 U / μl (Life Technolgies) . An aliquot of 10 μl of one of the synthesis reactions of the second strand was labeled by the addition of 10 μCi of 32P-adCTP to monitor the efficiency of the synthesis of the second strand. The reactions were incubated at 16 ° C for two hours followed by the addition of 1 μl of a 10 mM dNTP solution and 5.0 μl of T4 DNA polymerase (10 U / μl, Boehringer Mannheim Corporation; Indianapolis, IN) and incubated for an additional 10 minutes at 16 ° C. The unincorporated 32P-adCTP was removed in the labeled reaction by chromatography through a pore size 400 gel filtration column (CLONTECH) before analysis by agarose gel electrophoresis. The reaction was terminated by the addition of 10 μl of 0.5 M EDTA and extraction with phenol / chloroform and chloroform followed by precipitation with ethanol in the presence of 3.0 M sodium acetate and 2 μl of the Pellet Paint carrier (Novagen, Inc .; Madison, Wl). DNA production was estimated as approximately 2 μg of the start mRNA template of 10 μg. The EcoRI adapters were ligated into the 5 'ends of the cDNA described above to allow cloning into an expression vector. A 12.5 μl aliquot of the cDNA (approximately 2.0 μg) and 3 μl of 69 pmol / μl of the BcoRI adapter (Pharmacia LKB Biotechnology, Inc.) were mixed with 2.5 μl of the lOx 2.5 μl ligase buffer (660 mM Tris-HCl pH 7.5, 100 mM MgCl 2), 2.5 μl of 10 mM ATP, 3.5 μl of 0.1 M DTT and 1 μg of DNA ligase Tr 15 U / μl (Promega Corp, Madison Wl). The reaction was incubated 1 hour at 5 ° C, 2 hours at 7.5 ° C, 2 hours at 10 ° C, 2 hours at 12.5 ° C, and 16 hours at 10 ° C. The reaction was terminated by the addition of 65 ul of H20 and 10 μl of the H lOx buffer (Boehringer Mannheim) and incubation at 70 ° C for 20 minutes. To facilitate directional cloning of the cDNA into an expression vector, the cDNA was digested with XhoI, resulting in cDNA molecules having a 5 'BcoRI cohesive end and a 3' Xho cohesive end. The Xhol restriction site at the 3 'end of the cDNA had been previously introduced. Digestion in a restriction enzyme was performed in a reaction mixture by the addition of 1.0 μl of 40 U / μl Xhol (Boehringer Mannheim) at 37 ° C for 45 minutes. The reaction was terminated by incubation at 70 ° C for 20 minutes and chromatography through the pore size gel filtration column 400 (CLONTECH). The cDNA was precipitated with ethanol, washed with 70% ethanol, dried with air and resuspended in 13.5 μl of water, 2 μl of the 10X kinase buffer (660 mM Tris-HCl, pH 7.5, 100 mM MgCl 2), 0.5 μl of DTT 0.1 μm, 2 μL of 10 mM ATP, 2 μL of the T4 polynucleotide kinase (10 U / μL, Life Technologies). After incubation at 37 ° C for 30 minutes, the cDNA was precipitated with ethanol in the presence of 2.5 M ammonium acetate, and fractionated on an electrophoresis gel in 0.8% low melt agarose. Contamination adapters and cDNA below 0.6 kb in length were cut from the gel. The electrodes were inverted and the cDNA fractioned until it was concentrated close to the lane of origin. The area of the gel containing the concentrated cDNA was cut and placed in a microcentrifuge tube, and the approximate volume of the silica gel was determined. An aliquot of water of approximately three times the volume of the silica gel (300 μl) and 35 μl of the Iβ-agarose IO buffer (NEW ENGLAD BIOLABS) were added to the tube, and the agarose was heated to 65 ° C for 15 minutes. minutes The following equilibrium of the sample was added at 45 ° C, 3 μl ß-agarose I 1 U / μl (NEW ENGLAD BIOLABS), and the mixture was incubated for 60 minutes at 45 ° C to digest the agarose. After incubation, 40 μl of 3M sodium acetate was added, and the mixture was incubated on ice for 15 minutes. The sample was centrifuged at 14,000 x g for 15 minutes at room temperature to remove undigested agarose. The DNA was precipitated with ethanol, washed with 70% ethanol, dried in air and resuspended in 20 μl of water. After recovering the low melting point agarose gel, the DNA was cloned into the BcoRI and Xhol sites of the pBLUESCRIPT SK + vector (Life Technologies, Inc.) and electroporated into DH10B cells. Bacterial colonies containing the ESTs of the known genes were identified and removed from the sequence analysis by repeated cycles of probe hybridization to high density colony filter arrays (Genoma Systems, Inc.; St. Louis, MO) cDNAs of known genes were assembled into groups of 50-100 inserts and labeled with 32P using a MEGAPRIME labeling kit (AMERSHAM PHARMACIA BIOTECH, Inc., Piscataway, NJ). Colonies that did not hybridize to the probe mix were selected for sequencing. Sequencing was performed with an ABI 377 sequencer using either T3 or the reverse primer. The resulting data was analyzed which resulted in the identification of new nucleotide sequences, including a fragment having the following sequence: GCACGAGGGCT ATGTTACCCC ATGACAGTTG ACCTCTTCTG GGTGAATATT ATCTGATTGA TTTTGCAAAT TCCACACCTG TATGGATGAA GAGAATATAT ATTGTAGATG CTAGATCATG AGGACTCCCA ACACCTCATT TCTAGTCCTG GCTTCACAGC CTCTTCTGGT CCTCATCTCA CTATCAGCAT TGATCCTGGC ATCATATTCA AGTCCCCTCC TAACCCGGGT CTCTCTTGAG ACAGTGAGAA CTAAAGAAGA TGGAAGACAC AATGATTTCA ACAAGATTAA GGATAAGGAT GCAAGTAGAG CAGGCAGGGA ACGAGGTTAT AGAAACTTTT TATTCCATTT CCATCTGTCT CTCTTTCCTC ACAACAAGTC (SEQ ID NO: 6.). This EST was used to obtain the coding region of Zsig67.

