WO2001077152A2 - Proteine 4 a noyau disulfure mammifere - Google Patents

Proteine 4 a noyau disulfure mammifere Download PDF

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Publication number
WO2001077152A2
WO2001077152A2 PCT/US2001/011506 US0111506W WO0177152A2 WO 2001077152 A2 WO2001077152 A2 WO 2001077152A2 US 0111506 W US0111506 W US 0111506W WO 0177152 A2 WO0177152 A2 WO 0177152A2
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Prior art keywords
zdsc4
gin
leu
lys
cys
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PCT/US2001/011506
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WO2001077152A3 (fr
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Edward C. Thayer
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Zymogenetics, Inc.
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Priority to AU2001251471A priority Critical patent/AU2001251471A1/en
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Publication of WO2001077152A3 publication Critical patent/WO2001077152A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

  • Protein inhibitors are classified into a series of families based on extensive sequence homologies among the family members and the conservation of intrachain disulfide bridges, see Laskowski and Kato, Ann. Rev. Biochem. 49: 593-626 (1980).
  • An example of a serine proteinase inhibitor is the serine proteinase inhibitor aprotinin, which is used therapeutically in the treatment of acute pancreatitis, various states of shock syndrome, hyperfibrinolytic hemorrhage and myocardial infarction.
  • Administration of aprotinin in high doses significantly reduces blood loss in connection with cardiac surgery, including cardiopulmonary bypass operations.
  • aprotinin when administered in vivo, aprotinin has been found to have a nephrotoxic effect in rats, rabbits and dogs after repeated injections of relatively high doses.
  • the nephrotoxicity (appearing, i.e., in the form of lesions) observed for aprotinin might' be ascribed to the accumulation of aprotinin in the proximal tubulus cells of the kidneys as a result of the high positive net charge of aprotinin, which causes it to be bound to the negatively charged surfaces of the tubuli.
  • This nephrotoxicity makes aprotinin less suitable for clinical purposes, particularly in those uses requiring administration of large doses of the inhibitor (such as cardiopulmonary bypass operations).
  • aprotinin is a bovine protein, which may induce an immune response upon administration to humans.
  • serine proteinase inhibitors which are not toxic for the treatment of acute pancreatitis, various states of shock syndrome, hyperfibrinolytic hemorrhage and myocardial infarction.
  • Zdsc4 polypeptide a disulfide core proteinase inhibitor
  • Human Zdsc4 is represented by SEQ ID NOs: 1 & 2.
  • the signal sequence of the polypeptide of SEQ ED NO: 2 is comprised of amino acid residue 1 , a methionine, to and including amino acid residue 21, a glycine.
  • the mature sequence is comprised of the polypeptide extending from amino acid residue 22, a glycine, to and including amino acid residue 79, a leucine.
  • the mature human polypeptide is also represented by SEQ I D NO: 3.
  • polypeptide being comprised of a sequence of amino acids containing the sequence of SEQ ID NOs: 2, 3, 4, 5, 6 or 7.
  • an isolated polynucleotide which encodes a polypeptide comprised of a sequence of amino acids containing the sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7.
  • a polynucleotide sequence which hybridizes under stringent conditions to either SEQ ID NO: 1 or to a complementary sequence of SEQ ID NO: 1.
  • polynucleotide sequence which is at least 90% or 95% homologous to a polynucleotide sequence which encodes the polypeptide of SEQ ID NO: 3.
  • an expression vector comprising (a) a transcription promoter; (b) a DNA segment encoding a Zdsc4 polypeptide, containing an amino acid sequence as described above.
  • a cultured eukaryotic, bacterial, fungal or other cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses a mammalian Zdsc4 polypeptide encoded by the DNA segment.
  • a chimeric polypeptide consisting essentially of a first portion and a second portion joined by a peptide bond.
  • the first portion of the chimeric polypeptide consists essentially of a
  • the invention also provides expression vectors encoding the chimeric polypeptides and host cells transfected to produce the chimeric polypeptides.
  • the present invention also provides vectors and expression vectors comprising such nucleic acid molecules, recombinant host cells comprising such vectors and expression vectors, and recombinant viruses comprising such expression vectors. These expression vectors and recombinant host cells can be used to prepare Zdsc4 polypeptides.
  • the present invention provides pharmaceutical compositions, comprising a pharmaceutically acceptable carrier and at least one of such an expression vector or recombinant virus.
  • such pharmaceutical compositions comprise a human Zdsc4 gene, or a variant thereof.
  • the present invention further contemplates antibodies and antibody fragments that specifically bind with Zdsc4 polypeptides.
  • Such antibodies include polyclonal antibodies, murine monoclonal antibodies, humanized antibodies derived from murine monoclonal antibodies, and human monoclonal antibodies.
  • Examples of antibody fragments include F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, scFv, and minimal recognition units.
  • the present invention also provides methods for detecting the presence of
  • Zdsc4 RNA in a biological sample comprising the steps of :
  • contacting a Zdsc4 nucleic acid probe under hybridizing conditions with either (i) test RNA molecules isolated from the biological sample, or (ii) nucleic acid molecules synthesized from the isolated RNA molecules, wherein the probe has a nucleotide sequence comprising a portion of the nucleotide sequence selected from the group consisting of SEQ ID NO:l, or the complement of SEQ ID NO:l, and
  • the presence of Zdsc4 polypeptide in a biological sample can be detected by methods that comprise the steps of (a) contacting the biological sample with an antibody, or an antibody fragment, that specifically binds with a polypeptide having the amino acid sequence of either SEQ ID NOs: 2 - 7 wherein the contacting is performed under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibody or bound antibody fragment.
  • kits for detecting Zdsc4 nucleic acid molecules or Zdsc4 polypeptides may comprise a container that comprises a nucleic acid molecule, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of nucleotides 213-665 of SEQ ID NO: 1, (b) a nucleic acid molecule comprising the complement of the nucleotide sequence of SEQ ID NO: 1, (c) a nucleic acid molecule that is a fragment of (a) consisting of at least eight nucleotides, (d) a nucleic acid molecule that is a fragment of (b) consisting of at least eight nucleotides, (e) a nucleic acid molecule comprising the nucleotide sequence of nucleotides 64-237 of SEQ ID NO:l.
  • kits may further comprise a second container that comprises one or more reagents capable of indicating the presence of the nucleic acid molecule.
  • a kit for detection of Zdsc4 polypeptide may comprise a container that comprises an antibody, or an antibody fragment, that specifically binds with a polypeptide having the amino acid sequence of either SEQ ID NOs: 2 - 7.
  • the present invention also contemplates isolated nucleic acid molecules comprising a nucleotide sequence that encodes an Zdsc4 secretion signal sequence and a nucleotide sequence that encodes a biologically active polypeptide, wherein the Zdsc4 secretion signal sequence comprises an amino acid sequence of residues 1 to 21 of SEQ ID NO: 2.
  • Illustrative biologically active polypeptides include Factor Vila, proinsulin, insulin, follicle-stimulating hormone, tissue type plasminogen activator, tumor necrosis factor, interleukin, colony stimulating factor, interferon, erythropoietin, and thrombopoietin.
  • the present invention provides fusion proteins comprising an Zdsc4 secretion signal sequence and a polypeptide, wherein the Zdsc4 secretion signal sequence comprises an amino acid sequence of residues 1 to 21 of SEQ ID NO: 2. Also claimed is a genomic sequence that encodes a Zdsc4 polypeptide.
  • genomic sequence SEQ ID NOs: 8 and 10.
  • the present invention also contemplates anti-idiotype antibodies, or anti- idiotype antibody fragments, that specifically bind with an anti-Zdsc4 antibody or antibody fragment.
  • SEQ ID NO 1 is the coding region for Zdsc4.
  • SEQ ID NOs: 8 and 10 are genomic sequences that contain exons encoding Zdsc4. A first exon extends from nucleotide 347 to and including nucleotide 437 followed by an intron from nucleotide
  • SEQ ID NO: 10 only contains the promoter region and exons and introns of Zdsc4.
  • the promoter extends from nucleotide 1 to and including nucleotide 40 of SEQ ID NO: 10.
  • SEQ ID NO: 11 also depicts the Zdsc4 promoter.
  • Serine proteinase inhibitors regulate the proteolytic activity of target proteinases by occupying the active site and thereby preventing occupation by normal substrates.
  • serine proteinase inhibitors fall into several unrelated structural classes, they all possess an exposed loop (variously termed an “inhibitor loop", a “reactive core”, a “reactive site”, a “binding loop”) which is stabilized by intermolecular interactions between residues flanking the binding loop and the protein core.
  • an exposed loop (variously termed an "inhibitor loop", a “reactive core”, a “reactive site”, a “binding loop") which is stabilized by intermolecular interactions between residues flanking the binding loop and the protein core.
  • the protein Zdscl is a member of a new subfamily, which appears to be closely related the Chelonianin family.
  • the Chelonianin family is characterized by a common structural motif, which comprises two adjacent beta-hairpin motifs, each consisting of two antiparallel beta strands connected by a loop region. The secondary structure of this motif is depicted by beta-sheet topology K (Branden, C. and Tooze, J.
  • the beta strands are linked by intra-chain hydrogen bonding and by a network of four disulfide bonds. These disulfide bonds stabilize the structure of the proteinase inhibitor and render it less susceptible to degradation.
  • This structural feature has caused the Chelonianin family to be referred to as the "four-disulfide core" family of proteinase inhibitors.
  • This family includes human antileukoproteinase, human elafin, guinea pig caltrin-like protein, human kallman syndrome protein, sea turtle chelonianin, and human epididymal secretory protein E4, and trout TOP-2, and C. Elegans C08G9.
  • Serine proteinase inhibitor activity can be measured using the method essentially described by Norris et al. , Biol. Chem. Hoppe-Seyler 371: 37-42 ( 1990). Briefly, various fixed concentrations of the Kunitz-type inhibitor are incubated in the presence of serine proteinases at the concentrations listed in Table 1 in 100 mM NaCl, 50 mM Tris HCI, 0.01% TWEEN80 (Polyoxyethylenesorbitan monoleate) (pH 7.4) at 25 °C. After a 30 minute incubation, the residual enzymatic activity is measured by the degradation of a solution of the appropriate substrate as listed in Table 1 in assay buffer.
  • the samples are incubated for 30 minutes after which the absorbance of each sample is measured at 405 nm.
  • An inliibition of enzyme activity is measured as a decrease in absorbance at 405 nm or fluorescence Em at 460 nm. From the results, the apparent inhibition constant Kj is calculated.
  • Inhibition assays were performed in microtiter wells in a total volume of 300 Dl in 10 mM NaCl, 50 mM Tris-HCl (pH 7.4), 0.01% TWEEN80 (Polyoxyethylenesorbitan monoleate). Each reaction contained 1 DM of the sample inhibitor and one of the proteases at the concentration listed in Table 1. The reactions were incubated at 25 °C for ten minutes after which the appropriate chromogenic substrate was added to the final concentration listed in Table 1 and the final reaction was incubated for thirty minutes at 25°C. Amidolytic activity was measured at 405 nm or by fluorescence Em at 460 nm. Percent inhibition was determined relative to reactions carried out in the absence of inhibitor representing 100% activity or 0% inhibition.
  • the serine proteinase inhibitors of the present invention may be used in the disclosed methods for the treatment of, inter alia, acute pancreatitis, various states of shock syndrome, hyperfibrinolytic hemorrhage and myocardial infarction.
  • the amyloid protein precursor homologues of the present invention may be used, inter alia, to generate antibodies for use in demonstrating tissue distribution of the precursor or for use in purifying such proteins.
  • the 8 cysteines in the four-disulfide core are bonded according to the pattern:
  • nucleic acid or “nucleic acid molecule” refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • Nucleic acid molecules can be composed of monomers that are naturally- occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g., oc-enantiomeric forms of naturally-occurring nucleotides), or a combination of both.
  • Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties.
  • Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters.
  • the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.
  • modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well- known heterocyclic substitutes.
  • Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages.
  • nucleic acid molecule also includes so-called “peptide nucleic acids,” which comprise naturally occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.
  • nucleic acid molecule refers to a nucleic acid molecule having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence.
  • sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • the term "contig” denotes a nucleic acid molecule that has a contiguous stretch of identical or complementary sequence to another nucleic acid molecule. Contiguous sequences are said to "overlap" a given stretch of a nucleic acid molecule either in their entirety or along a partial stretch of the nucleic acid molecule.
  • the term “degenerate nucleotide sequence” denotes a sequence of nucleotides that includes one or more degenerate codons as compared to a reference nucleic acid 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 "structural gene” refers to a nucleic acid molecule that is transcribed into messenger RNA (mRNA), which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
  • an "isolated nucleic acid molecule” is a nucleic acid molecule that is not integrated in the genomic DNA of an organism.
  • a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule.
  • Another example of an isolated nucleic acid molecule is a chemically synthesized nucleic acid molecule that is not integrated in the genome of an organism.
  • a nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome from that species.
  • a "nucleic acid molecule construct" is a nucleic acid molecule; either single- or double-stranded, that has been modified through human intervention to contain segments of nucleic acid combined and juxtaposed in an arrangement not existing in nature.
  • Linear DNA denotes non-circular DNA molecules having free 5' and 3' ends.
  • Linear DNA can be prepared from closed circular DNA molecules, such as plasmids, by enzymatic digestion or physical disruption.
  • cDNA complementary DNA
  • cDNA is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription.
  • cDNA to refer to a double-stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand.
  • the term “cDNA” also refers to a clone of a cDNA molecule synthesized from an RNA template.
  • a “promoter” is a nucleotide sequence that directs the transcription of a structural gene.
  • a promoter is located in the 5' non-coding region of a gene, proximal to the transcriptional start site of a structural gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements [DSEs; McGehee et al, Mol. Endocrinol. 7:551 (1993)], cyclic AMP response elements (CREs), serum response elements [SREs; Treisman, Seminars in Cancer Biol.
  • CREs cyclic AMP response elements
  • GREs glucocorticoid response elements
  • binding sites for other transcription factors such as CRE/ATF [O'Reilly etal, J. Biol. Chem. 267:19938 (1992)]/AP2 [Ye etal, J. Biol. Chem. 269:25728 (1994)], SP1, cAMP response element binding protein (CREB; Loeken, GeneExpr. 3:253 (1993)) and octamer factors [see, in general, Watson et al, eds., Molecular Biology of the Gene, 4th ed. (The
  • a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter. Repressible promoters are also known.
  • a “core promoter” contains essential nucleotide sequences for promoter function, including the TATA box and start of transcription. By this definition, a core promoter may or may not have detectable activity in the absence of specific sequences that may enhance the activity or confer tissue specific activity.
  • a “regulatory element” is a nucleotide sequence that modulates.the activity of a core promoter. For example, a regulatory element may contain a nucleotide sequence that binds with cellular factors enabling transcription exclusively or preferentially in particular cells, tissues, or organelles. These types of regulatory elements are normally associated with genes that are expressed in a "cell-specific,” “tissue-specific,” or “organelle-specific” manner.
  • the Zdsc4 ⁇ regulatory element preferentially induces gene expression in placental, trachea!, and uterine tissues, as opposed to lung, brain, liver, kidney, spleen, thymus, prostate, testis, ovary, small intestine, and pancreas tissues.
  • An “enhancer” is a type of regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.
  • Heterologous DNA refers to a DNA molecule, or a population of DNA molecules, that does not exist naturally within a given host cell.
  • DNA molecules heterologous to a particular host cell may contain DNA derived from the host cell species (i.e., endogenous DNA) so long as that host DNA is combined with non-host DNA (i.e., exogenous DNA).
  • a DNA molecule containing a non-host DNA segment encoding a polypeptide operably linked to a host DNA segment comprising a transcription promoter is considered to be a heterologous DNA molecule.
  • a heterologous DNA molecule can comprise an endogenous gene operably linked with an exogenous promoter.
  • a DNA molecule comprising a gene derived from a wild-type cell is considered to be heterologous DNA if that DNA molecule is introduced into a mutant cell that lacks the wild-type gene.
  • a “polypeptide” is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides.”
  • a “protein” is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-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.
  • a peptide or polypeptide encoded by a non-host DNA molecule is a "heterologous" peptide or polypeptide.
  • integrated genetic element is a segment of DNA that has been incorporated into a chromosome of a host cell after that element is introduced into the cell through human manipulation.
  • integrated genetic elements are most commonly derived from linearized plasmids that are introduced into the cells by electroporation or other techniques. Integrated genetic elements are passed from the original host cell to its progeny.
  • a "cloning vector” is a nucleic acid molecule, such as a plasmid, cosmid, or bacteriophage that has the capability of replicating autonomously in a host cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites that allow insertion of a nucleic acid molecule in a determinable fashion without loss of an essential biological function of the vector, as well as nucleotide sequences encoding a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector.
  • Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
  • an “expression vector” is a nucleic acid molecule encoding a gene that is expressed in a host cell.
  • an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be “operably linked to” the promoter.
  • a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.
  • a “recombinant host” is a cell that contains a heterologous nucleic acid molecule, such as a cloning vector or expression vector.
  • a recombinant host is a cell that produces Zdsc4from an expression vector.
  • Zdsc4 can be produced by a cell that is a "natural source" of Zdsc4, and that lacks an expression vector.
  • “Integrative transformants” are recombinant host cells, in which heterologous DNA has become integrated into the genomic DNA of the cells.
  • a “fusion protein” is a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes.
  • a fusion protein can comprise at least part of a Zdsc4 polypeptide fused with a polypeptide that binds an affinity matrix.
  • Such a fusion protein provides a means to isolate large quantities of Zdsc4 using affinity chromatography.
  • Receptor denotes a cell-associated protein that binds to a bioactive molecule termed a "ligand.” This interaction mediates the effect of the ligand on the cell.
  • Receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).
  • Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal - - .. transduction. In certain membrane-bound receptors, the extracellular ligand-binding domain and the intracellular effector domain are located in separate polypeptides that comprise the complete functional receptor.
  • the binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell, which in turn leads to an alteration in the metabolism of the cell.
  • Metabolic events that are often linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.
  • secretory signal sequence denotes a DNA sequence that encodes a peptide (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.
  • secretory peptide a DNA sequence that encodes a 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.
  • isolated polypeptide is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature.
  • a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure.
  • SDS sodium dodecyl sulfate
  • amino-terminal or N-terminal and “carboxyl-terminal or C- terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • expression refers to the biosynthesis of a gene product.
  • expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.
  • 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 polypeptide encoded by a splice variant of an mRNA transcribed from a gene.
  • the term "immunomodulator” includes cytokines, stem cell growth factors, lymphotoxins, co-stimulatory molecules, hematopoietic factors, and synthetic analogs of these molecules.
  • complement/anti-complement pair denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions.
  • biotin and avidin are prototypical members of a complement/anti-complement pair.
  • Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like.
  • the complement/anti-complement pair preferably has a binding affinity of less than 10 9 M "1 .
  • an "anti-idiotype antibody” is an antibody that binds with the variable region domain of an immunoglobulin.
  • an anti-idiotype antibody binds with the variable region of an anti-Zdsc4 antibody, and thus, an anti-idiotype antibody mimics an epitope of Zdsc4.
  • an “antibody fragment” is a portion of an antibody such as F(ab') 2 , F(ab) 2 , Fab', Fab, and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by ' the intact antibody. For example, an anti-Zdsc4 monoclonal antibody fragment binds with an epitope of Zdsc4.
  • antibody fragment also includes a synthetic or a genetically engineered polypeptide that binds to a specific antigen, such as polypeptides consisting of the light chain variable region, "Fv” fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
  • scFv proteins peptide linker
  • a “chimeric antibody” is a recombinant protein that contains the variable domains and complementary determining regions derived from a rodent antibody, while the remainder of the antibody molecule is derived from a human antibody.
  • Humanized antibodies are recombinant proteins in which murine complementarity determining regions of a monoclonal antibody have been transferred from heavy and light variable chains of the murine immunoglobulin into a human variable domain.
  • a "therapeutic agent” is a molecule or atom that is conjugated to an antibody moiety to produce a conjugate that is useful for therapy.
  • therapeutic agents include drugs, toxins, i munomodulators, chelators, boron compounds, photoactive agents or dyes, and radioisotopes.
  • a "detectable label” is a molecule or atom that can be conjugated to an antibody moiety to produce a molecule useful for diagnosis. Examples of detectable labels include chelators, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, or other marker moieties.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
  • Affinity tags include a poly- histidine tract, protein A [Nilsson et al, EMBO J. 4: 1075 (1985); Nilsson et al, Methods Enzymo 198:3 (1991)], glutathione S transferase [Smith and Johnson, Gene 67:31 (1988)], Glu-Glu affinity tag [Grussenmeyer et al, Proc.
  • naked antibody is an entire antibody, as opposed to an antibody fragment, which is not conjugated with a therapeutic agent. Naked antibodies include both polyclonal and monoclonal antibodies, as well as certain recombinant antibodies, such as chimeric and humanized antibodies.
  • antibody component includes both an entire antibody and an antibody fragment.
  • an “immunoconjugate” is a conjugate of an antibody component with a therapeutic agent or a detectable label.
  • antibody fusion protein refers to a recombinant molecule that comprises an antibody component and a therapeutic agent.
  • therapeutic agents suitable for such fusion proteins include immunomodulators ("antibody-immunomodulator fusion protein”) and toxins ("antibody-toxin fusion protein”). ' " ⁇ • - .
  • tumor associated antigen is a protein normally not expressed, or expressed at lower levels, by a normal counterpart cell.
  • tumor-associated antigens include alpha-fetoprotein, carcinoembryonic antigen, and Her-2/neu. Many other illustrations of tumor-associated antigens are known to those of skill in the art. See, for example, Urban et al, Ann. Rev. Immunol. 10:617 (1992).
  • an "infectious agent” denotes both microbes and parasites.
  • a “microbe” includes viruses, bacteria, rickettsia, mycoplasma, protozoa, fungi and like microorganisms.
  • a “parasite” denotes infectious, generally microscopic or very small multicellular invertebrates, or ova or juvenile forms thereof, which are susceptible to immune-mediated clearance or lytic or phagocytic destruction, such as malarial parasites, spirochetes, and the like.
  • An “infectious agent antigen” is an antigen associated with an infectious agent.
  • a “target polypeptide” or a “target peptide” is an amino acid sequence that comprises at least one epitope, and that is expressed on a target cell, such as a tumor cell, or a cell that carries an infectious agent antigen.
  • T cells recognize peptide epitopes presented by a major histocompatibihty complex molecule to a target polypeptide or target peptide and typically lyse the target cell or recruit other immune cells to the site of the target cell, thereby killing the target cell.
  • antigenic peptide is a peptide that will bind a major histocompatibility complex molecule to form an MHC-peptide complex, which is recognized by a T cell, thereby inducing a cytotoxic lymphocyte response upon presentation to the T cell.
  • antigenic peptides are capable of binding to an appropriate major histocompatibility complex molecule and inducing a cytotoxic T cells response, such as cell lysis or specific cytokine release against the target cell that binds or expresses the antigen.
  • the antigenic peptide can be bound in the context of a class I or class U major histocompatibility complex molecule, on an antigen presenting cell or on a target cell.
  • RNA polymerase JJ catalyzes the transcription of a structural gene to produce mRNA.
