WO2001029223A2 - Type i membrane protein ztsl1 - Google Patents

Abstract

Novel polypeptides, polynucleotides encoding them, materials and methods for making them, antibodies that specifically bind to them, and methods of using the polypeptides, polynucleotides, and antibodies are disclosed. The polypeptides comprise at least nine contiguous amino acid residues of SEQ ID NO:2 and may be prepared as polypeptide fusions comprising heterologous sequences, such as affinity tags. The polypeptides and polynucleotides encoding them may be used within a variety of therepeutic, diagnostic, and research applications, including in vitro diagnosis and in vivo imaging of cancers and other sites of abnormal cell proliferation.

Description

Description TYPE I MEMBRANE PROTEIN ZTSL1

BACKGROUND OF THE INVENTION

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

Cytokines are physicochemically diverse, ranging in size from 5 kDa (TGF-α) to 140 kDa (Mullerian-inhibiting substance). They include single polypeptide chains, as well as disulfϊde-linked homodimers and heterodimers. Some cytokines (e.g., bFGF, IL-lβ, TNF, and TGF-α) occur in both secreted (soluble) and membrane-anchored forms. The secreted forms may result from proteolysis of their membrane-anchored counterparts (Aggarwal and Puri, "Common and Uncommon Features of Cytokines and Cytokine Receptors: An Overview", in Aggarwal and Puri, eds., Human Cytokines: Their Role in Disease and Therapy. Blackwell Science, Cambridge, MA, 1995, 3-24).

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

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

The role of cytokines in controlling cellular processes makes them likely candidates and targets for therapeutic intervention; indeed, a number of cytokines have been approved for clinical use. Interferon-alpha (EFN-α), for example, is used in the treatment of hairy cell leukemia, chronic myeloid leukemia, Kaposi's sarcoma, condylomata acuminata, chronic hepatitis C, and chronic hepatitis B (Aggarwal and Puri, ibid.). Platelet-derived growth factor (PDGF) has been approved in the United States and other countries for the treatment of dermal ulcers in diabetic patients. The hematopoietic cytokine erythropoietin has been developed for the treatment of anemias (e.g., EP 613,683). G-CSF, GM-CSF, IFN-β, IFN-γ, and IL-2 have also been approved for use in humans (Aggarwal and Puri, ibid.). Experimental evidence supports additional therapeutic uses of cytokines and their inhibitors. Inhibition of PDGF receptor activity has been shown to reduce intimal hyperplasia in injured baboon arteries (Giese et al., Restenosis Summit VUI, Poster Session #23, 1996; U.S. Patent No. 5,620,687). Vascular endothelial growth factors (VEGFs) have been shown to promote the growth of blood vessels in ischemic limbs (Isner et al., The Lancet 348:370-374, 1996), and have been proposed for use as wound-healing agents, for treatment of periodontal disease, for promoting endothelialization in vascular graft surgery, and for promoting collateral circulation following myocardial infarction (WIPO Publication No. WO 95/24473; U.S. Patent No. 5,219,739). A soluble VEGF receptor (soluble flt-1) has been found to block binding of VEGF to cell-surface receptors and to inhibit the growth of vascular tissue in vitro (Biotechnology News 16(17):5-6, 1996). Experimental evidence suggests that inhibition of angiogenesis may be used to block tumor development (Biotechnology News, Nov. 13, 1997) and that angiogenesis is an early indicator of cervical cancer (Br. J. Cancer 76:1410-1415, 1997). More recently, thrombopoietin has been shown to stimulate the production of platelets in vivo (Kaushansky et al., Nature 369:568-571, 1994) and has been the subject of several clinical trials (reviewed by von dem Borne et al., Bailliere's Clin. Haematol. 11:427-445, 1998).

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

DESCRIPTION OF THE INVENTION

Within one aspect of the invention there is provided an isolated polypeptide comprising at least nine contiguous amino acid residues of SEQ ID NO:2. Within one embodiment, the isolated polypeptide of claim 1 consists of from 15 to

1500 amino acid residues. Within another embodiment, the at least nine contiguous amino acid residues of SEQ ID NO: 2 are operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein, an immunoglobulin constant region, a polyhistidine tag, and a peptide as shown in SEQ ID NO:3. Within another embodiment, the isolated polypeptide comprises at least 30 contiguous residues of SEQ ID NO:2. Exemplary polypeptides of the invention include, without limitation, those comprising residues 22-200, 1-200, 22-240, or 1-240 SEQ ID NO:2.

Within a second aspect of the invention there is provided an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as disclosed above; and a transcription terminator. Within one embodiment, the DNA segment comprises nucleotides 64-600 or 64-720 of SEQ ID NO:4. Within another embodiment, the expression vector further comprises a secretory signal sequence operably linked to the DNA segment. An exemplary secretory signal sequence encodes residues 1-21 of SEQ ID NO:2. Within a third aspect of the invention there is provided a cultured cell into which has been introduced an expression vector as disclosed above, wherein the cell expresses the DNA segment. Within one embodiment, the expression vector comprises a secretory signal sequence operably linked to the DNA segment, and the polypeptide is secreted by the cell. The cultured cell of the invention can be used within a method of making a protein, wherein the cell is cultured under conditions whereby the DNA segment is expressed and the polypeptide is produced, and the protein is recovered from the cell. Within one embodiment, the expression vector comprises a secretory signal sequence operably linked to the DNA segment, the polypeptide is secreted by the cell, and the polypeptide is recovered from a medium in which the cell is cultured.

Within a fourth aspect of the invention there is provided a protein produced by the method disclosed above.

Within a fifth aspect of the invention there is provided an antibody that specifically binds to a protein as disclosed above. Within another aspect of the invention there is provided an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a fusion protein, the protein comprising residues 1-21 of SEQ ID NO:2 operably linked to a second polypeptide; and a transcription terminator. There is also provided a cultured cell into which the expression vector has been introduced, wherein the cell expresses the DNA segment. The cell can be used within a method of making a protein, the method comprising the steps of culturing the cell under conditions whereby the DNA segment is expressed and the protein is produced; and recovering the protein.

Within a sixth aspect of the invention there is provided a method of detecting, in a test sample, the presence of an antagonist of ztsll activity, comprising the steps of culturing a cell that is responsive to ztsll, exposing the cell to a ztsll polypeptide in the presence and absence of a test sample, comparing levels of response to the ztsll polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay, and determining from the comparison the presence of an antagonist of ztsll activity in the test sample. These and other aspects of the invention will become evident upon reference to the following detailed description of the invention and the accompanying figure.

The figure is a Hopp/Woods hydrophilicity profile of the amino acid sequence shown in SEQ ID NO:2. The profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored. These residues are indicated in the figure by lower case letters.

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

The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988), Glu-

Glu affinity tag (Grussenmeyer et al., Proc. Natl Acad. Sci. USA 82:7952-4, 1985) (SEQ ID NO:3), substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-1210, 1988), streptavidin binding peptide, maltose binding protein (Guan et al., Gene 67:21- 30, 1987), cellulose binding protein, thioredoxin, ubiquitin, T7 polymerase, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107, 1991. DNAs encoding affinity tags and other reagents are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ; New England Biolabs, Beverly, MA; Eastman Kodak, New Haven, CT).

The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene. A "complement" of a polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.

"Conservative amino acid substitutions" are defined by the BLOSUM62 scoring matrix of Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992, an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins. As used herein, the term "conservative amino acid substitution" refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least one 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).

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

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

An "inhibitory polynucleotide" is a DNA or RNA molecule that reduces or prevents expression (transcription or translation) of a second (target) polynucleotide. Inhibitory polynucleotides include antisense polynucleotides, ribozymes, and external guide sequences. The term "inhibitory polynucleotide" further includes DNA and RNA molecules that encode the actual inhibitory species, such as DNA molecules that encode ribozymes.

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

An "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. The isolated polypeptide or protein may be prepared substantially free of other polypeptides or proteins, particularly those of animal origin. For some purposes, the polypeptides and proteins will be prepared in a highly purified form, i.e. greater than 95% pure or greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide or protein in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms. "Operably linked" means that two or more entities are joined together such that they function in concert for their intended purposes. When referring to DNA segments, the phrase indicates, for example, that coding sequences are joined in the correct reading frame, and transcription initiates in the promoter and proceeds through the coding segment(s) to the terminator. When referring to polypeptides, "operably linked" includes both covalently (e.g., by disulfide bonding) and non-covalently (e.g., by hydrogen bonding, hydrophobic interactions, or salt-bridge interactions) linked sequences, wherein the desired function(s) of the sequences are retained.

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

A "polynucleotide" is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.

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

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

A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless, thus, a protein "consisting of, for example, from 15 to 1500 amino acid residues may further contain one or more carbohydrate chains.

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

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

The term "splice variant" is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene. Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to ±10%. The present invention is based on the discovery of a novel polynucleotide and protein encoded by the polynucleotide. The polynucleotide is expressed primarily in tumor tissues. The polynucleotide and protein are thus markers for the presence of cancer in a mammal, and also provide targets for diagnostic and therapeutic agents. The novel protein, termed "ztsll," is related (24% amino acid sequence identity) to the previously described human T1/ST2 ligand disclosed by Gayle et al., J. Biol. Chem. 271:5784-5789, 1996. The Tl gene, known as ST2, DER4, and Fit-1, encodes a member of the interleukin-1 (IL-1) receptor family (Mitcham et al., J. Biol. Chem. 271:5777-5783, 1996). The Tl gene is transcribed in two forms, a soluble form and a membrane-bound form. The classical IL-1 ligands (IL-lα, IL-lβ, and IL- lra) do not bind Tl. The T1/ST2 ligand binds Tl but is unable to initiate signal transduction by the membrane-bound form. The ligand is apparently a type I membrane protein. It has a predicted molecular weight (excluding the signal sequence and transmembrane domain) of about 22 kD, and has no sequence or hydrophobicity profile similarity to the beta-trefoil cytokines IL-1 or the FGFs.