EXAMPLE 2 Southern Analysis of the Zsig67 Gene Southern analysis was performed using one commercially prepared Interspecies Zoo-Blot from CLONTECH Laboratories, Inc. The hybridization probe was generated from a gel-purified PCR amplification product of approximately 140 base pairs. The probe was made using ZC20,973 (5 'ATGCT AGATC ATGAG GACTC CCAAC 3', SEQ ID NO: 7), and ZC20,974 (5 'CCATC TTCTT TAGTT CTCAC TGTCT 3', SEQ ID NO: 8), as primers and the cDNA of the pituitary gland as a template. The probe was radioactively labeled using a REDIPRIME II labeling kit (AMERSHAM PHARMACIA BIOTECH, Inc., Piscataway, NJ), in accordance with the manufacturer's protocols. The probe was purified using a NUCTRAP push column (STRATAGENE, La Jolla, CA). The EXPRESSHYB solution (CLONTECH) was used for the prehybridization and hybridization solutions for the Southern stains. Hybridization took place overnight at 65 ° C. After hybridization, the stains were washed four times in 2X SSC, 0.05% SDS at room temperature, followed by two washes in 0.1 X SSC and 0.1% SDS at 50 ° C. The stains were then exposed to a Kodak BioMax film at 80 ° C. Hybridizing fragments were observed in the genomic DNA samples from rabbits, cows, dogs, mice and humans.