  • a nucleic acid molecule can be designed to contain an RNA polymerase II template in which the RNA transcript has a sequence that is complementary to that of a specific mRNA.
  • the RNA transcript is termed an "anti- sense RNA” and a nucleic acid molecule that encodes the anti-sense RNA is termed an "anti-sense gene.”
  • Anti-sense RNA molecules are capable of binding to mRNA molecules, resulting in an inhibition of mRNA translation.
  • an "anti-sense oligonucleotide specific for Zdsc4" or a "Zdsc4 anti-sense oligonucleotide” is an oligonucleotide having a sequence (a) capable of forming, a stable triplex with a portion of the Zdsc4 gene, or (b) capable of forming a stable duplex with a portion of an mRNA transcript of the Zdsc4 gene.
  • a "ribozyme” is a nucleic acid molecule that contains a catalytic center.
  • the term includes RNA enzymes, self-splicing RNAs, self-cleaving RNAs, and nucleic acid molecules that perform these catalytic functions.
  • a nucleic acid molecule that encodes a ribozyme is termed a "ribozyme gene.”
  • An "external guide sequence” is a nucleic acid molecule that directs the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, resulting in the cleavage of the mRNA by RNase P.
  • a nucleic acid molecule that encodes an external guide sequence is termed an "external guide sequence gene.”
  • the term "variant, human Zdsc4 gene” refers to nucleic acid molecules that encode a polypeptide having an amino acid sequence that is a modification of SEQ JD NO: 2. Such variants include naturally-occurring polymorphisms of Zdsc4 genes, as well as synthetic genes that contain conservative amino acid substitutions of the amino acid sequence of SEQ ID NO: 2 or 3. Additional variant forms of Zdsc4 genes are nucleic acid molecules that contain insertions or deletions of the nucleotide sequences described herein.
  • a variant Zdsc4 gene can be identified by determining whether the gene hybridizes with a nucleic acid molecule having the nucleotide sequence of SEQ ED NO: 1, or its complement, under stringent conditions.
  • variant Zdsc4genes can be identified by sequence comparison. Two amino acid sequences have "100% amino acid sequence identity” if the amino acid residues of the two amino acid sequences are the same when aligned for maximal correspondence. Similarly, two nucleotide sequences have "100% nucleotide sequence identity” if the nucleotide residues of the two nucleotide sequences are the same when aligned for maximal correspondence. Sequence comparisons can be performed using standard software programs such as those included in the LASERGENE bioinformatics computing suite, which is produced by DNASTAR (Madison, Wisconsin).
  • variant Zdsc4 a variant gene or polypeptide encoded by a variant gene is functionally characterized by either its ability to bind specifically to an anti-Zdsc4 antibody.
  • allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • 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. "Paralogs" are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, ⁇ - globin, ⁇ -globin, and myoglobin are paralogs of each other.
  • the present invention includes functional fragments of Zdsc4gQnes.
  • a "functional fragment" of a Zdsc4 gene refers to a nucleic acid molecule that encodes a portion of a Zdsc4 polypeptide which either (1) possesses an anti- viral or anti-proliferative activity, or (2) specifically binds with an anti-
  • a functional fragment of a human Zdsc4 gene described herein comprises a portion of the nucleotide sequence of SEQ ED NO: 1 or SEQ ID NO: 6.
  • Nucleic acid molecules encoding a human Zdsc4gene can be obtained by screening a human cDNA or genomic library using polynucleotide probes based upon SEQ ED NO: 1. These techniques are standard and well established.
  • RNA isolation techniques must provide a method for breaking cells, a means of inhibiting RNase-directed degradation of RNA, and a method of separating RNA from DNA, protein, and polysaccharide contaminants.
  • total RNA can be isolated by freezing tissue in liquid nitrogen, grinding the frozen tissue with a mortar and pestle to lyse.
  • total RNA can be isolated by extracting ground tissue with guanidinium isothiocyanate, extracting with organic solvents, and separating RNA from contaminants using differential centrifugation (see, for example, Chirgwin et al, Biochemistry 18:52 (1979); Ausubel (1995) at pages 4-1 to 4-6; Wu (1997) at pages 33- 41).
  • poly(A) + RNA In order to construct a cDNA library, poly(A) + RNA must be isolated from a total RNA preparation. Poly(A) + RNA can be isolated from total RNA using the standard technique of oligo(dT)-cellulose chromatography (see, for example, Aviv and Leder, Proc. Nat'lAcad. Sci. USA 69:1408 (1972); Ausubel (1995) at pages 4-11 to 4-12). Double-stranded cDNA molecules are synthesized from poly(A) + RNA using techniques well-known to those in the art. (See, for example, Wu (1997) at pages 41-46). Moreover, commercially available kits can be used to synthesize double- stranded cDNA molecules. For example, such kits are available from Life Technologies, Inc. (Gaifhersburg, MD), CLONTECH Laboratories, Inc. (Palo Alto, CA), Promega Corporation (Madison, Wl) and STRATAGENE (La Jolla, CA).
  • a cDNA library can be prepared in a vector derived from bacteriophage, such as a ⁇ gtlO vector. See, for example, Huynh et al, "Constructing and Screening cDNA Libraries in ⁇ gtlO and ⁇ gtl 1," in DNA Cloning: A Practical Approach Vol I, Glover (ed.), page 49 (IRL Press, 1985); Wu (1997) at pages 47-52.
  • double-stranded cDNA molecules can be inserted into a plasmid vector, such as a PBLUESCRIPT vector (STRATAGENE; La Jolla, CA), a LAMDAGEM-4 (Promega Corp.) or other commercially available vectors.
  • a plasmid vector such as a PBLUESCRIPT vector (STRATAGENE; La Jolla, CA), a LAMDAGEM-4 (Promega Corp.) or other commercially available vectors.
  • Suitable cloning vectors also can be obtained from the American Type Culture Collection (Manassas, VA).
  • the cDNA library is inserted into a prokaryotic host, using standard techniques.
  • a cDNA library can be introduced into competent E. coli DH5 cells, which can be obtained, for example, from Life Technologies, Inc. (Gaifhersburg, MD).
  • a human genomic library can be prepared by means well known in the art
  • Genomic DNA can be isolated by lysing tissue with the detergent Sarkosyl, digesting the lysate with proteinase K, clearing insoluble debris from the lysate by centrifugation, precipitating nucleic acid from the lysate using isopropanol, and purifying resuspended DNA on a cesium chloride density gradient.
  • Genomic DNA fragments that are suitable for the production of a genomic library can be obtained by the random shearing of genomic DNA or by the partial digestion of genomic DNA with restriction endonucleases.
  • Genomic DNA fragments can be inserted into a vector, such as a bacteriophage or cosmid vector, in accordance with conventional techniques, such as the use of restriction enzyme digestion to provide appropriate termini, the use of alkaline phosphatase treatment to avoid undesirable joining of DNA molecules, and ligation with appropriate ligases. Techniques for such manipulation are well-known in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages 307- 327).
  • Nucleic acid molecules that encode a human Zdsc4gene can also be obtained using the polymerase chain reaction (PCR) with oligonucleotide primers having nucleotide sequences that are based upon the nucleotide sequences of the human Zdsc4 gene, as described herein.
  • PCR polymerase chain reaction
  • General methods for screening libraries with PCR are provided by, for example, Yu et al, "Use of the Polymerase Chain Reaction to Screen Phage Libraries," in Methods in Molecular Biology, Vol 15: PCR Protocols: Current Methods and Applications, White (ed.), pages 211-215 (Humana Press, Inc. 1993).
  • a library containing cDNA or genomic clones can be screened with one or more polynucleotide probes based upon SEQ ED NO: 1, using standard methods (see, for example, Ausubel (1995) at pages 6-1 to 6-11).
  • Anti-Zdsc4 antibodies produced as described below, can also be used to detect Zdsc4 polypeptides expressed from clones an then to isolate the DNA sequences that encode human Zdsc4gc s from cDNA libraries.
  • the antibodies can be used to screen ⁇ gtll expression libraries, or the antibodies can be used.for immunoscreening following hybrid selection and translation [see, for example, Ausubel
  • a Zdsc4g&a& can be obtained by synthesizing nucleic acid molecules using mutually priming long oligonucleotides and the nucleotide sequences described herein (see, for example, Ausubel (1995) at pages 8-8 to 8-9).
  • Established techniques using the polymerase chain reaction provide the ability to synthesize DNA molecules at least two kilobases in length (Adang et al, Plant Molec. Biol. 21:1131 (1993), Bambot et al, PCR Methods and Applications 2:266 (1993), Dillon et al, "Use of the Polymerase Chain Reaction for the Rapid Construction of Synthetic Genes," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), pages 263-268, (Humana Press, Lnc. 1993), and Holowachuk et ⁇ /., PCR Methods Appl 4:299 (1995)).
  • the nucleic acid molecules of the present invention can also be synthesized with "DNA synthesizers" using protocols such as the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately.
  • the production of short genes 60 to 80 base pairs) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them.
  • special strategies may be required, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%.
  • synthetic genes double-stranded are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length.
  • One method for building a synthetic gene requires the initial production of a set of overlapping, complementary oligonucleotides, each of which is between 20 to 60 nucleotides long.
  • the sequences of the strands are planned so that, after annealing, the two end segments of the gene are aligned to give blunt ends.
  • Each internal section of the gene has complementary 3' and 5' terminal extensions that are designed to base pair precisely with an adjacent section.
  • synthetic genes can be designed with terminal sequences that facilitate insertion into a restriction endonuclease sites of a cloning vector and other sequences should also be added that contain signals for the proper initiation and termination of transcription and translation.
  • An alternative way to prepare a full-size gene is to synthesize a specified set of overlapping oligonucleotides (40 to 100 nucleotides). After the 3' and 5' extensions
  • the duplex is completed and the gaps filled by enzymatic DNA synthesis with E. coli DNA polymerase I.
  • This enzyme uses the 3'-hydroxyl groups as replication initiation points and the single-stranded regions as templates. After the enzymatic synthesis is completed, the nicks are sealed with T4 DNA Iigase.
  • the complete gene sequence is usually assembled from double-stranded fragments that are each put together by joining four to six overlapping oligonucleotides (20 to 60 base pairs each).
  • each fragment is cloned into a vector to amplify the amount of DNA available.
  • the double-stranded constructs are sequentially linked to one another to form the entire gene sequence.
  • Each double-stranded fragment and the complete sequence should be characterized by DNA sequence analysis to verify that the chemically synthesized gene has the correct nucleotide sequence.
  • the sequence of a Zdsc4cDNA or Zdsc4 genomic fragment can be determined using standard methods. Moreover, the identification of genomic fragments containing a Zdsc4 promoter or regulatory element can be achieved using well- established techniques, such as deletion analysis (see, generally, Ausubel (1995)). Cloning of 5' flanking sequences also facilitates production of Zdsc4 proteins by "gene activation," following the methods disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous Zdsc4gene in a cell is altered by introducing into the Zdsc41ocus 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 Zdsc45' non-coding sequence that permits homologous recombination of the construct with the endogenous Zdsc4 locus, whereby the sequences within the construct become operably linked with the endogenous Zdsc4 coding sequence.
  • an endogenous Z sc ⁇ promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.
  • the present invention provides a variety of nucleic acid molecules, including DNA and RNA molecules that encode the Zdsc4 polypeptides disclosed herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules.
  • Table 1 sets forth the one-letter codes used to denote degenerate nucleotide positions. "Resolutions” are the nucleotides denoted by a code letter.
  • “Complement” indicates the code for the complementary nucleotide(s).
  • the code Y denotes either C or T
  • its complement R denotes A or G
  • A being complementary to T
  • G being complementary to C.
  • degenerate codons encompassing all possible codons for a given amino acid, are set forth in Table 2.
  • polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ED NO: 2 or SEQ ED NO: 3. Variant sequences can be readily tested for functionality as described herein.
  • preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2).
  • the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammahan cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art.
  • preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, a degenerate codon sequences can serve as templates for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed, herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
  • the present invention further provides variant polypeptides and nucleic acid molecules that represent counterparts from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are Zdsc4 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human Zdsc4 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
  • a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses Zdsc4 as disclosed herein. Suitable sources of mRNA can be identified by probing northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line.
  • a Zdsc4-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences.
  • a cDNA can also be cloned using the polymerase chain reaction with primers designed from the representative human Zdsc4 sequences disclosed herein.
  • 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 Zdsc4 polypeptide. Similar techniques can also be applied to the isolation of genomic clones, and to the isolation of nucleic molecules that encode murine Zdsc4.
  • SEQ ED NO:l represents a single allele of human Zdsc4, and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the nucleotide sequence shown in SEQ ED NO:l
  • amino acid sequence changes are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NOs: 2 - 22.
  • cDNA molecules generated from alternatively spliced mRNAs, which retain the properties of the Zdsc4 polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
  • Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.
  • isolated nucleic acid molecules that encode human Zdsc4 can hybridize to nucleic acid molecules haying the nucleotide sequence of SEQ ID NO: 1, or a sequence complementary thereto, under "stringent conditions.”
  • 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.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • a nucleic acid molecule encoding a variant Zdsc4 polypeptide can be hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ED NO: 1 (or its complement) at 42°C overnight in a solution comprising 50% formamide, 5xSSC (lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution (lOOx Denhardt's solution: 2% (w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin), 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA.
  • 5xSSC lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate
  • 50 mM sodium phosphate pH 7.6
  • 5x Denhardt's solution l
  • hybridization mixture can be incubated at a higher temperature, such as about 65°C, in a solution that does not contain formamide.
  • a higher temperature such as about 65°C
  • premixed hybridization solutions are available (e.g., EXPRESSHYB Hybridization Solution from CLONTECH Laboratories, Inc.), and hybridization can be performed according to the manufacturer's instructions.
  • nucleic acid molecules can be washed to remove non-hybridized nucleic acid molecules under stringent conditions, or under highly stringent conditions.
  • Typical stringent washing conditions include washing in a solution of 0.5x - 2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 55 - 65 °C.
  • nucleic acid molecules encoding a variant Zdsc4 polypeptide hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement) under stringent washing conditions, in which the wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, including 0.5x SSC with 0.1% SDS at 55°C, or 2xSSC with 0.1% SDS at 65°C.
  • wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, including 0.5x SSC with 0.1% SDS at 55°C, or 2xSSC with 0.1% SDS at 65°C.
  • SSPE for SSC in the wash solution.
  • Typical highly stringent washing conditions include washing in a solution of O.lx - 0.2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 50 - 65°C.
  • SDS sodium dodecyl sulfate
  • nucleic acid molecules encoding a variant Zdsc4 polypeptide hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to O.lx - 0.2x SSC with 0.1% SDS at 50 - 65°C, including O.lx SSC with 0.1% SDS at 50°C, or 0.2xSSC with 0.1% SDS at 65°C.
  • the present invention also provides isolated Zdsc4 polypeptides that have a substantially similar sequence identity to the polypeptides of SEQ ID NOs: 2 or 3, or their orthologs.
  • substantially similar sequence identity is used herein to denote polypeptides having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the sequences shown in SEQ ID NO: 2 or 3 or their orthologs.
  • the present invention also contemplates Zdsc4 variant nucleic acid molecules that can be identified using two criteria: a determination of the similarity between the encoded polypeptide with the amino acid sequence of SEQ ID NOs: 2 or 3, and a hybridization assay, as described above.
  • Such Zdsc4 variants include nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement) under stringent washing conditions, in which the wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, and (2) that encode a polypeptide having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ID NOs: 2 or 3.
  • Zdsc4 variants can be characterized as nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ED NO: 1 (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to O.lx - 0.2x SSC with 0.1% SDS at 50 - 65°C, and (2) that encode a polypeptide having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ED NOs: 2 or 3. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al, Bull. Math. Bio. 48:603 (1986), and Henikoff and Henikoff, Proc.
  • the "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative Zdsc4 variant.
  • the FASTA algorithm is described by Pearson and Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol 183:63 (1990).
  • the ten regions with the highest density of identities are then re-scored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score.
  • FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as described above.
  • the present invention includes nucleic acid molecules that encode a polypeptide having a conservative amino acid change, compared with the amino acid sequence of SEQ ED NO: 2 or3. That is, variants can be obtained that contain one or more amino acid substitutions of SEQ ED NOs: 2 or 3, in which an alkyl amino acid is substituted for an alkyl amino acid in an Zdsc4 amino acid sequence, an aromatic amino acid is substituted for an aromatic amino acid in an Zdsc4 amino acid sequence, a sulfur-containing amino acid is substituted for a sulfur-containing amino acid in an Zdsc4 amino acid sequence, a hydroxy-containing amino acid is substituted for a hydroxy-containing amino acid in an Zdsc4 amino acid sequence, an acidic amino acid is substituted for an acidic amino acid in an Zdsc4 amino acid sequence, a basic amino acid is substituted for a basic amino acid in an Zdsc4 amino acid sequence, or a dibasic monocarboxylic amino acid is substituted for a dibasic mono
  • a “conservative amino acid substitution” is illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.
  • variant Zdsc4 polypeptides that have an amino acid sequence that differs from either SEQ ED NO: 2 can be obtained by substituting a threonine residue for Ser 39 , by substituting a valine residue for He 64 , by substituting an aspartate residue for Glu 75 , or by substituting a valine residue for De 107 . Additional variants can be obtained by producing polypeptides having two or more of these amino acid substitutions.
  • the BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat'lAcad. Sci. USA 89: 10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed above), the language "conservative amino acid substitution” preferably 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. According to this system, preferred conservative amino acid substitutions are characterized by a ' ⁇
  • BLOSUM62 value of at least 1 e.g., 1, 2 or 3
  • more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • “conservative amino acid” variants can be obtained, for example, by oligonucleotide- directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22; and McPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL Press 1991)).
  • the ability of such variants to promote anti-viral or anti-proliferative activity can be determined using a standard method, such as the assay described herein.
  • a variant Zdsc4 polypeptide can be identified by the ability to specifically bind anti-Zdsc4 antibodies.
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methanoproline, s-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, /Zo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
  • E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4- fluorophenylalanine).
  • a natural amino acid that is to be replaced e.g., phenylalanine
  • desired non-naturally occurring amino acid(s) e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4- fluorophenylalanine.
  • non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al, Biochem. 33:7470 (1994). Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions [Wynn and Richards, Protein Sci. 2:395 (1993)].
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for Zdsc4 amino acid residues.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et al, Proc. Nat'lAcad. Sci. USA 88:4498 (1991), Coombs and Corey, "Site-Directed Mutagenesis and Protein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)).
  • Zdsc4 labeled with biotin or FITC can be used for expression cloning of Zdsc4 receptors.
  • variant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations.
  • This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
  • Mutagenesis methods as disclosed herein can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
  • Mutagenized DNA molecules that encode biologically active polypeptides, or polypeptides that bind with anti-Zdsc4 antibodies 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.
  • the present invention also includes "functional fragments" of Zdsc4 polypeptides and nucleic acid molecules encoding such functional fragments.
  • Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a Zdsc4 polypeptide.
  • DNA molecules having the nucleotide sequence of SEQ JD NO: 1 can be digested with a nuclease to obtain a series of nested deletions. The fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for the ability to bind anti-Zdsc4 antibodies.
  • exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired fragment.
  • particular fragments of a Zdsc4 gene can be synthesized using the , polymerase chain reaction.
  • the present invention also contemplates functional fragments of an Zdsc4 gene that has amino acid changes, compared with the amino acid sequence of SEQ ID NOs: 2 or3.
  • An alternative approach to identifying a variant gene on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant Zdsc4gene can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 as discussed above.
  • the present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of a Zdsc4 polypeptide described herein.
  • Such fragments or peptides may comprise an "immunogenic epitope," which is a part of a protein that elicits an antibody response when the entire protein is used as an immunogen.
  • Immunogenic epitope-bearing peptides can be identified using standard methods (see, for example, Geysen et al, Proc. Nat'lAcad. Sci. USA 81:3998 (1983)).
  • polypeptide fragments or peptides may comprise an "antigenic epitope," which is a region of a protein molecule to which an antibody can specifically bind.
  • Certain epitopes consist of a linear or contiguous stretch of amino acids, and the antigenicity of such an epitope is not disrupted by denaturing agents. It is known in the art that relatively short synthetic peptides that can mimic epitopes of a protein can be used to stimulate the production of antibodies against the protein [see, for example, Sutcliffe et al, Science 219:660 (1983)]. Accordingly, antigenic epitope- bearing peptides and polypeptides of the present invention are useful to raise antibodies that bind with the polypeptides described herein.
  • Antigenic epitope-bearing peptides and polypeptides preferably contain at least four to ten amino acids, at least ten to fifteen amino acids, or about 15 to about 30 or more amino acids of SEQ ED NO: 2 or 3.
  • Such epitope-bearing peptides and polypeptides can be produced by fragmenting a Zdsc4 polypeptide, or by chemical peptide synthesis, as described herein.
  • epitopes can be selected by phage display of random peptide libraries [see, for example, Lane and Stephen, Curr. Opin. Immunol. 5:268 (1993), and Cortese etal, Curr. Opin. Biotechnol. 7:616 (1996)].
  • variant Zdsc4gene encodes a polypeptide that is characterized by its ability to bind specifically to an anti-Zdsc4 antibody. More specifically, variant human Zdsc4genes encode polypeptides that exhibit at least 50%, and preferably, greater than 70, 80, or 90%, of the activity of polypeptide encoded by the human Zdsc4gene described herein.
  • any Zdsc4 polypeptide including variants and fusion proteins, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above.
  • the present invention includes a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ ED NOs: 1 -8.
  • a computer-readable medium can be encoded with a data structure that provides at least one of the following sequences: SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ED NO:4, SEQ ED NO:5, SEQ ED NO:6, SEQ ED NO:7 and SEQ ID NO:8.
  • Suitable forms of computer-readable media include magnetic media and optically readable media.
  • magnétique media examples 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-re-writable (RW), and CD-recordable), and digital versatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).
  • compact discs e.g., CD-read only memory (ROM), CD-re-writable (RW), and CD-recordable
  • DVD digital versatile/video discs
  • Fusion proteins of Zdsc4 can be used to express Zdsc4 in a recombinant host, and to isolate expressed Zdsc4. As described below, particular
  • Zd.sc4 fusion proteins also have uses in diagnosis and therapy.
  • fusion protein comprises a peptide that guides a Zdsc4 polypeptide from a recombinant host cell.
  • a secretory signal sequence also known as a signal peptide, a leader sequence, prepro sequence or pre sequence
  • the secretory signal sequence may be derived from Zdsc4
  • a suitable signal sequence may also be derived from another secreted • protein or synthesized de novo.
  • the secretory signal sequence is operably linked to a Zdsc4-encoding sequence such that the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell.
  • Secretory signal sequences are commonly positioned 5' to the nucleotide sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the nucleotide 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).
  • yeast signal sequence is preferred for expression in yeast cells.
  • suitable yeast signal sequences are those derived from yeast mating phermone -factor (encoded by the MF l gene), invertase (encoded by the SUC2 gene), or acid phosphatase (encoded by the PH05 gene). See, for example, Romanos et al. , "Expression of Cloned Genes in Yeast," in DNA Cloning 2: A Practical Approach, 2 nd Edition, Glover and Hames (eds.), pages 123-167 (Oxford University Press 1995).
  • Zdsc4 can be expressed as a fusion protein comprising a glutathione S-transferase polypeptide.