Ztsll is also related (22% amino acid sequence identity) to the human P24 protein (Blum et al., J. Biol. Chem. 271: 17183-17189, 1996). P24 is an endoplasmic reticulum (ER)-bound protein predicted to have a role in secretory protein sorting and transport from the ER to the Golgi apparatus. A representative human ztsll DNA sequence is shown in SEQ ID

NO:l, and the encoded amino acid sequence is shown in SEQ ID NO:2. Those skilled in the art will recognize that the illustrated sequences represent a single allele of ztsll, and that allelic variation is expected to exist. Those skilled in the art will also recognize that many proteins are produced in alternatively spliced forms. For example, some cytokines are produced in both soluble and membrane-anchored forms.

Ztsll may also exist in multiple forms, for example soluble and membrane-anchored forms.

The sequence shown in SEQ ID NO: 2 includes a putative secretory peptide comprising residues 1-21. Residues 201-223 include characteristics of a transmembrane domain. Alignment with related protein sequences shows the presence of three conserved residues in ztsll: Cys 55, Cys 119, and Asp 81. The two conserved cysteines may be disulfide-bonded. Those skilled in the art will recognize that predicted domain boundaries are somewhat imprecise and may vary by up to ± 5 amino acid residues.

While not wishing to be bound by theory, ztsll may be an antagonist that binds IL-1 receptor and regulates the activity of an as yet undiscovered IL-1 homolog. The presence of a transmembrane domain-like sequence at the carboxyl terminus of ztsl suggests that the protein can exist in a membrane-anchored form. Thus, ztsll may modulate the proliferation, differention, or metabolism of responsive cell types, particularly IL-1 responsive cells. Polypeptides of the present invention comprise at least 9 or at least 15 contiguous amino acid residues of SEQ ID NO: 2. Within certain embodiments of the invention, the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of SEQ ID NO:2, up to the entire predicted mature polypeptide (residues 22 to 240 of SEQ ID NO:2) the primary translation product (residues 1 to 240 of SEQ ID NO:2), or a soluble form (residues 1 to 200 or 22 to 200). As disclosed in more detail below, these polypeptides can further comprise additional, non-ztsll, polypeptide sequence(s).

Within the polypeptides of the present invention are polypeptides that comprise an epitope-bearing portion of a protein as shown in SEQ ID NO:2. An "epitope" is a region of a protein to which an antibody can bind. See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 8L3998-4002, 1984. Epitopes can be linear or conformational, the latter being composed of discontinuous regions of the protein that form an epitope upon folding of the protein. Linear epitopes are generally at least 6 amino acid residues in length. Relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, Sutcliffe et al., Science 219:660-666. 1983. Antibodies that recognize short, linear epitopes are particularly useful in analytic and diagnostic applications that employ denatured protein, such as Western blotting (Tobin, Proc. Natl. Acad. Sci. USA 76:4350-4356, 1979), or in the analysis of fixed cells or tissue samples. Antibodies to linear epitopes are also useful for detecting fragments of ztsll, such as might occur in body fluids or cell culture media.

Antigenic, epitope-bearing polypeptides of the present invention are useful for raising antibodies, including monoclonal antibodies, that specifically bind to^~ a ztsll protein. Antigenic, epitope-bearing polypeptides contain a sequence of at least six, generally at least nine, often from 15 to about 30 contiguous amino acid residues of a ztsll protein (e.g., SEQ ID NO:2). Polypeptides comprising a larger portion of a ztsll protein, i.e. from 30 to 50 residues up to the entire sequence, are included. It is preferred that the amino acid sequence of the epitope-bearing polypeptide is selected to provide substantial solubility in aqueous solvents, that is the sequence includes relatively hydrophilic residues, and hydrophobic residues are substantially avoided. Such regions include those comprising residues 148-153, 234-239, 146-151, 233-238, and 144-149 of SEQ ID NO:2. Larger hydrophilic peptides include, for example, residues 144-153.

Polypeptides of the present invention can be prepared with one or more amino acid substitutions, deletions or additions as compared to SEQ ID NO:2. These changes are preferably of a minor nature, that is, conservative amino acid substitutions and other changes that do not significantly affect the folding or activity of the protein or polypeptide, and include amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue, an amino or carboxyl-terminal cysteine residue to facilitate subsequent linking to maleimide-activated keyhole limpet hemocyanin, a small linker peptide of up to about 20-25 residues, or an extension that facilitates purification (an affinity tag) as disclosed above. Two or more affinity tags may be used in combination. Polypeptides comprising affinity tags can further comprise a polypeptide linker and/or a proteolytic cleavage site between the ztsll polypeptide and the affinity tag. Such cleavage sites include, for example, thrombin cleavage sites and factor Xa cleavage sites.

The present invention further provides a variety of other polypeptide fusions. For example, a ztsll polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584. Dimerizing proteins in this regard include immunoglobulin constant region domains, which can be used in combination with immunoglobulin hinge regions to create a ztsll -Fc fusion protein. For example, residues 22-200 of SEQ ID NO: 2 can be fused to an immunoglobulin Fc molecule to produce a dimeric form of the ztsll protein. Immunoglobulin-ztsll polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric ztsll analogs. In addition, a ztsll polypeptide can be joined to another bioactive molecule, such as a cytokine, to provide a multifunctional molecule. One or more helices of a ztsll polypeptide can be joined to another cytokine to enhance or otherwise modify its biological properties. Auxiliary domains can be fused to ztsll polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen). For example, a ztsll polypeptide or protein can be targeted to a predetermined cell type by fusing a ztsll polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell. In the alternative, a ztsll polypeptide can be used as a targetting moiety within a polypeptide fusion. In this way, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. A ztsll polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain. Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34:1-9, 1996.

A secretory peptide of a ztsll protein can be used to direct the secretion of other proteins of interest from a host cell. Thus, the present invention provides, inter alia, fusions comprising such a secretory peptide operably linked to another protein of interest. The secretory peptide can be used to direct the secretion of other proteins of interest by joining a polynucleotide sequence encoding it, in the correct reading frame, to the 5' end of a sequence encoding the other protein of interest. Those skilled in the art will recognize that the resulting fused sequence may encode additional residues of a ztsll protein at the amino terminus of the protein to be secreted. In the extreme case, the fusion may comprise an entire ztsll protein fused to the amino terminus of a second protein, whereby secretion of the fusion protein is directed by the ztsll secretory peptide. It will often be desirable to include a proteolytic cleavage site between the ztsll sequence and the other protein of interest. When such fusions are designed so that the secreted protein retains a portion of the ztsll protein, the fusion protein can be purified by means that exploit the properties of the remaining ztlsl sequence. Typical of such methods is immunoaffinity chromatography using an antibody directed against ztsll. When such a fusion is engineered to contain a cleavage site at the fusion point, the fusion can be cleaved and the protein of interest recovered free of extraneous (ztsll) sequence. Polypeptide fusions of the present invention will generally contain not more than about 1,500 amino acid residues, usually not more than about 1,200 residues, more commonly not more than about 1,000 residues, and will in many cases be considerably smaller. For example, a ztsll polypeptide of 219 residues (residues 22-240 of SEQ ID NO:2) can be fused to E. coli /3-galactosidase (1,021 residues; see Casadaban et al., J. Bacteriol. 143:971-980, 1980), a 10-residue spacer, and a 4- residue factor Xa cleavage site to yield a polypeptide of 1254 residues. In a second example, residues 22-240 of SEQ ID NO:2 can be fused to maltose binding protein (approximately 370 residues), a 4-residue cleavage site, and a 6-residue polyhistidine tag. The proteins of the present invention can also comprise non-naturally occuring amino acid residues. Non-naturally occuring amino acids include, without limitation, tr n-f-3-methylproline, 2,4-methanoproline, c.-.-4-hydroxyproline, trans-A- hydroxyproline, N-methylglycine, α/Zo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occuring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRΝAs. Methods for synthesizing amino acids and aminoacylating tRΝAs are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., /. Am. Chem. Soc. 113:2722, 1991; Εllman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-809, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-10149, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRΝA and chemically aminoacylated suppressor tRΝAs (Turcatti et al., J. Biol. Chem. 271:19991-19998. 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occuring amino acid(s) (e.g., 2-azaphenylalanine, 3- azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occuring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994. Naturally occuring amino acid residues can be converted to non-naturally occuring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395- 403, 1993).

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

A hydrophilicity profile of SEQ ID NO: 2 is shown in the attached figure. Those skilled in the art will recognize that hydrophilicity will be taken into account when designing alterations in the amino acid sequence of a ztsll polypeptide, so as not to disrupt the overall profile.