It is noted that with respect to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. * LIST OF SEQUENCE < 110 > Zy oGenetics. Inc. < 120 > ZSIG67: A MEMBER OF THE HUMAN HORMONE FAMILY OF SECRETINA-GLUCAGON- VASOACTIVE INTESTINAL PEPTIDE < 130 > 98-70PC < 160 > 8 < 170 > FastSEQ for Windows Version 3.0 < 210 > 1 < 211 > 1682 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (111) ... (422) < 400 > 1 gctatgttac cccatgacag ttgacctctt ctgggtgaat attatctgat tgattttgca 60 aattccacac ctgtatggat gaagagaata tatattgtag atgctagatc atg agg 116 Met Arg 1 act ccc aac ttact tta cta gtc ctg gct tca cag cct ctt ctg gtc 164 Thr Pro Asn Thr Ser Phe Leu Val Leu Ala Ser Gln Pro Leu Leu Val 5 10 15 ctc ate tca cta tca gca ttg ate ctg gca tca tat tca agt ccc ctc 212 Leu He Ser Leu Ser Ala Leu lie Leu Ala Ser Tyr Ser Ser Pro Leu 20 25 30 cta acc cgg gtc tet ctt gag aca gtg aga act aaa gaa gat ggá aga 260 Leu Thr Arg Val Ser Leu Glu Thr Val Arg Thr Lys Glu Asp Gly Arg 35 40 45 50 falls aat gat ttc aac aag aga gat aag gat gca agt aga gca ggc 308 His Asn Asp Phe Asn Lys lie Lys Asp Lys Asp Wing Arg Ala Gly 55 60 65 agg gaga cga ggt tat aga aac ttt tta ttc cat ttc cat ctg cet tet ctc 356 Arg Glu Arg Gly Tyr Arg Asn Phe Leu Phe His Phe His Leu Ser Leu 70 75 80 ttt cct falls aac agt ccc aat age att tca aag ggt ttc aag ttc cat 404 Phe Pro His Asn Ser Pro Asn Be He Ser Lys Gly Phe Lys Phe His 85 90 95 gta agt tat aga aaa aaa taaagcacaa gttctctgtg acatgttcag 452 Val Ser Tyr Arg Lys Lys 100 cttatctatg aatataacgg aactgaaacc atatttcaat aaattattaa cagcaatccc 512 ggatatcage ttgccaagaa acagataaca gggcaaatcc attataaetc ttaagcaaat 572 cctggaattt taaaaaaatt gaatagtgta tcaaataagt gcactgttag ctgggacttc 632 attaaacagc attttttttc ctttctttcc tgttcctccc cctttcccac cagcttccag 692 ccagaataat tgataactaa atctcaccat gaatttgttg ggaatttacc cacaatagtt 752 etcaatttat tttaagattt aaaataatat atttttaaat gac agtagattaa tttacag 812 aaatttcaga taggataata cagaacettg aaaagactta ttttttacaa acttccttaa 872 aaattcttaa ttgttctaac agtcacagtt ttgtttatgt atacaattgg agttctgcag 932 taagacactg tggggttatt agtacatata tcaaattcat ttcctgtata gaggctccta 992 ttgttacatja gctgttcttg tatattgatg tttaatgata tggggcegaa ttgttattca 1052 aataattaca gcccattcct gattatttag atgctgatta actagtcaat gaettattec 1112 ctttgacaaa atttcccacc ctctggcttt cctttgatgt taatgaggaa gaggatccag 1172 ttttcatccc atttggcaag gttggtttca tggactgagc cactctggat gtaagatcag 1232 gtagacttta tagetactet agttaggtta tggtaatgga ggttgcatca gaacaetcaa 1292 tatcaccata aagtcaagcc tttgagaaga aactcacagt ccttagtgga atttgatata 1352 tttgaaacaa catettatgt tacaaaaaaa tagcatttta aaaattgggt