  • Glutathione S-transferease fusion proteins are typically soluble, and easily purifiable from E. coli lysates on immobilized glutathione columns.
  • a Zdsc4 fusion protein comprising a maltose binding protein polypeptide can be isolated with an amylose resin column, while a fusion protein comprising the C-terminal end of a truncated Protein A gene can be purified using IgG-Sepharose.
  • Established techniques for expressing a heterologous polypeptide as a fusion protein in a bacterial cell are described, for example, by Williams et al, "Expression of Foreign Proteins in E. coli Using Plasmid Vectors and Purification of Specific Polyclonal Antibodies," in DNA Cloning 2: A Practical Approach, 2 nd Edition, Glover and Hames (Eds.), pages 15-58 (Oxford University Press 1995).
  • commercially available expression systems are available. For example, the PINPOINT Xa protein purification system (Promega).
  • Peptide tags that are useful for isolating heterologous polypeptides -expressed by either prokaryotic or eukaryotic cells include polyHistidine tags (which have an affinity for nickel-chelating resin), c-myc tags, calmodulin binding protein (isolated with calmodulin affinity chromatography), substance P, the RYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which binds with anti-FLAG antibodies). See, for example, Luo et al, Arch. Biochem. Biophys. 329:215 (1996), Morganti et al, Biotechnol. Appl. Biochem. 23:67 (1996), and Zheng et al, Gene 186:55 (1997). Nucleic acid molecules encoding such peptide tags are available, for example, from Sigma- Aldrich Corporation (St. Louis, MO).
  • the present invention also contemplates that the use of the secretory signal sequence contained in the Zdsc4 polypeptides of the present invention to direct other polypeptides into the secretory pathway.
  • a signal fusion polypeptide can be made wherein a secretory signal sequence derived from amino acid residues 1 to 21 of SEQ ID NO: 2 is operably linked to another polypeptide using methods known in the art and disclosed herein.
  • the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
  • Such constructs have numerous applications known in the art.
  • these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as a receptor.
  • a normally non-secreted protein such as a receptor.
  • Such fusions may be used in a transgenic animal or in a cultured recombinant host to direct peptides through the secretory pathway.
  • exemplary polypeptides include pharmaceutically active molecules such as Factor V ⁇ a, proinsulin, insulin, follicle stimulating hormone, tissue type plasminogen activator, tumor necrosis factor, interleukins [e.g., interleukin-1 (E -1), JL-2, IL-3, IL-4, JL-5, IL-6, EL-7, JL-8, IL-9, EL- 10, EL- 11, EL- 12, EL- 13, EL- 14, and EL- 15), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)], interferons (e.g., interferons- ⁇ , - ⁇ , - ⁇ , -co, - ⁇ , and - ⁇ ), the stem cell growth factor designated "SI factor,” erythropoietin, and thrombopoietin.
  • Zdsc4 secretory signal sequence can be constructed using standard techniques.
  • fusion protein comprises a Zdsc4 polypeptide and an immunoglobulin heavy chain constant region, typically an F c fragment, which contains two or three constant region domains and a hinge region but lacks the 'variable region.
  • an immunoglobulin heavy chain constant region typically an F c fragment
  • F c fragment an immunoglobulin heavy chain constant region
  • Chang et al U.S. Patent No. 5,723,125
  • a fusion protein comprising a human interferon and a human immunoglobulin Fc fragment.
  • the C-terminal of the interferon is linked to the N-terminal of the Fc fragment by a peptide linker moiety.
  • An example of a peptide linker is a peptide comprising primarily a T cell inert sequence, which is immunologically inert.
  • An exemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGG S (SEQ JD NO: 9).
  • a preferred Fc moiety is a human ⁇ 4 chain, which is stable in solution and has little or no complement activating activity.
  • the present invention contemplates a Zdsc4 fusion protein that comprises a Zdsc4 moiety and a human Fc fragment, wherein the C-terminus of the Zdsc4 moiety is attached to the N-terminus of the Fc fragment via a peptide linker.
  • the Zdsc4 moiety can be a Zdsc4 molecule or a fragment thereof.
  • an Zdsc4 fusion protein comprises an IgG sequence, an Zdsc4 moiety covalently joined to the aminoterminal end of the IgG sequence, and a signal peptide that is covalently joined to the aminoterminal of the Zdsc4 moiety, wherein the IgG sequence consists of the following elements in the following order: a hinge region, a CH 2 domain, and a CH 3 domain. Accordingly, the IgG sequence lacks a CHi domain.
  • the Zdsc4 moiety displays a Zdsc4 activity, as described herein, such as the ability to bind with a Zdsc4 receptor.
  • Fusion proteins comprising a Zdsc4 moiety and an Fc moiety can be used, for example, as an in vitro assay tool.
  • the presence of a Zdsc4 receptor in a biological sample can be detected using a Zdsc4-immunoglobulin fusion protein, in which the Zdsc4 moiety is used to target the cognate receptor, and a macromolecule, such as Protein A or anti-Fc antibody, is used to detect the bound fusion protein-receptor complex.
  • fusion proteins can be used to identify agonists and antagonists that interfere with the binding of Zdsc4 to its receptor.
  • fusion proteins can be constructed that comprise a murine Zdsc4 polypeptide and an immunoglobulin heavy chain constant region
  • antibody-Zdsc4 fusion proteins comprising antibody variable domains
  • Methods of making antibody-cytokine fusion proteins are known to those of skill in the art. For example, antibody fusion proteins comprising an interleukin-2 moiety are described by Boleti etal,. Ann. Oncol. 6:945 (1995), Nicolet etal, Cancer Gene Ther. 2:161 (1995), Becker etal, Proc. Nat'l
  • cytokine-antibody fusion proteins include IL-8, IL-12, or Zdsc4as the cytokine moiety (Holzer et al, Cytokine 8:214 (1996); Gillies et al, J. Immunol. 160:6195 (1998); Xiang et al, Hum. Antibodies Hybridomas 7:2 (1996)). Also see, Gillies, U.S. Patent No. 5,650,150.
  • hybrid Zdsc4 proteins can be constructed using regions or domains of the inventive [see, for example, Picard, Cur. Opin. Biology 5:511 (1994)]. These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest. Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure. Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them.
  • a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein.
  • fusion proteins may exhibit other properties as disclosed herein.
  • General methods for enzymatic and chemical cleavage of fusion proteins are described, for example, by Ausubel (1995) at pages 16-19 to 16-25.
  • the present invention also contemplates chemically modified Zdsc4 compositions, in which a Zdsc4 polypeptide is linked with a polymer.
  • the polymer is water-soluble so that the Zdsc4 conjugate does not precipitate in an aqueous environment, such as a physiological environment.
  • An example of a suitable polymer is one that has been modified to have a single reactive group, such as an active ester for acylation, or an aldehyde for alkylation. In this way, the degree of polymerization can be controlled.
  • a reactive aldehyde is polyethylene glycol propionaldehyde, or mono-(Cl-ClO) alkoxy, or aryloxy derivatives thereof (see, for example, Harris, et al , U.S. Patent No. 5,252,714).
  • the polymer may be branched or unbranched.
  • a mixture of polymers can be used to produce Zdsc4 conjugates.
  • Zdsc4 conjugates used for therapy should preferably comprise pharmaceutically acceptable water-soluble polymer moieties. Conjugation of interferons with water-soluble polymers has been shown to enhance the circulating half-life of the interferon, and to reduce the immunogenicity of the polypeptide (see, for example, Nieforth et al, Clin. Pharmacol. Ther. 59:636 (1996), and Monkarsh et al, Anal Biochem. 247:434 (1997)).
  • Suitable water-soluble polymers include polyethylene glycol (PEG), monomethoxy-PEG, mono-(Cl-C10)alkoxy-PEG, aryloxy-PEG, poly-(N- vinyl pyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde, bz ' s-succinimidyl carbonate PEG, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyefhylated polyols (e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or other carbohydrate-based polymers.
  • Suitable PEG may have a molecular weight from about 600 to about 60,000, including, for example, 5,000, 12,000, 20,000 and 25,000.
  • a Zdsc4 conjugate can also comprise a mixture of such water-soluble polymers.
  • Zdsc4 conjugate comprises a Zdsc4 moiety and a polyalkyl oxide moiety attached to the N-terminus of the Zdsc4 moiety.
  • PEG is one suitable polyalkyl oxide.
  • Zdsc4 can be modified with PEG, a process known as "PEGylation.” PEGylation of Zdsc4 can be carried out by any of the PEGylation reactions known in the art (see, for example, EP 0 154316, Delgado et al, Critical Reviews in Therapeutic Drug Carrier Systems 9:249 (1992), Duncan and Spreafico, Clin. Pharmacokinet.
  • PEGylation can be performed by an acylation reaction or by an alkylation reaction with a reactive polyethylene glycol molecule.
  • Zdsc4 conjugates are formed by condensing activated PEG, in which a terminal hydroxy or amino group of PEG has been replaced by an activated linker (see, for example, Karasiewicz et al, U.S. Patent No. 5,382,657).
  • PEGylation by acylation typically requires reacting an active ester derivative of PEG with a Zdsc4 polypeptide.
  • An example of an activated PEG ester is PEG esterified to N-hydroxysuccinimide.
  • acylation includes the following types of linkages between Zdsc4 and a water-soluble polymer: amide, carbamate, urethane, and the like.
  • Methods for preparing PEGylated Zdsc4 by acylation will typically comprise the steps of (a) reacting an Zdsc4 polypeptide with PEG (such as a reactive ester of an aldehyde derivative of PEG) under conditions whereby one or more PEG groups attach to Zdsc4, and (b) obtaining the reaction product(s).
  • PEG such as a reactive ester of an aldehyde derivative of PEG
  • the optimal reaction conditions for acylation reactions will be • determined based upon known parameters and desired results. For example, the larger the ratio of PEG: Zdsc4, the greater the percentage of polyPEGylated Zdsc4 product.
  • the product of PEGylation by acylation is typically a polyPEGylated
  • the resulting Zdsc4 product wherein the lysine ⁇ -amino groups are PEGylated via an acyl linking group.
  • An example of a connecting linkage is an amide.
  • the resulting Zdsc4 will be at least 95% mono-, di-, or tri-pegylated, although some species with higher degrees of PEGylation may be formed depending upon the reaction conditions.
  • PEGylated species can be separated from unconjugated Zdsc4 polypeptides using standard purification methods, such as dialysis, ultrafiltration, ion exchange chromatography, affinity chromatography, and the like.
  • PEGylation by alkylation generally involves reacting a terminal aldehyde derivative of PEG with Zdsc4 in the presence of a reducing agent.
  • PEG groups are preferably attached to the polypeptide via a -CH 2 -NH group.
  • Derivatization via reductive alkylation to produce a monoPEGylated product takes advantage of the differential reactivity of different types of primary amino groups available for derivatization.
  • the reaction is performed at a pH that allows one to take advantage of the pKa differences between the ⁇ -amino groups of the lysine residues and the ⁇ -amino group of the N-terminal residue of the protein.
  • a water-soluble polymer that contains a reactive group such as an aldehyde
  • the conjugation with the polymer occurs predominantly at the N-terminus of the protein without significant modification of other reactive groups such as the lysine side chain amino groups.
  • the present invention provides a substantially homogenous preparation of Zdsc4 monopolymer conjugates.
  • Reductive alkylation to produce a substantially homogenous population of monopolymer Zdsc4 conjugate molecule can comprise the steps of: (a) reacting an Zdsc4 polypeptide with a reactive PEG under reductive alkylation conditions at a pH suitable to permit selective modification of the ⁇ -amino group at the amino terminus of the Zdsc4, and (b) obtaining the reaction product(s).
  • the reducing agent used for reductive alkylation should be stable in aqueous solution and preferably be able to reduce only the Schiff base formed in the initial process of reductive alkylation.