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

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and

Sauer (Science 241:53-57, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). These authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region- directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

Variants of the disclosed ztsll DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391 , 1994 and Stemmer, Proc. Natl. Acad. Sci. USA 9J,: 10747- 10751, 1994. Briefly, variant genes are generated by in vitro homologous recombination by random fragmentation of a parent gene followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent genes, such as allelic variants or genes from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes. In many cases, the structure of the final polypeptide product will result from processing of the nascent polypeptide chain by the host cell, thus the final sequence of a ztsll polypeptide produced by a host cell will not always correspond to the full sequence encoded by the expressed polynucleotide. For example, expressing the complete ztsll sequence in a cultured mammalian cell is expected to result in removal of at least the secretory peptide, while the same polypeptide produced in a prokaryotic host would not be expected to be cleaved. Differential processing of individual chains may result in heterogeneity of expressed polypeptides. ztsll proteins of the present invention are expected to modulate the proliferation, differentiation, migration, adhesion, or metabolism of responsive cell types. Biological activity of ztsll proteins is assayed using in vitro or in vivo assays designed to detect cell proliferation, differentiation, migration or adhesion; or changes in cellular metabolism (e.g., production of other growth factors or other macromolecules). Many suitable assays are known in the art, and representative assays are disclosed herein. Assays using cultured cells are most convenient for screening, such as for determining the effects of amino acid substitutions, deletions, or insertions. However, in view of the complexity of developmental processes (e.g., angiogenesis, wound healing), in vivo assays will generally be employed to confirm and further characterize biological activity. Certain in vitro models, such as the three- dimensional collagen gel matrix model of Pepper et al. (Biochem. Biophys. Res. Comm. 189:824-831, 1992), are sufficiently complex to assay histological effects. Assays can be performed using exogenously produced proteins, or can be carried out in vivo or in vitro using cells expressing the polypeptide(s) of interest. Assays can be conducted using ztsll proteins alone or in combination with other cytokines, such as members of the IL-1 family or hematopoietic cytokines (e.g., EPO, TPO, G-CSF, stem cell factor). Representative assays are disclosed below. Mutagenesis methods as disclosed above can be combined with high volume or high-throughput screening methods to detect biological activity of ztsll variant polypeptides. Assays that can be scaled up for high throughput include mitogenesis assays, which can be run in a 96-well format. Mutagenized DNA molecules that encode active ztsll polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art can prepare a variety of polypeptide fragments or variants of SEQ ID NO:2 that retain the activity of wild-type ztsl 1.

The present invention also provides ztsll polynucleotide molecules. These polynucleotides include DNA and RNA, both single- and double-stranded, the former encompassing both the sense strand and the antisense strand. A representative DNA sequence encoding the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:l. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. SEQ ID NO:4 is a degenerate DNA sequence that encompasses all DNAs that encode the ztsll polypeptide of SEQ ID NO: 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO:4 also provides all RNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus, ztsll polypeptide-encoding polynucleotides comprising nucleotides 1-720 or nucleotides 64-720 of SEQ ID NO:4, and their RNA equivalents are contemplated by the present invention, as are segments of SEQ ID NO:4 encoding other ztsll polypeptides disclosed herein. Table 1 sets forth the one-letter codes used within SEQ ID NO:4 to denote degenerate nucleotide positions. "Resolutions" are the nucleotides denoted by a code letter. "Complement" indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.

Table 1

Nucleotide Resolutions Complement Resolutions

A A T T

C C G G

G G C C

T T A A

R A|G Y C|T

Y C|T R A|G

M A|C K G|T

K G|T M A|C

S C|G S C|G

W A|T w A|T

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

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

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

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

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

The degenerate codons used in SEQ ID NO:4, encompassing all possible codons for a given amino acid, are set forth in Table 2, below.

Table 2

Amino One-Letter Degenerate

Acid Code Codons Codon

Cys C TGC TGT TGY

Ser s AGC AGT TCA TCC TCG TCT WSN

Thr T ACA ACC ACG ACT CAN

Pro P CCA CCC CCG CCT CCN

Ala A GCA GCC GCG GCT GCN

Gly G GGA GGC GGG GGT GGN

Asn N AAC AAT AAY

Asp D GAC GAT GAY

Glu E GAA GAG GAR

Gin Q CAA CAG CAR

His H CAC CAT CAY

Arg R AGA AGG CGA CGC CGG CGT MGN

Lys K AAA AAG AAR

Met M ATG ATG lie I ATA ATC ATT ATH

Leu L CTA CTC CTG CTT TTA TTG YTN

Val V GTA GTC GTG GTT GTN

Phe F TTC TTT TTY

Tyr Y TAC TAT TAY

Trp V _W TGG TGG

Ter TAA TAG TGA TRR

Asn|Asp B RAY

Glu|Gln Z SAR

Any X NNN

Gap - —

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

One of ordinary skill in the art will also appreciate that different species can exhibit preferential codon usage. See, in general, Grantham et al., Nuc. Acids Res. 8:1893-912, 1980; Haas et al. Curr. Biol. 6:315-24, 1996; Wain-Hobson et al., Gene 13:355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res. 14:3075-87, 1986; and Dcemura, J. Mol Biol. 158:573-97, 1982. Introduction of preferred codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NO:4 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein.

Within certain embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:l or a sequence complementary thereto under stringent conditions. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C. As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of ztsll RNA. Cells from pancreas, thyroid, and various tumor tissues are preferred. Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94. 1979). Poly (A)⁺ RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972). Complementary DNA (cDNA) is prepared from poly(A)⁺ RNA using known methods. In the alternative, genomic DNA can be isolated. Polynucleotides encoding ztsll polypeptides are then identified and isolated by, for example, hybridization or PCR.

Full-length clones encoding ztsll can be obtained by conventional cloning procedures. Complementary DNA (cDNA) clones are usually preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron. Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library. Expression libraries can be probed with antibodies to ztsll, receptor fragments, or other specific binding partners. ztsll polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a ztsll gene. Promoter elements from a ztsll gene can thus be used to direct the expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy. Cloning of 5' flanking sequences also facilitates production of ztsll proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous ztsll gene in a cell is altered by introducing into the ztsll locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site. The targeting sequence is a ztsll 5' non-coding sequence that permits homologous recombination of the construct with the endogenous ztsll locus, whereby the sequences within the construct become operably linked with the endogenous ztsll coding sequence. In this way, an endogenous ztsll promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue- specific, or otherwise regulated expression. A 5'-flanking ztsll genomic sequence is shown in SEQ ID NO:5.

Those skilled in the art will recognize that the sequences disclosed in SEQ ID NOS:l and 2 represent a single allele of human ztsll. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. The present invention further provides counterpart polypeptides and polynucleotides from other species ("orthologs"). Of particular interest are ztsll polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. These non-human ztsll polypeptides and polynucleotides, as well as antagonists thereof and other related molecules, can be used, inter alia, in veterinary medicine. Orthologs of human ztsll can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses ztsll as disclosed above. A library is then prepared from mRNA of a positive tissue or cell line. A ztsll -encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequence. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human ztsll sequence disclosed herein. Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to ztsll polypeptide. Similar techniques can also be applied to the isolation of genomic clones.

For any ztsll polypeptide, including variants and fusion proteins, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2, above. Moreover, those of skill in the art can use standard software to devise ztsll variants based upon the nucleotide and amino acid sequences described herein. The present invention thus provides a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ ED NO:l, SEQ ID NO:2, SEQ ID NO:4, and portions thereof. Suitable forms of computer-readable media include magnetic media and optically-readable media. Examples of magnetic media include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT), a disk cache, and a ZIP™ disk. Optically readable media are exemplified by compact discs (e.g., CD-read only memory (ROM), CD- rewritable (RW), and CD-recordable), and digital versatile/video discs (DVD) (e.g.,

DVD-ROM, DVD-RAM, and DVD+RW).

The ztsll polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides can be produced according to conventional techniques using cells into which have been introduced an expression vector encoding the polypeptide. As used herein, "cells into which have been introduced an expression vector" include both cells that have been directly manipulated by the introduction of exogenous DNA molecules and progeny thereof that contain the introduced DNA. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987. In general, a DNA sequence encoding a ztsll polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers can be provided on separate vectors, and replication of the exogenous DNA is provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.

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

(see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).

Cultured mammalian cells can be used as hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al., ibid.), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993). The production of recombinant polypeptides in cultured mammalian cells is disclosed by, for example, Levinson et al., U.S. Patent No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al.,

J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1, ATCC No. CCL 61; or CHO DG44, Chasin et al., Som. Cell Molec. Genet. 12:555, 1986) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, VA. Suitable promoters include those from metallothionein genes (U.S. Patent Nos. ), the adenovirus major late promoter, and promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patents Nos. 4,579,821; 4,601,978; and 4,956,288. Expression vectors for use in mammalian cells include pZP-1 and pZP-9, which have been deposited with the American Type Culture Collection, Manassas, VA USA under accession numbers 98669 and 98668, respectively, and derivatives thereof. Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." An exemplary selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. An exemplary amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that produce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, and placental alkaline phosphatase, can be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.

The adenovirus system (disclosed in more detail below) can also be used for protein production in vitro. By culturing adenovirus-infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division. In an alternative method, adenovirus vector-infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (See Gamier et al., Cytotechnol. 15:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the disposition of the expressed protein in the cell. Within the infected 293 cell production protocol, non-secreted proteins can also be effectively obtained.

Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV) according to methods known in the art, such as the transposon-based system described by Luckow et al. (J. Virol. 67:4566-4579, 1993). This system, which utilizes transfer vectors, is commercially available in kit form (Bac-to-Bac™ kit; Life Technologies, Rockville, MD). The transfer vector (e.g., pFastBacl™; Life Technologies) contains a Tn7 transposon to move the DNA encoding the protein of interest into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid." See, Hill-Perkins and Possee, /. Gen. Virol 7 971-976, 1990; Bonning et al., J. Gen. Virol. 75: 1551- 1556, 1994; and Chazenbalk and Rapoport, J. Biol. Chem. 270: 1543-1549, 1995. In addition, transfer vectors can include an in-frame fusion with DNA encoding a polypeptide extension or affinity tag as disclosed above. Using techniques known in the art, a transfer vector containing a ztsll -encoding sequence is transformed into E. coli host cells, and the cells are screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, such as Sf9 cells. Recombinant virus that expresses ztsll protein is subsequently produced. Recombinant viral stocks are made by methods commonly used the art. For protein production, the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., High Five™ cells; Invitrogen, Carlsbad, CA). See, for example, U.S. Patent No. 5,300,435. Serum-free media are used to grow and maintain the cells. Suitable media formulations are known in the art and can be obtained from commercial suppliers. The cells are grown up from an inoculation density of approximately 2-5 x 10⁵ cells to a density of 1-2 x 10⁶ cells, at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3. Procedures used are generally known in the art.