ggggcactgt 1412 ggatcactcc tgtaatccca cacttgggg aggccagggt gggcagatca cttgaggccg 1472 ggatttcgag gctagcctgg ccaacgtggt gaagccccat etetattaga actacaggaa 1532 ttagctgggc atggtggtgc acgcctgtgg tgccagccac tcgggtggtt gagacatgag 1592 aattgctttg acccgggagg cggaggttgc gatcatgeca agtgagccaa ctgcactcta 1652 1682 cagageaaga gcctgggcga ctctgtctca < 210 > 2 < 211 > 104 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Met Arg Thr Pro Asn Thr Ser Phe Leu Val Leu Wing Ser Gln Pro Leu 1 5 10 15 Leu Val Leu lie Ser Leu Be Wing Leu He Leu Wing Being Tyr Being Ser Pro Leu Leu Thr Arg Val Being Leu Glu Thr Val Arg Thr Lys Glu Asp 35 40 45 Gly Arg His Asn Asp Phe Asn Lys He Lys Asp Lys Asp Wing Ser Arg 50 55 60 Wing Gly Arg Glu Arg Gly Tyr Arg Asn Phe Leu Phe His Pfae Hi's Leu 65 70 75 80 Ser Leu Phe Pro His Asn Ser Pro Asn Ser Be Ser Lys Gly Phe Lys 85 90 95 Phe His Val Ser Tyr Arg Lys Lys 100 < 2? 0 > 3 < 211 > 312 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > This degenerate sequence encodes the amino acid sequence of SEQ ID NO: 2. < 221 > Variation < 222 > (1) . .. (312) < 223 > , N is any neucleotide < 400 > 3 atgmgnacnc cnaayacnws nttyytngtn ytngcnwsnc arccnytnyt ngtnytnath 60 wsnytnwsng cnytnathyt ngcnwsntay wsnwsnccny tnytnacnmg ngtnwsnytn 120 garacngtnm gnacnaarga rgayggnmgn cayaaygayt tyaayaarat haargayaar 180 gaygcnwsnm gngcnggn g ngar gnggn taymgnaayt .tyytnttyca ttycayytn 240 wsnytnttyc cncayaayws nccnaaywsn athwsnaarg gnttyaartt ycaygtnwsn taymgnaara ar 300312 < 210 > 4 < 211 > 16 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Linked Peptide < 400 > 4 Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 m 10 115c < 210 > 5 < 211 > 48 < 212 > DNA < 213 > Sequential Artificial < 220 > < 223 > Oligonucleotide primer < 400 > 5 gtctgggttc gctactcgag gcggccgcta tttttttttt tttttttt 48 < 210 > 6 < 211 > 381 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > EST < 400 > 6 gcacgagggc tatgttaccc catgacagtt gacctcttct gggtgaatat tatetgattg 60 attttgcaaa ttccacacct gtatggatga agagaatata tattgtagat gctagatcat 120 gaggactccc aacacctcat ttctagtcct ggetteacag cctcttctgg tcctcatctc 180 actatcagea ttgatcctgg catcatatte aagtcccctc ctaacccggg tctctcttga 240 gacagtgaga actaaagaag atggaagaca aacaagatta caatgatttc aggataagga 300 tgeaagtaga geaggeaggg aacgaggtta tagaaaettt ttattccatt tccatctgtc tctctttcct cacaacaagt c 381 360 < 210 > 7 < 211 > 25 < 212 > DNA < 213 Artificial sequence < 220 > < 223 > PCR priming < 400 > 7 atgetagate atgaggactc ccaac 25 < 210 > 8 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Cevador PCR. < 400 > 8 ccatcttctt tagttetcae tgtct 25