  • Preferred reducing agents include sodium borohydride, sodium cyanoborohydride, dimethylamine borane, trimethylamine borane, and pyridine borane.
  • the reductive alkylation reaction conditions are those that permit the selective attachment of the water-soluble polymer moiety to the N-terminus of Zdsc4.
  • Such reaction conditions generally provide for pKa differences between the lysine , amino groups and the ⁇ -amino group at the N-terminus.
  • the pH also affects the ratio of polymer to protein to be used. In general, if the pH is lower, a larger excess of polymer to protein will be desired because the less reactive the N-terminal ⁇ -group, the more polymer is needed to achieve optimal conditions. If the pH is higher, the polymer:Zdsc4 need not be as large because more reactive groups are available.
  • the pH will fall within the range of 3 - 9, or 3 - 6.
  • Another factor to consider is the molecular weight of the water-soluble polymer. Generally, the higher the molecular weight of the polymer, the fewer number of polymer molecules which may be attached to the protein. For PEGylation reactions, the typical molecular weight is about 2 kDa to about 100 kDa, about 5 kDa to about 50 kDa, or about 12 kDa to about 25 kDa.
  • the molar ratio of water-soluble polymer to Zdsc4 will generally be in the range of 1:1 to 100:1. Typically, the molar ratio of water-soluble polymer to Zdsc4 will be 1: 1 to 20:1 for polyPEGylation, and 1:1 to 5:1 for monoPEGylation.
  • polypeptides of the present invention can be produced in recombinant host cells following conventional techniques.
  • a nucleic acid molecule encoding the polypeptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then, introduced into a host cell.
  • expression vectors can include translational regulatory sequences and a marker gene that is suitable for selection of cells that carry the expression vector.
  • Expression vectors that are suitable for production of a foreign protein in eukaryotic cells typically contain (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of transcripts, such as a transcription • . termination/polyadenylation sequence.
  • expression vectors can also include nucleotide sequences encoding a secretory sequence that directs the heterologous polypeptide into the secretory pathway of a host cell.
  • a Zdsc4 expression vector may comprise a Zdsc4 gene and a secretory sequence derived from a Zdsc4 gene or another secreted gene.
  • Zdsc4 proteins of the present invention may be expressed in mammalian cells.
  • suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293- HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44 [Chasin et al, Som. Cell. Molec. Genet.
  • GH1 rat pituitary cells
  • H-4-U-E rat hepatoma cells
  • COS-1 SV40- transformed monkey kidney cells
  • NIH-3T3 murine embryonic cells
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian vims, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression.
  • Suitable transcriptional and translational regulatory sequences also can be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes.
  • Transcriptional regulatory sequences include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene [Hamer et al, J. Molec. Appl. Genet.
  • a prokaryotic promoter such as the bacteriophage T3
  • RNA polymerase promoter can be used to control Zdsc4 gene expression in mammalian cells if the prokaryotic promoter is regulated by a eukaryotic promoter [Zhou et al, Mol Cell Biol. 10:4529 (1990), and Kaufman et al, Nucl Acids Res. 19:4485 (1991)].
  • An expression vector can be introduced into host cells using a variety of standard techniques including calcium phosphate transfection, Uposome-mediated transfection, microprojectile-mediated delivery, electroporation, and the like.
  • the transfected cells are selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome. Techniques for introducing vectors into eukaryotic cells and techniques for selecting such stable transformants using a dominant selectable marker are described, for example, by Ausubel (1995) and by Murray (ed.), Gene Transfer and Expression Protocols (Humana Press 1991).
  • one suitable selectable marker is a gene that provides resistance to the antibiotic neomycin.
  • selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like.
  • Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • drugs resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • markers that introduce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
  • Zdsc4 polypeptides can also be produced by cultured mammalian cells using a viral delivery system.
  • viruses for this purpose include adenovirus, herpesvirus, vaccinia virus and adeno-associated virus (AAV).
  • Adenovirus a double- stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid [for a review, see Becker et al, Meth. Cell Biol. 43:161 (1994), and Douglas and Curiel, Science & Medicine 4:44 (1997)].
  • Advantages of the adenovirus system include the accommodation of relatively large DNA inserts, the ability to grow to high-titer, the ability to infect a broad range of mammalian cell types, and flexibility that allows use with a large number of available vectors containing different promoters.
  • Adenovirus vector-infected human 293 cells ATCC Nos. CRL-1573, 45504, 45505
  • Zdsc4 genes may also be expressed in other higher eukaryotic cells, such as avian, fungal, insect, yeast, or plant cells.
  • the baculovirus system provides an efficient means to introduce cloned Zdsc4 genes into insect cells.
  • Suitable expression vectors are based upon the Autographa calif ornica multiple nuclear polyhedrosis virus (AcMNPV), and contain well-known promoters such as Drosoph ⁇ la heat shock protein (hsp) 70 promoter, Autographa calif ornica nuclear polyhedrosis virus immediate-early gene promoter (ie-1) and the delayed early 39K promoter, baculovirus plO promoter, and the Drosoph ⁇ la metallothionein promoter.
  • hsp Drosoph ⁇ la heat shock protein
  • ie-1 Autographa calif ornica nuclear polyhedrosis virus immediate-early gene promoter
  • baculovirus plO promoter the Drosoph ⁇ la metallothi
  • a second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow [Luckow, et al, J. Virol. 67:4566 (1993)].
  • This system which utilizes transfer vectors, is sold in the BAC-to-BAC kit (Life Technologies, Rockville, MD).
  • This system utilizes a transfer vector, PFASTBAC (Life Technologies) containing a Tn7 transposon to move the DNA encoding the Zdsc4 ⁇ polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990), Bonning, et al, J.
  • transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed Zdsc4 polypeptide, for example, a Glu-Glu epitope tag [Grussenmeyer et al, Proc. Nat'lAcad. Sci. 82:7952 (1985)].
  • a transfer vector containing a Zdsc4 gene is transformed into E. coli, and screened for bacmids that contain an interrupted lacZ gene indicative of recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculovirus genome is then isolated using common techniques.
  • the illustrative PFASTBAC vector can be modified to a considerable degree.
  • the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins (see, for example, Hill-Perkins and Possee, /. Gen. Virol 71:971 (1990), Bonning, et al, J. Gen. Virol. 75:1551 (1994), and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995).
  • a short or long version of the basic protein promoter can be used.
  • transfer vectors can be constructed that replace the native Zdsc4 secretory signal sequences with secretory signal sequences derived from insect proteins.
  • a secretory signal sequence from ⁇ cdysteroid Glucosyltransferase ( ⁇ GT), honeybee Melittin (Invitrogen Corporation; Carlsbad, CA), or baculovirus gp67 (PharMingen: San Diego, CA) can be used in constructs to replace the native Zdsc4 secretory signal sequence.
  • the recombinant virus or bacmid is used to transfect host cells.
  • suitable insect host cells include cell lines derived from JPLB-5/-21, a Spodoptera frugiperda pupal ovarian cell line, such as S ⁇ (ATCC CRL 1711), 5 21 AE, and Sf21 (Invitrogen Corporation; San Diego, CA), as well as Drosophila Schneider-2 cells, and the HIGH FTVEO cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No. 5,300,435).
  • S ⁇ ATCC CRL 1711
  • 5 21 AE Adrosophila Schneider-2
  • Sf21 Invitrogen Corporation
  • Drosophila Schneider-2 cells Drosophila Schneider-2 cells
  • HIGH FTVEO cell line Invitrogen
  • Commercially available serum-free media can be used to grow and to maintain the cells.
  • Suitable media are Sf900 HTM (Life Technologies) or ESF 921TM (Expression Systems) for the Sf9 cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, KS) or Express FiveOTM (Life Technologies) for the T. ni cells.
  • the cells are typically grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 6 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.
  • MOI multiplicity of infection
  • yeast cells can also be used to express the genes described herein.
  • Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichiapastoris, and Pichia methanolica.
  • Suitable promoters for expression in yeast include promoters from GAL1 (galactose), PGK ' (phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1 (alcohol ox ⁇ dase), HIS4 (histidinol dehydrogenase), and the like.
  • GAL1 galactose
  • PGK ' phosphoglycerate kinase
  • ADH alcohol dehydrogenase
  • AOX1 alcohol ox ⁇ dase
  • HIS4 histidinol dehydrogenase
  • These vectors include Yip-based vectors, such as YIp5, YRp vectors, such as YRpl7, YEp vectors such as YEpl3 and YCp vectors, such as YCp 19.
  • Yip-based vectors such as YIp5
  • YRp vectors such as YRpl7
  • YEp vectors such as YEpl3
  • YCp vectors such as YCp 19.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
  • a preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No.
  • 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, Schizosaccharomycespom.be, 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 (1986), and Cregg, U.S. Patent No.4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al, U.S. Patent No. 4,935,349.
  • Pichia methanolica as host for the production of recombinant proteins is disclosed by Raymond, U.S. Patent No. 5,716,808, Raymond, U.S. Patent No. 5,736,383, Raymond et al, Yeast 14:11-23 (1998), and in international publication Nos. WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.
  • DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
  • the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 or AUG2).
  • Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase
  • FMD catalase
  • CAT catalase
  • a preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5- aminoimidazole carboxylase (AE .C; EC 4.1.1.21), and which allows ade2 host cells to grow in the absence of adenine.
  • AE .C phosphoribosyl-5- aminoimidazole carboxylase
  • ade2 host cells For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) ate deleted.
  • P. methanolica cells can be transformed by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
  • Expression vectors can also be introduced into plant protoplasts, intact plant tissues, or isolated plant cells.
  • Methods for introducing expression vectors into plant tissue include the direct infection or co-cultivation of plant tissue with Agrobacterium tumefaciens, microprojectile-mediated delivery, DNA injection, electroporation, and the like. See, for example, Horsch et al, Science 227:1229 (1985), Klein et al, Biotechnology 10:268 (1992), and Miki et al, "Procedures for Introducing Foreign DNA into Plants," in Methods in Plant Molecular Biology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press, 1993).
  • Zdsc4 genes can be expressed in prokaryotic host cells.
  • Suitable promoters that can be used to express Zdsc4 polypeptides in a prokaryotic host are well-known to those of skill in the art and include promoters capable of recognizing the T4, T3, Sp6 and T7 polymerases, the P R and P L promoters of bacteriophage lambda, the trp, recA, heat shock, lacUV5, tac, Ipp-lacSpr, phoA, and lacZ promoters of E. coli, promoters of 5.
  • subtilis subtilis, the promoters of the bacteriophages of Bacillus, Streptomyces promoters, the int promoter of bacteriophage lambda, the bla promoter of pBR322, and the CAT promoter of the chloramphenicol acetyl trans- ferase gene.
  • Prokaryotic promoters have been reviewed by Glick, /. Ind. Microbiol 1:277 (1987), Watson et al, Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), and by Ausubel etal. (1995).
  • Preferred prokaryotic hosts include E. coli and Bacillus subtilus. Suitable strains of E. coli include BL21(DE3), BL21(DE3) ⁇ LysS, BL21(DE3) ⁇ LysE,
  • Suitable strains of Bacillus subtilus include BR151, YB886, MI119, MI120, and B 170 [see, for example, Hardy, "Bacillus Cloning Methods," in DNA Cloning: A Practical Approach, Glover (ed.) (IRL Press 1985)].
  • the polypeptide When expressing a Zdsc4 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.
  • 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.
  • polypeptides of the present invention it is preferred to purify the polypeptides of the present invention to at least about 80% purity, more preferably to at least about 90% purity, even more preferably to at least about 95% purity, or even greater than 95% purity with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
  • the polypeptides of the present invention may also be purified to a pharmaceutically pure state, which is greater than 99.9% pure.