Other higher eukaryotic cells can also be used as hosts, including plant cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47- 58, 1987.

Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). An exemplary vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-3465, 1986; Cregg, U.S. Patent No. 4,882,279; and Raymond et al., Yeast \Λ, 11-23, 1998. Aspergillus cells can be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533. Production of recombinant proteins in Pichia methanolica is disclosed in U.S. Patents Nos. 5,716,808, 5,736,383, 5,854,039, and 5,888,768. Prokaryotic host cells, including strains of the bacteria Escherichia coli,

Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.). When expressing a ztsll polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding. Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. ztsll polypeptides can also be prepared through chemical synthesis according to methods known in the art, including exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, IL, 1984;

Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989. In vitro synthesis is particularly advantageous for the preparation of smaller polypeptides.

Using methods known in the art, ztsll proteins can be prepared as monomers or multimers; glycosylated or non-glycosylated; pegylated or non- pegylated; and may or may not include an initial methionine amino acid residue.

Depending upon the intended use, the polypeptides and proteins of the present invention can be purified to >80% purity, >90% purity, >95% purity, or to a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Within certain embodiments, the polypeptide or protein is substantially free of other polypeptides or proteins, particularly those of animal origin.

Ztsll proteins (including chimeric polypeptides and multimeric proteins) are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See, in general, Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice. Springer- Verlag, New York, 1994. Proteins comprising a polyhistidine affinity tag (typically about 6 histidine residues) are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bio/Technol. 6: 1321-1325, 1988. Proteins comprising a glu-glu tag can be purified by immunoaffinity chromatography according to conventional procedures. See, for example, Grussenmeyer et al., ibid. Maltose binding protein fusions are purified on an amylose column according to methods known in the art. Target cells for use in ztsll activity assays include, without limitation, fibroblasts, smooth muscle cells, endothelial cells, T cells, hepatocytes, and mesangial cells. Target cells include both primary cells and cell lines.

Activity of ztsll proteins can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to an appropriate animal model. Assays measuring cell proliferation or differentiation are well known in the art. For example, assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990), incorporation of radiolabeled nucleotides (as disclosed by, e.g., Raines and Ross, Methods Enzymol 109:749-773, 1985; Wahl et al., Mol. Cell Biol. 8:5016-5025, 1988; and Cook et al., Analytical Biochem. 179:1-7, 1989), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., /. Immunol. Methods 82:169-179, 1985), and use of tetrazolium salts (Mosmann, J. Immunol Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res. 48:4827-4833, 1988). Differentiation can be assayed using suitable precursor cells that can be induced to differentiate into a more mature phenotype. Assays measuring differentiation include, for example, measuring cell-surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; and Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses. 161-171, 1989). Ztsll activity may also be detected using assays designed to measure ztsll modulation of production of one or more additional growth factors or other macromolecules. Such assays include those for determining the presence of hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor alpha (TGFα), interleukin-6 (IL-6), VEGF, acidic fibroblast growth factor (aFGF), angiogenin, and other macromolecules. Assays of IL-1 activity include, for example, gel-shift assays for NF-κB activation, Thr-669 kinase activity assays, and IL- 8 promoter activation assays. See, Mitcham et al., ibid. Suitable assays include mitogenesis assays, receptor-binding assays, competition binding assays, immunological assays (e.g., ELISA), and other formats known in the art. Metalloprotease secretion is measured from treated primary human dermal fibroblasts, synoviocytes and chondrocytes. The relative levels of collagenase, gelatinase and stromalysin produced in response to culturing in the presence of a ztsll protein is measured using zymogram gels (Loita and Stetler-Stevenson, Cancer Biology 1:96- 106, 1990). Procollagen collagen synthesis by dermal fibroblasts and chondrocytes in response to a test protein is measured using ³H-proline incorporation into nascent secreted collagen. ³H-labeled collagen is visualized by SDS-PAGE followed by autoradiography (Unemori and Amento, J. Biol. Chem. 265: 10681-10685, 1990). Glycosaminoglycan (GAG) secretion from dermal fibroblasts and chondrocytes is measured using a 1,9-dimethylmethylene blue dye binding assay (Farndale et al., Biochim. Biophys. Ada 883:173-177, 1986). Inhibition of cytokine activity is assayed by including ztsll with one or more cytokines known to be active in a given assay. Collagen and GAG assays, for example, are carried out in the presence of IL-1 β or TGF-β to examine the ability of ztsll protein to modify the established responses to these cytokines.

Monocyte activation assays are carried out (1) to look for the ability of ztsll proteins to modulate monocyte activation, including attachment-induced or endotoxin-induced monocyte activation (Fuhlbrigge et al., J. Immunol. 138: 3799- 3802, 1987). IL-lβ and TNFα levels produced in response to activation are measured by ELISA (Biosource, Inc. Camarillo, CA). Monocyte/macrophage cells, by virtue of

CD14 (LPS receptor), are exquisitely sensitive to endotoxin, and proteins with moderate levels of endotoxin-like activity will activate these cells.

In vitro assays for pro- and anti-inflammatory activity are known in the art. Exemplary activity assays include mitogenesis assays in which JJL-l responsive cells (e.g., the D10.N4.M murine T cell line) are incubated in the presence of IL-1 or a test protein for 72 hours at 37°C in a 5% CO atmosphere. IL-2 (and optionally IL-4) is added to the culture medium to enhance sensitivity and specificity of the assay. ³H- thymidine is then added, and incubation is continued for six hours. The amount of label incorporated is indicative of agonist activity. See, Hopkins and Humphreys, J. Immunol. Methods 120:271-276, 1989; Greenfeder et al., J. Biol. Chem. 270:22460- 22466, 1995. Stimulation of cell proliferation can also be measured using thymocytes cultured in a test protein in combination with phytohemagglutinin. IL-1 is used as a control. Proliferation is detected as ³H-thymidine incorporation or metabolic breakdown of (MTT) (Mosman, ibid.).

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

Cell migration is assayed essentially as disclosed by Kahler et al. (Arteriosclerosis, Thrombosis, and Vascular Biology 17:932-939, 1997). A protein is considered to be chemotactic if it induces migration of cells from an area of low protein concentration to an area of high protein concentration. A typical assay is performed using modified Boyden chambers with a polystryrene membrane separating the two chambers (Transwell®; Corning Costar® Corp.). The test sample, diluted in medium containing 1% BSA, is added to the lower chamber of a 24-well plate containing Transwells. Cells are then placed on the Transwell insert that has been pretreated with 0.2% gelatin. Cell migration is measured after 4 hours of incubation at 37°C. Non-migrating cells are wiped off the top of the Transwell membrane, and cells attached to the lower face of the membrane are fixed and stained with 0.1% crystal violet. Stained cells are then extracted with 10% acetic acid and absorbance is measured at 600 nm. Migration is then calculated from a standard calibration curve. Cell migration can also be measured using the matrigel method of Grant et al. ("Angiogenesis as a component of epithelial-mesenchymal interactions" in Goldberg and Rosen, Epithelial-Mesenchymal Interaction in Cancer, Birkhauser Verlag, 1995, 235-248; aatout, Anticancer Research 17:451-456, 1997). Cell adhesion activity is assayed essentially as disclosed by LaFleur et al. (J. Biol Chem. 272:32798-32803, 1997). Briefly, microtiter plates are coated with the test protein, non-specific sites are blocked with BSA, and cells (such as smooth muscle cells, leukocytes, or endothelial cells) are plated at a density of approximately 10⁴ - 10⁵ cells/well. The wells are incubated at 37°C (typically for about 60 minutes), then non-adherent cells are removed by gentle washing. Adhered cells are quantitated by conventional methods (e.g., by staining with crystal violet, lysing the cells, and determining the optical density of the lysate). Control wells are coated with a known adhesive protein, such as fibronectin or vitronectin.

Other metabolic effects of ztsll proteins can be measured by culturing target cells in the presence and absence of a protein and observing changes in adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, or the like. Suitable assays are known in the art.

The activity of ztsll proteins can be measured with a silicon-based biosensor microphysiometer that measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses. An exemplary such device is the Cytosensor™ Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA. A variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell et al., Science 257:1906-1912, 1992; Pitchford et al., Meth. Enzymol. 228:84-108, 1997; Arimilli et al., J. Immunol. Meth. 212:49-59, 1998; and Van Liefde et al., Ewr. J. Pharmacol. 346:87-95, 1998. The microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including ztsll proteins, their agonists, and antagonists.

Expression of ztsll polynucleotides in animals provides models for further study of the biological effects of overproduction or inhibition of protein activity in vivo. Ztsll -encoding polynucleotides and antisense polynucleotides can be introduced into test animals, such as mice, using viral vectors or naked DNA, or transgenic animals can be produced.

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

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

An alternative method of gene delivery comprises removing cells from the body and introducing a vector into the cells as a naked DNA plasmid. The transformed cells are then re-implanted in the body. Naked DNA vectors are introduced into host cells by methods known in the art, including transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter. See, Wu et al., J. Biol Chem. 263:14621-14624, 1988; Wu et al., J. Biol Chem. 267:963- 967, 1992; and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994.