Claims (20)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. An asylated polypeptide, characterized in that it comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2, wherein the isolated polypeptide specifically binds to an antibody that specifically binds to a polypeptide that consists of the amino acid sequence of SEQ ID NO: 2, and wherein any difference between the amino acid sequence of the isolated polypeptide and the amino acid sequence corresponding to SEQ ID NO: 2 is due to a conservative amino acid substitution. The isolated polypeptide of claim 1, characterized in that the isolated polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:

2.

3. The isolated polypeptide of the claim 1, characterized in that the isolated polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2.

4. An isolated polypeptide selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence of amino acid residues 29 to 45 of SEQ ID NO: 2, (b), a polypeptide comprising the amino acid sequence of residues of amino acid 51 to 63 of SEQ ID NO: 2, (c) a polypeptide comprising the amino acid sequence of amino acid residues 52 to 101 of SEQ ID NO: 2, (d) a polypeptide comprising the amino acid sequence of amino acid residues 68 to 92 of SEQ ID NO: 2, (e) a polypeptide comprising the amino acid sequence of amino acid residues 70 to 101 of SEQ ID NO: 2, and (f) a polypeptide comprising the amino acid sequence of maino acid residues 73 to 92 of SEQ ID NO: 2.

5. The isolated polypeptide of the claim 4, characterized in that it is either a polypeptide comprising the amino acid sequence of amino acid residues 1 to 40 of SEQ ID NO: 2, or a polypeptide comprising the amino acid sequence of amino acid residues 1 to 101 of SEQ ID NO: 2. NO: 2. The isolated polypeptide of claim 4, characterized in that the isolated polypeptide is selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence of amino acid residues 29 to 48 of SEQ ID NO : 2, (b) a polypeptide comprising the amino acid sequence of amino acid residues 29 to 48 of SEQ ID NO: 2, wherein the last amino acid residue of the mentioned sequence is subjected to amidation, (c) a polypeptide comprising the amino acid sequence of amino acid residues 29 to 50 of SEQ ID NO: 2, (d) a polypeptide comprising the amino acid sequence of amino acid residues 29 to 50 of SEQ ID NO: 2, (e) a polypeptide comprising the amino acid sequence of amino acid residues 29 to 63 of SEQ ID NO: 2, (f) a polypeptide comprising the amino acid sequence of amino acid residues 29 to 68 of SEQ ID NO: 2, (g) a polypeptide which comprises the amino acid sequence of amino acid residues 29 to 101 of SEQ ID NO: 2, wherein the last amino acid residue of said sequence is subjected to amidation, and (h) a polypeptide comprising the amino acid residue sequence of amino acids 29 to 104 of SED ID NO: 2. 7. The isolated polypeptide of claim 4, characterized in that the isolated polypeptide is selected from the group consisting of (a) a polypeptide comprising the amino acid sequence of amino acid residues 51 to 65 of SEQ ID NO: 2, wherein the last amino acid residue of the said sequence is subjected to amidation, (b) a polypeptide comprising the amino acid sequence of amino acid residues 51 to 67 of SEQ ID NO: 2. DO NOT : 2, (c) a polypeptide comprising the amino acid sequence of amino acid residues 51 to 92 of SEQ ID NO: 2, (d) a polypeptide comprising the amino acid sequence of amino acid residues 51 to 104 of SEQ ID NO: 2, (e) a polypeptide comprising the amino acid sequence of amino acid residues 52 to 101 of SEQ ID NO. : 2, wherein the last amino acid residue of the mentioned sequence is subjected to amidation, (f) a polypeptide comprising the amino acid sequence of amino acid residues 52 to 104 of SEQ ID NO: 2, (g) a polypeptide comprising the amino acid sequence of amino acid residues 68-101 of SEQ ID NO: 2, (h) a polypeptide comprising the amino acid sequence of amino acid residues 68-104 of SED ID NO: 2, (i) a polypeptide comprising the amino acid sequence of amino acid residues 73 to 101 of SEQ ID NO: 2, and (j) a polypeptide comprising the amino acid sequence of amino acid residues 73 to 104 of SEQ ID NO: 2. 8. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 3, and (b) a nucleic acid molecule that remains hybridized after the conditions of severe washing to a nucleic acid molecule consisting of a nucleotide sequence of 111-422 of SEQ ID NO: 1, or the nucleotide complement 111-422 of SEQ ID NO: 1. 9. The isolated nucleic acid molecule of claim 8, characterized in that any difference between the amino acid sequence encoded by the nucleic acid molecule and the corresponding amino acid sequence of SEQ ID NO: 2 is due to a conservative amino acid substitution . The isolated nucleic acid molecule of claim 8, characterized in that the nucleic acid molecule comprises either the nucleotide sequence of nucleotides 195 to 422 of SEQ ID NO: 1, or the nucleotide sequence of nucleotides 111 to 422 of SEQ ID NO: 1. 11. A vector, characterized in that it comprises the isolated nucleic acid molecule of claim 10. 12. An expression vector, characterized in that it comprises the isolated nucleic acid molecule of claim 10, a transcription promoter, and a. transcription terminator, wherein the promoter is operably linked to the nucleic acid molecule, and wherein the nucleic acid molecule is operably linked to the transcription terminator. 13. A recombinant host cell, characterized in that it comprises the expression vector of claim 12, characterized in that the host cell is selected from the group consisting of bacteria cells, yeast cells, fungal cells, insect cells, mammalian cells, and plant cells. A method for using the expression vector of claim 12 to produce the Zsig67 protein, characterized in that it comprises culturing recombinant host cells comprising the expression vector and producing the Zsig67 protein. 15. An antibody or antibody fragment, characterized in that it specifically binds to the polypeptide of claim 4. 1

6. A method for detecting the presence of RNA of Zsig67 in a biological sample, characterized in that it comprises the steps of: (a) contacting a Zsig67 nucleic acid probe under hybridization conditions with either (i) testing of RNA molecules isolated from the biological sample, or ( ii) nucleic acid molecules synthesized from isolated RNA molecules, wherein the probe comprises a nucleotide sequence comprising either a portion of the nucleic acid molecule of claim 10, or its complement, and (b) detecting the formation of hybrid nucleic acid probe and either test the RNA probe molecules or the synthesized nucleic acid molecules, wherein the presence of the hybrids indicate the presence of Zsig67 RNA in the biological sample. 1

7. A method for detecting the presence of Zsig67 in a biological sample, characterized in that it comprises the steps of: (a) contacting the biological sample with an antibody, or an antibody fragment, of claim 15, wherein the contact it is carried out under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibodies or linked antibody fragments. 1

8. An anti-idiotype antibody, or anti-idiotype antibody fragment, that specifically binds to the antibody or antibody fragment of claim 15. 1

9. A fusion protein, characterized in that it comprises a polypeptide of claim 4. 20. The fusion protein of claim 19, characterized in that the fusion protein further comprises an immunoglobulin portion.