  • a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • Fractionation and/or conventional purification methods can be used to obtain preparations of Zdsc4 purified from natural sources (e.g, uterine tissue), and recombinant Zdsc4 polypeptides and fusion Zdsc4 polypeptides purified from recombinant host cells.
  • ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl- Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica- based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
  • Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Selection of a particular method for polypeptide isolation and purification is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988), and Doonan, Protein Purification Protocols (The Humana Press 1996).
  • Zdsc4 isolation and purification can be devised by those of skill in the art.
  • anti-Zdsc4 antibodies obtained as described below, can be used to isolate large quantities of protein by immunoaffinity purification.
  • the use of monoclonal antibody columns to purify interferons from recombinant cells and from natural sources has been described, for example, by Staehelin et al, J. Biol Chem. 256:9750 (1981), and by Adolf et al, J. Biol. Chem. 265:9290 (1990).
  • methods for binding ligands, such as Zdsc4, to receptor polypeptides bound to support media are well known in the art.
  • polypeptides of the present invention can also be isolated by exploitation of particular properties.
  • immobilized metal ion adsorption (EMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate [Sulkowski, Trends in Biochem. 3:1 (1985)]. Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • EMC immobilized metal ion adsorption
  • Zdsc4 polypeptides or fragments thereof may also be prepared through chemical synthesis, as described below.
  • Zdsc4 polypeptides may be monomers or multimers; glycosylated or non-glycosylated; PEGylated or non-PEGylated; and may or may not include an initial methionine amino acid residue.
  • Peptides and polypeptides of the present invention comprise at least at least 15, preferably at least 30 or 50contiguous amino acid residues of SEQ ED NOs: 2 or 3. Nucleic acid molecules encoding such peptides and polypeptides are useful as polymerase chain reaction primers and probes.
  • Zdsc4 polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis.
  • the polypeptides are preferably prepared by solid phase peptide synthesis, for example as described by Merrifield, /. Am. Chem. Soc. 85:2149 (1963).
  • the synthesis is carried out with amino acids that are protected at the alpha- amino terminus.
  • Trifunctional amino acids with labile side-chains are also protected with suitable groups to prevent undesired chemical reactions from occurring during the assembly of the polypeptides.
  • the alpha-amino protecting group is selectively removed to allow subsequent reaction to take place at the amino-terminus. The conditions for the removal of the alpha-amino protecting group do not remove the side-chain protecting groups.
  • N- terminal cysteine-containing peptide is chemically ligated to a peptide having a C- terminal thioester group to form a normal peptide bond at the ligation site.
  • the "expressed protein ligation” method is a semi-synthesis variation of the ligation approach (see, for example, Muir et al, Proc. Nat'lAcad. Sci. USA 95:6705 (1998); Severinov and Muir, /. Biol Chem. 273:16205 (1998)).
  • synthetic peptides and protein cleavage fragments are linked to form the desired protein product.
  • This method is particularly useful for the site-specific incorporation of unnatural amino acids (e.g., amino acids comprising biophysical or biochemical probes) into proteins.
  • the gene or gene fragment is expressed in frame fused with a chitin binding domain sequence, and a Pro-Gly is appended to the native C terminus of the protein of interest.
  • the presence of a C-terminal glycine reduces the chance of side reactions, because the glycine residue accelerates native chemical ligation.
  • Affinity chromatography with a chitin resin is used to purify the expressed fusion protein, and the chemical ligation step is initiated by incubating the resin-bound protein with thiophenol and synthetic peptide in buffer. This mixture produces the in situ generation of a highly reactive phenyl ⁇ thioester derivative of the protein that rapidly ligates with the synthetic peptide to produce the desired semi-synthetic protein.
  • the disclosed polypeptides can be used to construct Zdsc4 variants.
  • Zdsc4 variants can be initially identified on the basis of hybridization analysis, sequence identity determination, or by the ability to specifically bind anti-Zdsc4 antibody.
  • Zdsc4 its agonists and antagonists are valuable in both in vivo and in vitro uses.
  • cytokines can be used as components of defined cell culture media, alone or in combination with other cytokines and hormones, to replace serum that is commonly used in cell culture.
  • Antagonists are also useful as research reagents for characterizing sites of interaction between Zdsc4 and its receptor.
  • pharmaceutical compositions comprising Zdsc4 antagonists can be used to inhibit Zdsc4 activity.
  • Zdsc4 analogs are agonists or antagonists having an amino acid sequence that is a mutation of the amino acid sequences disclosed herein.
  • Another general class of Zdsc4 analogs is provided by anti-idiotype antibodies, and fragments thereof, as described below.
  • recombinant antibodies comprising anti-idiotype variable domains can be used as analogs [see, for example, Monfardini et al., Proc. Assoc. Am. Physicians 108:420 (1996)]. Since the variable domains of anti-idiotype Zdsc4 antibodies mimic Zdsc4, these domains can provide either Zdsc4 agonist or antagonist activity.
  • a third approach to identifying Zdsc4 analogs is provided by the use of combinatorial libraries.
  • the activity of Zdsc4 can be measured by a silicon-based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent cellular responses.
  • An exemplary device is the CYTOSENSOR Microphysiometer manufactured by Molecular Devices Corp. (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 (1992), Pitchford et al, Meth. Enzymol. 228:84 (1997), Arimilli et al, J. Immunol. Meth.
  • microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells.
  • the microphysiometer directly measures cellular responses to various stimuli, including Zdsc4, its agonists, or antagonists.
  • the microphysiometer is used to measure responses of a Zdsc4 responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to Zdsc4 polypeptide.
  • Zdsc4 responsive eukaryotic cells comprise cells into which a receptor for Zdsc4 has been transfected to create a cell that is responsive to Zdsc4, or cells that are naturally responsive to Zdsc4 Accordingly, a microphysiometer can be used to identify cells, tissues, or cell lines which respond to an Zdsc4 stimulated pathway, and which express a functional Zdsc4 receptor.
  • cells that express a functional Zdsc4 receptor can be identified by (a) providing test cells, (b) incubating a first portion of the test cells in the absence of Zdsc4, (c) incubating a second portion of the test cells in the presence of Zdsc4, and (d) detecting a change (e.g., an increase or decrease in extracellular acidification rate, as measured by a microphysiometer) in a cellular response of the second portion of the test cells, as compared to the first portion of the test cells, wherein such a change in cellular response indicates that the test cells express a functional Zdsc4 receptor.
  • a change e.g., an increase or decrease in extracellular acidification rate, as measured by a microphysiometer
  • An additional negative control may be included in which a portion of the test cells is incubated with Zdsc4 and an anti-Zdsc4 antibody to inhibit the binding of Zdsc4 with its cognate receptor.
  • the microphysiometer also provides one means to identify Zdsc4 agonists.
  • agonists of Zdsc4 can be identified by a method, comprising the steps of (a) providing cells responsive to Zdsc4, (b) incubating a first portion of the cells in the absence of a test compound, (c) incubating a second portion of the cells in the presence of a test compound, and (d) detecting a change, for example, an increase or diminution, in a cellular response of the second portion of the cells as compared to the first portion of the cells, wherein such a change in cellular response indicates that the test compound is an Zdsc4 agonist.
  • An illustrative change in cellular response is a measurable change in extracellular acidification rate, as measured by a microphysiometer.
  • incubating a third portion of the cells in the presence of Zdsc4 and in the absence of a test compound can be used as a positive control for the Zdsc4 responsive cells, and as a control to compare the agonist activity of a test compound with that of Zdsc4.
  • An additional control may be included in which a portion of the cells is incubated with a test compound (or Zdsc4) and an anti-Zdsc4 antibody to inhibit the binding of the test compound (or Zdsc4) with the Zdsc4 receptor.
  • the microphysiometer also provides a means to identify Zdsc4 antagonists.
  • Zdsc4 antagonists can be identified by a method, comprising the steps of (a) providing cells responsive to Zdsc4, (b) incubating a first portion of the cells in the presence of Zdsc4 and in the absence of a test compound, (c) incubating a second portion of the cells in the presence of both Zdsc4 and the test compound, and (d) comparing the cellular responses of the first and second cell portions, wherein a decreased response by the second portion, compared with the response of the first portion, indicates that the test compound is an Zdsc4 antagonist.
  • An illustrative change in cellular response is a measurable change extracellular acidification rate, as measured by a microphysiometer.
  • Zdsc4 its analogs, and anti-iodiotype Zdsc4 antibodies can be used to identify and to isolate Zdsc4 receptors.
  • proteins and peptides of the present invention can be immobilized on a column and used to bind receptor proteins from membrane preparations that are run over the column (Hermanson et al. (eds.), Immobilized Affinity Ligand Techniques, pages 195-202 (Academic Press 1992)).
  • Radiolabeled or affinity labeled Zdsc4 polypeptides can also be used to identify or to localize Zdsc4 receptors in a biological sample [see, for example, Deutscher (ed.), Methods in Enzymol, vol.
  • a solid phase system can be used to identify a Zdsc4 receptor, or an agonist or antagonist of a Zdsc4 receptor.
  • a Zdsc4 polypeptide or Zdsc4 fusion protein can be immobilized onto the surface of a receptor chip of a commercially available biosensor instrument (BIACORE, Biacore AB; Uppsala, Sweden). The use of this instrument is disclosed, for example, by Karlsson, Immunol Methods 145:229 (1991), and Cunningham and Wells, /. Mol. Biol 234:554 (1993).
  • a Zdsc4 polypeptide or fusion protein is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within a flow cell.
  • a test sample is then passed through the cell.
  • a receptor is present in the sample, it will bind to the immobilized polypeptide or fusion protein, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
  • This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
  • This system can also be used to examine antibody-antigen interactions, and the interactions of other complement/anti- complement pairs.
  • Antibodies to Zdsc4 can be obtained, for example, using the product of a Zdsc4 expression vector or Zdsc4 isolated from a natural source as an antigen. Particularly useful anti-Zdsc4 antibodies "bind specifically" with Zdsc4. Antibodies are considered to be specifically binding if the antibodies exhibit at least one of the following two properties: (1) antibodies bind to Zdsc4 with a threshold level of binding activity, and (2) antibodies do not significantly cross-react with polypeptides related to Zdsc4.
  • antibodies specifically bind if they bind to a Zdsc4 polypeptide, peptide or epitope with a binding affinity (K a ) of 10 6
  • antibodies do not significantly cross-react with related polypeptide .molecules, for example, if they detect Zdsc4, but not known related polypeptides using a standard Western blot analysis. Examples of known related polypeptides are orthologs and proteins from the same species that are members of a protein family.
  • Anti-Zdsc4 antibodies can be produced using antigenic Zdsc4 epitope- bearing peptides and polypeptides.
  • Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least nine, preferably between 15 to about 30 amino acids contained within SEQ ID NOs: 2 or 3.
  • peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that bind with Zdsc4.
  • amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues, while hydrophobic residues are preferably avoided). Moreover, amino acid sequences containing proline residues may be also be desirable for antibody production.
  • amino acids 24 to 46 of SEQ ID NO: 2 (SEQ ID NO: 4), amino acids 24 to 64 (SEQ ID NO: 5), 30 to 64 (SEQ ID NO: 6), and amino acids 25 to 59 (SEQ ID NO: 7).
  • the present invention contemplates the use of any one of antigenic peptides to generate antibodies to Zdsc4.
  • the present invention also contemplates polypeptides comprising at least one of the above-described antigenic peptides.
  • Polyclonal antibodies to recombinant Zdsc4 protein or to Zdsc4 isolated from natural sources can be prepared using methods well known to those of skill in the art. See, for example, Green et al., "Production of Polyclonal Antisera,” in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al., "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford University Press 1995).
  • the immunogenicity of a Zdsc4 polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • 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 Zdsc4 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.
  • 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.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid
  • an anti- Zdsc4 antibody of the present invention may also be derived from a subhuman primate antibody.