Transgenic mice, engineered to express a ztsll gene, and mice that exhibit a complete absence of ztsll gene function, referred to as "knockout mice" (Snouwaert et al., Science 257:1083, 1992), can also be generated (Lowell et al., Nature 366:740-742, 1993). These mice can be employed to study the ztsll gene and the protein encoded thereby in an in vivo system. Transgenic mice are particularly useful for investigating the role of ztsll proteins in early development in that they allow the identification of developmental abnormalities or blocks resulting from the over- or underexpression of a specific factor. See also, Maisonpierre et al., Science 277:55-60, 1997 and Hanahan, Science 277:48-50, 1997. Promoters for transgenic expression include promoters from metallothionein and albumin genes. Antisense methodology can be used to inhibit ztsll gene transcription to examine the effects of such inhibition in vivo. Polynucleotides that are complementary to a segment of a ztsll -encoding polynucleotide (e.g., a polynucleotide as set forth in SEQ ID NO:l) are designed to bind to ztsll -encoding mRNA and to inhibit translation of such mRNA. Such antisense oligonucleotides can also be used to inhibit expression of ztsll polypeptide-encoding genes in cell culture.

The polypeptides, nucleic acids and antibodies of the present invention may be used in diagnosis or treatment of disorders associated with cell loss or abnormal cell proliferation (including cancer). Analysis of gene expression has shown that ztsll is expressed in a variety of tumor tissues, including breast, colon, kidney, brain, liver, prostate, and pancreas (islet cell hyperplasia) tumors. Ztsll is thus a diagnostic marker of these tumors. Those skilled in the art will recognize that assays can be performed on body fluids (e.g., plasma, serum, urine), tissue samples, or isolated cells. In addition, ztsll provides a target for therapeutic agents. Assays for ztsll can be used to detect soluble protein in body fluids

(e.g., plasma, serum, urine) or cell-associated protein in isolated cells or tissue samples. General methods for collecting samples and assaying for the presence and amount of a protein are known in the art. Assays will commonly employ an anti-ztsll antibody or other specific binding partner (e.g., soluble receptor). The antibody or binding partner can itself be labeled, thereby directly providing a detectable signal, or a labeled second antibody or binding partner can be used to provide the signal.

Labeled anti-ztsll antibodies or other binding partners may be used in vivo for imaging tumors or other sites of abnormal cell proliferation. Imaging agents will commonly include a radionuclide, electron-dense compound, or other detectable molecule.

Anti-ztsll antibodies or other binding partners can be directly or indirectly conjugated to radionuclides or other detectable molecules, and these conjugates used for diagnostic or therapeutic applications. For in vivo use, an anti- ztsll antibody or other binding partner can be directly or indirectly coupled to a detectable molecule and delivered to a mammal having cells, tissues, or organs that express ztsll. Suitable detectable molecules include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles, electron-dense compounds, heavy metals, and the like. These can be either directly attached to the antibody or other binding partner, or indirectly attached according to known methods, such as through a chelating moiety. For indirect attachment of a detectable molecule, the detectable molecule can be conjugated with a first member of a complementary/anticomplementary pair, wherein the second member of the pair is bound to the anti-ztsll antibody or other binding partner. Biotin/streptavidin is an exemplary complementary/anticomplementary pair; others will be evident to those skilled in the art. Toxin-conjugated antibodies or other binding partners may be used for targeted cell or tissue inhibition or ablation, such as in cancer therapy. Of particular interest in this regard are conjugates of a ztsll polypeptide and a cytotoxin, which can be used to target the cytotoxin to a tumor or other abnormal tissue that is expressing ztsll. For such therapeutic use, anti-ztsll antibodies or other binding partners are directly or indirectly coupled to cytotoxic molecules as generally disclosed above. Suitable cytotoxic molecules include bacterial and plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin, saporin, and the like); therapeutic radionuclides, such as iodine-131, rhenium- 188, and yttrium-90; and cytotoxic drugs, such as adriamycin. In another embodiment, a cytokine is linked to an anti-ztsll antibody or other binding partner, and the resulting conjugate is used for enhancing in vitro cytotoxicity (for instance, that mediated by monoclonal antibodies against tumor targets) and for enhancing in vivo killing of target tissues (for example, blood and bone marrow cancers). See, generally, Hornick et al., Blood 89:4437-4447, 1997). In general, cytokines are toxic if administered systemically. The antibody or other binding partner conjugate enables targeting of a cytokine to a desired site of action, such as a cell having cell-surface ztsll, thereby providing an elevated local concentration of cytokine. Suitable cytokines for this purpose include, for example, interleukin-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Such conjugates may be used to cause cytokine-induced killing of tumors and other tissues undergoing angiogenesis or neovascularization. Antibody-cytokine conjugates can be produced as fusion proteins. Those skilled in the art will recognize that antibody fragments are effective as targeting agents.

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

As used herein, the term "antibodies" includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and Fab fragments, single chain antibodies, and the like, including genetically engineered antibodies. Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non- human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered" antibody). In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. One skilled in the art can generate humanized antibodies with specific and different constant domains (i.e., different Ig subclasses) to facilitate or inhibit various immune functions associated with particular antibody constant domains. Antibodies are defined to be specifically binding if they bind to a ztsll polypeptide or protein with an affinity at least 10-fold greater than the binding affinity to control (non-ztsll) polypeptide or protein. The affinity of a monoclonal antibody can be readily determined by one of ordinary skill in the art (see, for example, Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949). Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see for example, Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982). As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats. The immunogenicity of a ztsll polypeptide may be increased through the use of an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of a ztsll polypeptide or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

Alternative techniques for generating or selecting antibodies include in vitro exposure of lymphocytes to ztsll polypeptides, and selection of antibody display libraries in phage or similar vectors (e.g., through the use of immobilized or labeled ztsll polypeptide). Human antibodies can be produced in transgenic, non-human animals that have been engineered to contain human immunoglobulin genes as disclosed in WIPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination. A variety of assays known to those skilled in the art can be utilized to detect antibodies that specifically bind to ztsll polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include concurrent immunoelectrophoresis, radio-immunoassays, radio- immunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, Western blot assays, inhibition or competition assays, and sandwich assays.

In addition to the diagnostic and therapeutic uses disclosed above, anti- ztsll antibodies can be used for affinity purification of the protein, for immunolocalization within whole animals or tissue sections, for immunohistochemistry, and as antagonists to block protein activity in vitro and in vivo. Antibodies to ztsll can also be used in analytical methods employing fluorescence-activated cell sorting (FACS), for screening expression libraries, and for generating anti-idiotypic antibodies. For pharmaceutical use, ztsll proteins, anti-ztsll antibodies, and other bioactive compounds are formulated for topical or parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. In general, pharmaceutical formulations will include a ztsll polypeptide, antibody, or other compound in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water, or the like. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995. Ztsll will commonly be used in a concentration of about 10 to 100 μg/ml of total volume, although concentrations in the range of 1 ng/ml to 1000 μg/ml may be used. For topical application the protein will be applied in the range of 0.1-10 μg/cm² of surface area. The exact dose will be determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art. Dosing is daily or intermittently over the period of treatment. intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. Sustained release formulations can also be employed.

Ztsll proteins, agonists, and antagonists may be used for modulating the expansion, proliferation, activation, differentiation, migration, or metabolism of responsive cell types, which include both primary cells and cultured cell lines as disclosed above. Ztsll polypeptides are added to tissue culture media for these cell types at a concentration of about 10 pg/ml to about 100 ng/ml. Those skilled in the art will recognize that ztsll proteins can be advantageously combined with other growth factors in culture media. Within the laboratory research field, ztsll proteins can also be used as molecular weight standards or as reagents in assays for determining circulating levels of the protein, such as in the diagnosis of disorders characterized by over- or underproduction of ztsll protein or in the analysis of cell phenotype.

Polynucleotides and polypeptides of the present invention will additionally find use as educational tools as a laboratory practicum kits for courses related to genetics and molecular biology, protein chemistry and antibody production and analysis. Due to its unique polynucleotide and polypeptide sequence molecules of ztsll can be used as standards or as "unknowns" for testing purposes. For example, ztsll polynucleotides can be used as an aid, such as, for example, to teach a student how to prepare expression constructs for bacterial, viral, and or mammalian expression, including fusion constructs, wherein ztsll is the gene to be expressed; for experimentally determining the restriction endonuclease cleavage sites of the polynucleotides (which can be determined from the sequence using conventional computer software, such as MapDraw™ (DNASTAR, Madison, WI)); determining mRNA and DNA localization of ztsll polynucleotides in tissues (i.e., by Northern and Southern blotting as well as polymerase chain reaction); and for identifying related polynucleotides and polypeptides by nucleic acid hybridization.

Ztsll polypeptides can be used educationally as an aid to teach preparation of antibodies; identifying proteins by Western blotting; protein purification; determining the weight of expressed ztsll polypeptides as a ratio to total protein expressed; identifying peptide cleavage sites; coupling amino and carboxyl terminal tags; amino acid sequence analysis, as well as, but not limited to monitoring biological activities of both the native and tagged protein (i.e., receptor binding, signal transduction, proliferation, and differentiation) in vitro and in vivo, ztsll polypeptides can also be used to teach analytical skills such as mass spectrometry, circular dichroism to determine conformation, in particular the locations of the disulfide bonds, x-ray crystallography to determine the three-dimensional structure in atomic detail, nuclear magnetic resonance spectroscopy to reveal the structure of proteins in solution. For example, a kit containing the ztsll can be given to the student to analyze. Since the amino acid sequence would be known by the professor, the protein can be given to the student as a test to determine the skills or develop the skills of the student, the teacher would then know whether or not the student has correctly analyzed the polypeptide. Since every polypeptide is unique, the educational utility of ztsll would be unique unto itself.

Ztsll proteins can also be used to identify inhibitors of their activity. Test compounds are added to the assays disclosed above to identify compounds that inhibit the activity of ztsll protein. In addition to those assays disclosed above, samples can be tested for inhibition of ztsll activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of ztsll -dependent cellular responses. For example, ztsll -responsive cell lines can be transfected with a reporter gene construct that is responsive to a ztsll -stimulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a ztsll -activated serum response element (SRE) operably linked to a gene encoding an assayable protein, such as luciferase. Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of ztsll on the target cells as evidenced by a decrease in ztsll stimulation of reporter gene expression. Assays of this type will detect compounds that directly block ztsll binding to cell-surface receptors, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding. In the alternative, compounds or other samples can be tested for direct blocking of ztsll binding to receptor using ztsll tagged with a detectable label (e.g., ¹²⁵I, biotin, horseradish peroxidase, FTTC, and the like). Within assays of this type, the ability of a test sample to inhibit the binding of labeled ztsll to the receptor is indicative of inhibitory activity, which can be confirmed through secondary assays. Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors. Polypeptides and proteins of the present invention can be used to identify and isolate receptors. Ztsll receptors may be involved in growth regulation in the liver, blood vessel formation, and other developmental processes. For example, ztsll proteins and polypeptides can be immobilized on a column, and membrane preparations run over the column (as generally disclosed in Immobilized Affinity Ligand Techniques, Hermanson et al., eds., Academic Press, San Diego, CA, 1992, pp.195-202). Proteins and polypeptides can also be radiolabeled (Methods Enzymol., vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Academic Press, San Diego, 1990, 721-737) or photoaffinity labeled (Brunner et al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedan et al., Biochem. Pharmacol. 33:1167-1180, 1984) and used to tag specific cell-surface proteins. In a similar manner, radiolabeled ztsll proteins and polypeptides can be used to clone the cognate receptor in binding assays using cells transfected with an expression cDNA library.

The polynucleotides of the present invention can be used in diagnostic applications. For example, the ztsll gene, a probe comprising ztsll DNA or RNA, or a subsequence thereof can be used to determine the presence of mutations at or near the ztsll locus. Detectable chromosomal aberrations at the ztsll gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes, and rearrangements. These aberrations can occur within the coding sequence, within introns, or within flanking sequences, including upstream promoter and regulatory regions, and may be manifested as physical alterations within a coding sequence or changes in gene expression level. Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14- 17 nucleotides) can be used. PCR primers are at least 5 nucleotides in length, preferably 15 or more nt, more preferably 20-30 nt. Short polynucleotides can be used when a small region of the gene is targetted for analysis. For gross analysis of genes, a polynucleotide probe may comprise an entire exon or more. Probes will generally comprise a polynucleotide linked to a signal-generating moiety such as a radionucleotide. In general, these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (c) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient. Genetic samples for use within the present invention include genomic DNA, cDNA, and RNA. The polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO:l, the complement of SEQ ID NO:l, or an RNA equivalent thereof. Suitable assay methods in this regard include molecular genetic techniques known to those in the art, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, ligation chain reaction

(Barany, PCR Methods and Applications 1:5-16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; A.J. Marian, Chest 108:255-65, 1995). Ribonuclease protection assays (see, e.g., Ausubel et al., ibid., ch. 4) comprise the hybridization of an RNA probe to a patient RNA sample, after which the reaction product (RNA-RNA hybrid) is exposed to RNase. Hybridized regions of the RNA are protected from digestion. Within PCR assays, a patient genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in size or amount of recovered product are indicative of mutations in the patient. Another PCR-based technique that can be employed is single strand conformational polymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications 1:34-38, 1991).

Sequence tagged sites (STSs) can also be used independently for chromosomal localization. An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome. An STS is defined by a pair of oligonucleotide primers that are used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Since STSs are based solely on DNA sequence they can be completely described within an electronic database, for example, Database of Sequence Tagged Sites (dbSTS), GenBank (National Center for Biological information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), and can be searched with a gene sequence of interest for the mapping data contained within these short genomic landmark STS sequences.

The ztsll gene maps to human chromosome 16 at 16q22. This region is associated with several disorders, including acute myelogenous leukemia, acute myeloid leukemia (M4Eo subtype), macular corneal dystrophy (associated with mutations in the sulfotransferase gene CHST6), cirrhosis (North American Indian childhood type), and hypertension. See, OMIM™ Database, Johns Hopkins University, 2000 (http://www.ncbi. nlm.nih.gov/entrez/query.fcgi?db=OMIM).

Inhibitors of ztsll activity (ztsll antagonists) include anti-ztsll antibodies, inactive receptor-binding fragments of ztsll polypeptides, soluble ztsll receptors, and other peptidic and non-peptidic agents (including inhibitory polynucleotides and small molecule inhibitors). Such antagonists can be used to block the effects of ztsll on cells or tissues. Antagonists are formulated for pharmaceutical use as generally disclosed above, taking into account the precise chemical and physical nature of the inhibitor and the condition to be treated. The relevant determinations are within the level of ordinary skill in the formulation art.

Polynucleotides encoding ztsll polypeptides and inhibitory polynucleotides are useful within gene therapy applications where it is desired to increase or inhibit ztsll activity. If a mammal has a mutated or absent ztsll gene, a ztsll gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a ztsll polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno- associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. A defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-330, 1991); an attenuated adenovirus vector, such as the vector described by Stratford- Perricaudet et al., /. Clin. Invest. 90:626-630, 1992; and a defective adeno-associated virus vector (Samulski et al., J. Virol. 61:3096-3101, 1987; Samulski et al., J. Virol. 63:3822-3888, 1989). Within another embodiment, a ztsll gene can be introduced in a retroviral vector as described, for example, by Anderson et al., U.S. Patent No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., J. Virol. 62:1120, 1988; Temin et al., U.S. Patent No. 5,124,263; Dougherty et al., WIPO Publication WO 95/07358; and Kuo et al., Blood 82:845, 1993. Alternatively, the vector can be introduced by liposome-mediated transfection ("lipofection"). Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-8031, 1988). The use of lipofection to introduce exogenous polynucleotides into specific organs in vivo has certain practical advantages, including molecular targeting of liposomes to specific cells. Directing transfection to particular cell types is particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the purpose of targeting. Peptidic and non- peptidic molecules can be coupled to liposomes chemically. Within another embodiment, cells are removed from the body, a vector is introduced into the cells as a naked DNA plasmid, and the transformed cells are re-implanted into the body as disclosed above.

Antisense polynucleotides can be used to inhibit ztsll gene transcription. Polynucleotides that are complementary to a segment of a ztsll - encoding polynucleotide (e.g., a polynucleotide as set forth in SEQ ID NO:l) are designed to bind to ztsll -encoding mRNA and to inhibit translation of such mRNA. Antisense polynucleotides can be targetted to specific tissues using a gene therapy approach with specific vectors and/or promoters, such as viral delivery systems. Ribozymes can also be used as ztsll antagonists. Ribozymes are RNA molecules that contain a catalytic center and a target RNA binding portion. The term includes RNA enzymes, self-splicing RNAs, self-cleaving RNAs, and nucleic acid molecules that perform these catalytic functions. A ribozyme selectively binds to a target RNA molecule through complementary base pairing, bringing the catalytic center into close proximity with the target sequence. The ribozyme then cleaves the target RNA and is released, after which it is able to bind and cleave additional molecules. A nucleic acid molecule that encodes a ribozyme is termed a "ribozyme gene." Ribozymes can be designed to express endonuclease activity that is directed to a certain target sequence in a 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). An expression vector can be constructed in which a regulatory element is operably linked to a nucleotide sequence that encodes a ribozyme. In another approach, 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 that encode a ztsll polypeptide. An external guide sequence is constructed for directing 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., U.S. Patent No. 5,168,053; Yuan et al., Science 263:1269, 1994; Pace et al., WIPO Publication No. WO 96/18733; George et al., WIPO Publication No. WO 96/21731; and Werner et al., WIPO Publication No. WO 97/33991). An external guide sequence generally comprises a ten- to fifteen- nucleotide sequence complementary to ztsll mRNA, and a 3'-NCCA nucleotide sequence, wherein N is preferably a purine. The external guide sequence transcripts bind to the targeted mRNA species by the formation of base pairs between the mRNA and the complementary external guide sequences, thus promoting cleavage of mRNA by RNase P at the nucleotide located at the 5 '-side of the base-paired region.

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

Examples Example 1

A panel of cDNAs from human tissues was screened for ztsll expression using PCR. The panel contained 80 cDNA samples from various normal and cancerous human tissues and cell lines is shown in Table 3, below. The cDNAs were made using a commercially available kit (Marathon™ kit; Clontech Laboratories, Inc., Palo Alto, CA), tested with clathrin primers ZC21,195 (SEQ ID NO:6) and ZC21,196 (SEQ ID NO:7), and diluted based on the intensity of the clathrin band. To assure quality of the panel samples, three tests for quality control (QC) were run. First, to assess the RNA quality used for the libraries, certain of the cDNAs were tested for average insert size by PCR with vector oligos that were specific for the vector sequences for an individual cDNA library. Second, standardization of the concentration of the cDNA in panel samples was achieved using standard PCR methods to amplify full length alpha tubulin or G3PDH cDNA using a 5' vector oligonucleotide primer ZC14,063 (SEQ ID NO:8) and 3' alpha tubulin specific oligonucleotide primer ZC17,574 (SEQ ID NO:9) or 3' G3PDH specific oligonucleotide primer ZC17,600 (SEQ ID NO: 10). Third, a sample was sequenced to check for possible ribosomal or mitochondrial DNA contamination. The panel was set up in a 96-well format that included a human genomic DNA (obtained from Clontech Laboratories, Inc.) positive control sample. Each well contained approximately 0.2- 100 pg/μl of cDNA. The PCR reactions were set up using oligonucleotide primers ZC37,244 (SEQ ID NO: 11) and ZC37,245 (SEQ ID NO: 12), DNA polymerase (Ex Taq™; TAKARA Shuzo Co. Ltd., Biomedicals Group, Japan), and a density increasing agent and tracking dye (RediLoad, Research Genetics, Inc., Huntsville, AL). The amplification was carried out as follows: inclubation at 94°C for 2 minutes;

35 cycles of 94°C for 30 seconds, 60.2°C for 30 seconds, and 72°C for 30 seconds; followed by incubation at 72°C for 5 minutes. About 10 μl of the PCR reaction product was electrophoresed on a 4% agarose gel. The predicted DNA fragment size of -461 bp was observed in adrenal gland, cervix, colon, fetal kidney, fetal liver, kidney, liver, lung, lymph node, mammary gland, ovary, pancreas, placenta, prostate, rectum, salivary gland, small intestine, stomach, testis, thymus, adipocyte, islet, RPMI 1788 cells, thyroid, esophagus tumor, liver tumor, lung tumor, rectum tumor, stomach tumor, bone marrow, CD3+ cells, HaCAT cells, and MG63 cells.

The DNA fragments for adipocyte, islet, kidney, and RPMI 1788 cells were excised and purified using a commercially available gel extraction kit (obtained from Qiagen, Chatsworth, CA) according to the manufacturer's instructions. Fragments were sequenced to to confirm their identity as ztsll. Table 3

Tissue/Cell line #samples Tissue/Cell line #samples

Adrenal gland ] Bone marrow 2

Bladder 1 Fetal brain 2

Bone Marrow i Islet 1

Brain Prostate 2

Cervix 1 [ RPMI #1788 (ATCC # CCL-156) 2

Colon ] Testis 3

Fetal brain Thyroid

Fetal heart 2 WI38 (ATCC # CCL-75)

Fetal kidney ] Spinal cord

Fetal liver HaCat - human keratinocytes

Fetal lung I HPV (ATCC # CRL-2221)

Fetal muscle I MG63

Fetal skin I Prostate SM

HPVS (ATCC # CRL- ] [ CD3+ selected PBMC's

2221) - selected lonomycin + PMA stimulated

Heart 2 Heart

K562 (ATCC # CCL-243) ] Pituitary

Kidney Placenta 2

Liver Salivary gland

Lung ] Mammary gland

Lymph node Ovary

Melanoma ] Adipocyte 1

Pancreas ] Esophagus tumor

Pituitary ] Stomach tumor

Placenta 1 Liver tumor

Prostate Lung tumor

Rectum ] Ovarian tumor

Salivary Gland Rectal tumor

Skeletal muscle ] Uterus tumor 2

Small intestine ] Thymus

Spinal cord Thyroid

Spleen Trachea

Stomach Uterus

Testis . > Example 2

Recombinant ztsll is produced in E. coli using a His₆ tag/maltose binding protein (MBP) double affinity fusion system as generally disclosed by Pryor and Leiting, Prot. Expr. Pur. 10:309-319, 1997. A thrombin cleavage site is placed at the junction between the affinity tag and ztsll sequences.

The fusion construct is assembled in the vector pTAP98, which comprises sequences for replication and selection in E. coli and yeast, the E. coli tac promoter, and a unique Smal site just downstream of the MBP-His₆-thrombin site coding sequences. The ztsll cDNA (SEQ ID NO:l) is amplified by PCR using primers each comprising 40 bp of sequence homologous to vector sequence and 25 bp of sequence that anneals to the cDNA. The reaction is run using Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN) for 30 cycles of 94°C, 30 seconds; 60°C, 60 seconds; and 72°C, 60 seconds. One microgram of the resulting fragment is mixed with 100 ng of Smal-cut pTAP98, and the mixture is transformed into yeast to assemble the vector by homologous recombination (Oldenburg et al., Nucl Acids. Res. 25:451-452, 1997). Ura⁺ transformants are selected.

Plasmid DNA is prepared from yeast transformants and transformed into E. coli MCI 061. Pooled plasmid DNA is then prepared from the MCI 061 transformants by the miniprep method after scraping an entire plate. Plasmid DNA is analyzed by restriction digestion.

E. coli strain BL21 is used for expression of ztsll. Cells are transformed by electroporation and grown on minimal glucose plates containing casamino acids and ampicillin. Protein expression is analyzed by gel electrophoresis. Cells are grown in liquid glucose media containing casamino acids and ampicillin. After one hour at 37°C, IPTG is added to a final concentration of lmM, and the cells are grown for an additional 2-3 hours at 37°C. Cells are disrupted using glass beads, and extracts are prepared.

Example 3

Larger scale cultures of ztsll transformants are prepared by the method of Pryor and Leiting (ibid.). 100-ml cultures in minimal glucose media containing casamino acids and 100 μg/ml ampicillin are grown at 37°C in 500-ml baffled flasks to OD₆₀₀ = 0.5. Cells are harvested by centrifugation and resuspended in 100 ml of the same media at room temperature. After 15 minutes, IPTG is added to 0.5 mM, and cultures are incubated at room temperature (ca. 22.5°C) for 16 to 20 hours with shaking at 125 rpm. The culture is harvested by centrifugation, and cell pellets are stored at -70°C.

Example 4

For larger-scale protein preparation, 500-ml cultures of E. coli BL21 expressing the ztsll -MB P-His₆ fusion protein are prepared essentially as disclosed in Example 3. Cell pellets are resuspended in 100 ml of binding buffer (20 mM Tris, pH 7.58, 100 mM NaCl, 20 mM NaH₂PO₄, 0.4 mM 4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride [Pefabloc® SC; Boehringer-Mannheim, Indianapolis, IN], 2 μg/ml Leupeptin, 2 μg/ml Aprotinin). The cells are lysed in a French press at 30,000 psi, and the lysate is centrifuged at 18,000 x g for 45 minutes at 4°C to clarify it. Protein concentration is estimated by gel electrophoresis with a BSA standard.

Recombinant ztsll fusion protein is purified from the lysate by affinity chromatography. immobilized cobalt resin (Talon® metal affinity resin; Clontech Laboratories, Inc., Palo Alto, CA) is equilibrated in binding buffer. One ml of packed resin per 50 mg protein is combined with the clarified supernatant in a tube, and the tube is capped and sealed, then placed on a rocker overnight at 4°C. The resin is then pelleted by centrifugation at 4°C and washed three times with binding buffer. Protein is eluted with binding buffer containing 0.2M imidazole. The resin and elution buffer are mixed for at least one hour at 4°C, the resin is pelleted, and the supernatant is removed. An aliquot is analyzed by gel electrophoresis, and concentration is estimated. Amylose resin is equilibrated in amylose binding buffer (20 mM Tris-HCl, pH 7.0, 100 mM NaCl, 10 mM EDTA) and combined with the supernatant from the Talon resin at a ratio of 2 mg fusion protein per ml of resin. Binding and washing steps are carried out as disclosed above. Protein is eluted with amylose binding buffer containing 10 mM maltose using as small a volume as possible to minimize the need for subsequent concentration. The eluted protein is analyzed by gel electrophoresis and staining with Coomassie blue using a BSA standard, and by Western blotting using an anti-MBP antibody.

Example 5

An expression plasmid containing all or part of a polynucleotide encoding ztsll is constructed via homologous recombination. A fragment of ztsll cDNA is isolated by PCR using primers that comprise, from 5' to 3' end, 40 bp of flanking sequence from the vector and 17 bp corresponding to the amino and carboxyl termini from the open reading frame of ztsll. The resulting PCR product includes flanking regions at the 5' and 3' ends corresponding to the vector sequences flanking the ztsll insertion point. Ten μl of the 100 μl PCR reaction mixture is run on a 0.8% low-melting-temperature agarose (SeaPlaque GTG®; FMC BioProducts, Rockland, ME) gel with 1 x TBE buffer for analysis. The remaining 90 μl of the reaction mixture is precipitated with the addition of 5 μl 1 M NaCl and 250 μl of absolute ethanol.

The plasmid pZMP6, which has been cut with Smal, is used for recombination with the PCR fragment. Plamid pZMPό is a mammalian expression vector containing an expression cassette having the cytomegalovirus immediate early promoter, multiple restriction sites for insertion of coding sequences, a stop codon, and a human growth hormone terminator; an E. coli origin of replication; a mammalian selectable marker expression unit comprising an SV40 promoter, enhancer and origin of replication, a DHFR gene, and the SV40 terminator; and URA3 and CEN-ARS sequences required for selection and replication in S. cerevisiae. It was constructed from pZP9 (deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, under Accession No. 98668) with the yeast genetic elements taken from pRS316 (available from the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, under Accession No. 77145), an internal ribosome entry site (IRES) element from poliovirus, and the extracellular domain of CD8 truncated at the C-terminal end of the transmembrane domain.

One hundred microliters of competent yeast (S. cerevisiae) cells are combined with 10 μl of the DNA preparations from above and transferred to a 0.2-cm electroporation cuvette. The yeast/DNA mixture is electropulsed using power supply

(BioRad Laboratories, Hercules, CA) settings of 0.75 kV (5 kV/cm), ∞ ohms, 25 μF. To each cuvette is added 600 μl of 1.2 M sorbitol, and the yeast is plated in two 300- μl aliquots onto two URA-D plates and incubated at 30°C. After about 48 hours, the Ura⁺ yeast transformants from a single plate are resuspended in 1 ml H2O and spun briefly to pellet the yeast cells. The cell pellet is resuspended in 1 ml of lysis buffer

(2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundred microliters of the lysis mixture is added to an Eppendorf tube containing 300 μl acid-washed glass beads and 200 μl phenol-chloroform, vortexed for 1 minute intervals two or three times, and spun for 5 minutes in an Eppendorf centrifuge at maximum speed. Three hundred microliters of the aqueous phase is transferred to a fresh tube, and the DNA is precipitated with 600 μl ethanol (EtOH), followed by centrifugation for 10 minutes at 4°C. The DNA pellet is resuspended in 10 μl H2O.

Transformation of electrocompetent E. coli host cells (Electromax DH10B™ cells; obtained from Life Technologies, Inc., Gaithersburg, MD) is done with 0.5-2 ml yeast DNA prep and 40 μl of cells. The cells are electropulsed at 1.7 kV, 25 μF, and 400 ohms. Following electroporation, 1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, MI), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KC1, 10 mM MgCl₂, 10 mM MgSO₄, 20 mM glucose) is plated in 250-μl aliquots on four LB AMP plates (LB broth (Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/L Ampicillin).

Individual clones harboring the correct expression construct for ztsll are identified by restriction digest to verify the presence of the ztsll insert and to confirm that the various DNA sequences have been joined correctly to one another. The inserts of positive clones are subjected to sequence analysis. Larger scale plasmid DNA is isolated using a commercially available kit (QIAGEN Plasmid Maxi Kit, Qiagen, Valencia, CA) according to manufacturer's instructions. The correct construct is designated pZMP6/ztsll.

Example 6 CHO DG44 cells (Chasin et al., Som. Cell. Molec. Genet. 12:555-666,

1986) are plated in 10-cm tissue culture dishes and allowed to grow to approximately 50% to 70% confluency overnight at 37°C, 5% CO2, in Ham's F12/FBS media (Ham's F12 medium (Life Technologies), 5% fetal bovine serum (Hyclone, Logan, UT), 1% L-glutamine (JRH Biosciences, Lenexa, KS), 1% sodium pyruvate (Life Technologies)). The cells are then transfected with the plasmid ztsll/pZMP6 by liposome-mediated transfection using a 3:1 (w/w) liposome formulation of the polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l- propaniminium-trifluoroacetate and the neutral lipid dioleoyl phosphatidylethanolamine in membrane-filetered water (Lipofectamine™ Reagent, Life Technologies), in serum free (SF) media formulation (Ham's F12, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate). Ztsll /pZMP6 is diluted into 15 -ml tubes to a total final volume of 640 μl with SF media. 35 μl of Lipofectamine™ is mixed with 605 μl of SF medium. The resulting mixture is added to the DNA mixture and allowed to incubate approximately 30 minutes at room temperature. Five ml of SF media is added to the DNA:Lipofectamine™ mixture. The cells are rinsed once with 5 ml of SF media, aspirated, and the DNA: Lipofectamine™ mixture is added. The cells are incubated at 37°C for five hours, then 6.4 ml of Ham's F12/10% FBS, 1% PSN media is added to each plate. The plates are incubated at 37°C overnight, and the DNA: Lipofectamine™ mixture is replaced with fresh 5% FBS Ham's media the next day. On day 3 post- transfection, the cells are split into T-175 flasks in growth medium. On day 7 postransfection, the cells are stained with FITC-anti-CD8 monoclonal antibody (Pharmingen, San Diego, CA) followed by anti-FITC-conjugated magnetic beads (Miltenyi Biotec). The CD8-positive cells are separated using commercially available columns (mini-MACS columns; Miltenyi Biotec) according to the manufacturer's directions and put into DMEM/Ham's F12/5% FBS without nucleosides but with 50 nM methotrexate (selection medium).

Cells are plated for subcloning at a density of 0.5, 1 and 5 cells per well in 96-well dishes in selection medium and allowed to grow out for approximately two weeks. The wells are checked for evaporation of medium and brought back to 200 μl per well as necessary during this process. When a large percentage of the colonies in the plate are near confluency, 100 μl of medium is collected from each well for analysis by dot blot, and the cells are fed with fresh selection medium. The supernatant is applied to a nitrocellulose filter in a dot blot apparatus, and the filter is treated at 100°C in a vacuum oven to denature the protein. The filter is incubated in 625 mM Tris-glycine, pH 9.1, 5mM β-mercaptoethanol, at 65°C, 10 minutes, then in 2.5% nonfat dry milk Western A Buffer (0.25% gelatin, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.05% Igepal CA-630) overnight at 4°C on a rotating shaker. The filter is incubated with the antibody-HRP conjugate in 2.5% non-fat dry milk Western A buffer for 1 hour at room temperature on a rotating shaker. The filter is then washed three times at room temperature in PBS plus 0.01% Tween 20, 15 minutes per wash. The filter is developed with chemiluminescence reagents (ECL™ direct labelling kit; Amersham Corp., Arlington Heights, IL) according to the manufacturer's directions and exposed to film (Hyperfilm ECL, Amersham Corp.) for approximately 5 minutes. Positive clones are trypsinized from the 96-well dish and transferred to 6-well dishes in selection medium for scaleup and analysis by Western blot.

Example 7

Full-length ztsll protein is produced in BHK cells transfected with pZMP6/ztsll (Example 5). BHK 570 cells (ATCC CRL-10314) are plated in 10-cm tissue culture dishes and allowed to grow to approximately 50 to 70% confluence overnight at 37°C, 5% CO₂, in DMEM/FBS medium (DMEM, Gibco/BRL High Glucose; Life Technologies supplemented with 5% fetal bovine serum (Hyclone, Logan, UT), 1 mM L-glutamine (JRH Biosciences, Lenexa, KS), and 1 mM sodium pyruvate (Life Technologies)). The cells are then transfected with pZMP6/ztsll by liposome-mediated transfection (using Lipofectamine™; Life Technologies), in serum free (SF) medium (DMEM supplemented with 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate). The plasmid is diluted into 15-ml tubes to a total final volume of 640 μl with SF medium. 35 μl of the lipid mixture is mixed with 605 μl of SF medium, and the resulting mixture is allowed to incubate approximately 30 minutes at room temperature. Five milliliters of SF medium is then added to the DNA:lipid mixture. The cells are rinsed once with 5 ml of SF medium, aspirated, and the DNA:lipid mixture is added. The cells are incubated at 37°C for five hours, then 6.4 ml of DMEM/10% FBS, 1% PSN media is added to each plate. The plates are incubated at 37°C overnight, and the DNA:lipid mixture is replaced with fresh 5% FBS/DMEM medium the next day. On day 5 post- transfection, the cells are split into T-162 flasks in selection medium (DMEM + 5% FBS, 1% L-Gln, 1% NaPyr, 1 μM methotrexate). Approximately 10 days post- transfection, two 150-mm culture dishes of methotrexate-resistant colonies from each transfection are trypsinized, and the cells are pooled and plated into a T-162 flask and transferred to large-scale culture.

Example 8

Polyclonal anti-peptide antibodies are prepared by immunizing two female New Zealand white rabbits with a peptide comprising residues 144-153 of SEQ ID NO:2 with an N-terminal cys residue. The peptide is synthesized using an Applied Biosystems Model 431 A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA) according to the manufacturer's instructions and conjugated to the carrier protein maleimide-activated keyhole limpet hemocyanin (KLH) through the cysteine residue (Pierce Chemical Co., Rockford, IL). The rabbits are each given an initial intraperitoneal (IP) injection of 200 μg of conjugated peptide in complete

Freund's Adjuvant (Pierce Chemical Co.) followed by booster IP injections of 100 μg conjugated peptide in incomplete Freund's Adjuvant every three weeks. Seven to ten days after the administration of the third booster injection, the animals are bled, and the serum is collected. The rabbits are then boosted and bled every three weeks. Antibodies are affinity purified from the rabbit serum using a CNBr-

Sepharose® 4B peptide column (Pharmacia Biotech) prepared using 10 mg of peptide per gram CNBr-Sepharose®, followed by dialysis in PBS overnight. Antibodies are characterized by an ELISA titer check using 1 μg/ml of the appropriate peptide as an antibody target.

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

Claims

CLAIMSWhat is claimed is:

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

2. The polypeptide of claim 1 wherein said at least nine conguous amino acid residues comprise residues 148-153, 234-239, 146-151, 233-238, or 144-149 of SEQ ID NO:2.

3. The isolated polypeptide of claim 1 or claim 2 which is from 15 to 1500 amino acid residues in length.

4. The isolated polypeptide of claim 1 or claim 2 wherein said at least nine contiguous amino acid residues of SEQ ID NO:2 are operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein, an immunoglobulin constant region, a polyhistidine tag, and a peptide as shown in SEQ ID NO:3.

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

6. The isolated polypeptide of claim 1 comprising: residues 22-200 of SEQ ID NO:2; residues 1-200 of SEQ ID NO:2; residues 22-240 of SEQ ID NO:2; or residues 1-240 of SEQ ID NO:2.

An expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide comprising residues 22-200 of SEQ ID NO:2; and a transcription terminator.

8. The expression vector of claim 7 wherein the DNA segment comprises nucleotides 64-600 or 64-720 of SEQ ID NO:4.

9. The expression vector of claim 7 or claim 8 further comprising a secretory signal sequence operably linked to the DNA segment.

10. The expression vector of claim 9, wherein the secretory signal sequence encodes residues 1-21 of SEQ ID NO: 2.

11. The expression vector of claim 7 wherein the polypeptide comprises residues 22-240 of SEQ ID NO:2.

12. A cultured cell into which has been introduced the expression vector of any of claims 7-11, wherein the cell expresses the DNA segment.

13. A method of making a polypeptide comprising: culturing the cell of claim 12 under conditions whereby the DNA segment is expressed and the polypeptide is produced; and recovering the polypeptide.

14. A protein produced by the method of claim 13.

15. An antibody that specifically binds to the protein of claim 14.

16. An expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a fusion protein, said protein comprising residues 1- 21 of SEQ JO NO:2 operably linked to a second polypeptide; and a transcription terminator.

17. A cultured cell into which has been introduced the expression vector of claim 16, wherein the cell expresses the DNA segment.

18. A method of making a protein comprising: culturing the cell of claim 17 under conditions whereby the DNA segment is expressed and the protein is produced; and recovering the protein.