  • General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al, international patent publication No. WO 91/11465, and in Losman et al, Int. J. Cancer 46:310 , (1990).
  • monoclonal anti-Zdsc4 antibodies can be generated.
  • Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art [see, for example, Kohler et al, Nature 256:495 (1975), Coligan et al (eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1- 2.6.7 (John Wiley & Sons 1991) ["Coligan”], Picksley et al, "Production of monoclonal antibodies against proteins expressed in E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)].
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising an Zdsc4 gene product, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B- lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • an anti-Zdsc4 antibody of the present invention may be derived from a human monoclonal antibody.
  • Human monoclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas.
  • Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al, Nature Genet. 7:13 (1994), Lonberg et al, Nature 368:856 (1994), and Taylor et al, Int. Immun. 6:579 (1994).
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography [see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al, "Purification of Immunoglobulin G (IgG)," in
  • antibody fragments can be obtained, for example, byproteolytic hydrolysis of the antibody.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 - This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab' monovalent fragments.
  • the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages.
  • an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly.
  • These methods are described, for example, by Goldenberg, U.S. patent No. 4,331,647, Nisonoff et al, Arch Biochem. Biophys. 89:230 (1960), Porter, Biochem. J. 73:119 (1959), Edelman et al, in Methods in Enzymology Vol 1, page 422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and 2.10.- 2.10.4.
  • Fv fragments comprise an association of VH and V L chains. This association can be noncovalent, as described by Inbar et al, Proc. Nat'l Acad. Sci. USA 69:2659 (1972).
  • the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech. 12:437 (1992)).
  • the Fv fragments may comprise V H and V chains which are connected by a peptide linker.
  • These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • scFvs Methods for producing scFvs are described, for example, by Whitlow et al, Methods: A Companion to Methods in Enzymology 2:97 (1991) (also see, Bird et al, Science 242:423 (1988), Ladner et al, U.S. Patent No. 4,946,778, Pack etal, Bio Technology 11:1271 (1993), and Sandhu, supra).
  • a scFV can be obtained by exposing lymphocytes to lymphocytes.
  • Zdsc4 polypeptide in vitro and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Zdsc4 protein or peptide).
  • Genes encoding polypeptides having potential Zdsc4 polypeptide-binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli. Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • random peptide display libraries can be used to screen for peptides that interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • a known target can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • Techniques for creating and screening such random peptide display libraries are known in the art [Ladner et al, U.S. Patent No. 5,223,409, Ladner ⁇ t al., U.S. Patent No. 4,946,778, Ladner et al, U.S. Patent No. 5,403,484, Ladner et al, U.S. Patent No. 5,571,698, and Kay et al, Phage Display of Peptides and Proteins (Academic Press, Inc.
  • Random peptide display libraries can be screened using the Zdsc4 sequences disclosed herein to identify proteins that bind to Zdsc4.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells [see, for example, Larrick et al, Methods: A Companion to Methods in Enzymology 2:106 (1991), Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in
  • an anti-Zdsc4 antibody may be derived from a
  • humanized monoclonal antibody Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain. Typical residues of human antibodies are then substituted in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al, Proc. Nat'lAcad. Sci. USA 86:3833 (1989).
  • Patent No. 5,693,762 Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-Zdsc4 antibodies or antibody fragments, using standard techniques. See, for example, Green et al, "Production of Polyclonal Antisera,” in Methods In Molecular Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, see Coligan at pages 2.4.1-2.4.7.
  • monoclonal anti- idiotype antibodies can be prepared using anti-Zdsc4 antibodies or antibody fragments as immunogens with the techniques, described above.
  • humanized anti-idiotype antibodies or subhuman primate anti-idiotype antibodies can be prepared using the above-described techniques. Methods for producing anti- idiotype antibodies are described, for example, by.Irie, U.S. Patent No. 5,208,146,
  • Nucleic acid molecules can be used to detect the expression of a Zdsc4 gene in a biological sample.
  • probe molecules can include murine Zdsc4 encoding sequences
  • preferred probe molecules include double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequence of SEQ ID NO: 1 , or a fragment thereof.
  • Probe molecules may be DNA, RNA, oligonucleotides, and the like.
  • RNA isolated from a biological sample
  • RNA isolated from a biological sample
  • RNA isolated from a biological sample
  • ionic strength that promote base pairing between the probe and target Zdsc4 RNA species.
  • the amount of hybrids is detected.
  • Illustrative biological samples include blood, urine, saliva, tissue biopsy, and autopsy material.
  • RNA detection includes northern analysis and dot/slot blot hybridization [see, for example, Ausubel (1995) at pages 4-1 to 4-27, and Wu et al. (eds.), "Analysis of Gene Expression at the RNA
  • Nucleic acid probes can be detectably labeled with radioisotopes such as 32 P or 35 S.
  • Zdsc4 RNA can be detected with a nonradioactive hybridization method [see, for example, Isaac (ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes (Humana Press, Inc. 1993)].
  • nonradioactive detection is achieved by enzymatic conversion of chromogenic or chemiluminescent substrates.
  • Illustrative nonradioactive moieties include biotin, fluorescein, and digoxigenin.
  • Z sc ⁇ oligonucleotide probes are also useful for in vivo diagnosis.
  • F-labeled oligonucleotides can be administered to a subject and visualized by positron emission tomography [Tavitian et al, Nature Medicine 4:467 (1998)].
  • Numerous diagnostic procedures take advantage of the polymerase chain reaction (PCR) to increase sensitivity of detection methods.
  • Standard techniques for performing PCR are well-known [see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methods and Applications (Humana Press, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press, Inc.
  • PCR reverse transcriptase
  • RNA is isolated from a biological sample, reverse transcribed to cDNA, and the cDNA is incubated with Zdsc4 primers [see, for example, Wu et al. (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR,” in Methods in Gene Biotechnology, pages 15-28 (CRC Press, Inc. 1997)].
  • Zdsc4 primers see, for example, Wu et al. (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR," in Methods in Gene Biotechnology, pages 15-28 (CRC Press, Inc. 1997).
  • PCR is then performed and the products are analyzed using standard techniques.
  • RNA is isolated from biological sample using, for example, the gunadinium-thiocyanate cell lysis procedure described above.
  • a solid-phase technique can be used to isolate mRNA from a cell lysate.
  • a reverse transcription reaction can be primed with the isolated RNA using random oligonucleotides, short homopolymers of dT, or Zdsc4anti-sense oligomers.
  • Oligo-dT primers offer the advantage that various mRNA nucleotide sequences are amplified that can provide control target sequences.
  • Zdsc4 sequences are amplified by the polymerase chain reaction using two flanking oligonucleotide primers that are typically 20 bases in length. ' .
  • PCR amplification products can be detected using a variety of " approaches.
  • PCR products can be fractionated by gel electrophoresis, and visualized by ethidium bromide staining.
  • fractionated PCR products can be transferred to a membrane, hybridized with a detectably-labeled Zdsc4 probe, and examined by autoradiography.
  • Additional alternative approaches include the use of digoxigenin-labeled deoxyribonucleic acid triphosphates to provide chemiluminescence detection, and the C-TRAK colorimetric assay.
  • CPT cycling probe technology
  • NASBA nucleic acid sequence-based amplification
  • CATCH cooperative amplification of templates by cross-hybridization
  • LCR Iigase chain reaction
  • Zdsc4 probes and primers can also be used to detect and to localize Zdsc4 gene expression in tissue samples.
  • Methods for such in situ hybridization are well-known to those of skill in the art (see, for example, Choo (ed.), In situ hybridization
  • Nucleic acid molecules comprising Zdsc4 nucleotide sequences can also be used to determine whether a subject's chromosomes contain a mutation in the Zdsc4 gene. Detectable chromosomal aberrations at the Zdsc4 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. Of particular interest are genetic ' '*
  • Zdsc4 maps to chromosome 20ql2. In this area maps the protooncogene src, the mutation of which induces colon cancer, and hepatocyte nuclear factor 4-alpha, a gene linked to noninsulin-dependent diabetes mellitus.
  • Aberrations associated with the Zdsc4 locus can be detected using nucleic acid molecules of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, amplification-refractory mutation system analysis (ARMS), single-strand conformation polymorphism (SSCP) detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis (FAMA), and other genetic analysis techniques known in the art [see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc.
  • RNA is isolated from a biological sample, and used to synthesize cDNA. PCR is then used to amplify the Zdsc4 target sequence and to introduce an RNA polymerase promoter, a translation initiation sequence, and an in- frame ATG triplet. PCR products are transcribed using an RNA polymerase, and the transcripts are translated in vitro with a T7-coupled reticulocyte lysate system.
  • the translation products are then fractionated by SDS-PAGE to determine the lengths of the translation products.
  • the protein truncation test is described, for example, by Dracopoli et ⁇ l (eds.), Current Protocols in Human Genetics, pages 9.11.1 - 9.11.18
  • Zdsc4 protein is isolated from a subject, the molecular weight of the isolated Zdsc4 protein is determined, and then compared with the molecular weight a normal Zdsc4 protein, such as a protein having the amino acid sequence of SEQ ID NO: 2.
  • a normal Zdsc4 protein such as a protein having the amino acid sequence of SEQ ID NO: 2.
  • Zdsc4 protein is indicative that the protein is truncated.
  • substantially lower molecular weight refers to at least about 10 percent lower, and preferably, at least about 25 percent lower.
  • the Zdsc4 protein may be isolated by various procedures known in the art including immunoprecipitation, solid phase radioimmunoassay, enzyme-linked immunosorbent assay, or Western blotting. The molecular weight of the isolated Zdsc4 protein can be determined using standard techniques, such as SDS-polyacrylamide gel electrophoresis.
  • kits for performing a diagnostic assay for Zdsc4 gene expression or to detect mutations in the Zdsc4 gene comprise nucleic acid probes, such as double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequence of SEQ JD NO: 1, or a fragment thereof.
  • Probe molecules may be DNA, RNA, oligonucleotides, and the like.
  • Kits may comprise nucleic acid primers for performing PCR.
  • kits contains all the necessary elements to perform a nucleic acid diagnostic assay described above.
  • a kit will comprise at least one container comprising a Zdsc4 probe or primer.
  • the kit may also comprise a second container comprising one or more reagents capable of indicating the presence of Zdsc4 sequences. Examples of such indicator reagents include detectable labels such as radioactive labels, fluorochromes, chemiluminescent agents, and the like.
  • a kit may also comprise a means for conveying to the user that the Zdsc4 probes and primers are used to detect Zdsc4 gene expression.
  • written instructions may state that the enclosed nucleic acid molecules can be used to detect either a nucleic acid molecule that encodes Zdsc4, or a nucleic acid molecule having a nucleotide sequence that is complementary to a Zdsc4-encoding nucleotide sequence.
  • the written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert.
  • the present invention contemplates the use of anti-Zdsc4 antibodies to screen biological samples in vitro for the presence of Zdsc4.
  • anti-Zdsc4 antibodies are used in liquid phase.
  • the presence of Zdsc4 in a biological sample can be tested by mixing the biological sample with a trace amount of labeled Zdsc4 and an anti-Zdsc4 antibody under conditions that promote binding between Zdsc4 and its antibody.
  • Complexes of Zdsc4 and anti-Zdsc4 in the sample can be separated from the reaction mixture by contacting the complex with an immobilized protein which binds with the antibody, such as an Fc antibody or Staphylococcus protein A.
  • the concentration of Zdsc4 in the biological sample will be inversely proportional to the amount of labeled Zdsc4 bound to the antibody and directly related to the amount of free-labeled Zdsc4.
  • Illustrative biological samples include blood, urine, saliva, tissue biopsy, and autopsy material.
  • in vitro assays can be performed in which anti-Zdsc4 antibody is bound to a solid-phase carrier.
  • antibody can be attached to a polymer, such as aminodextran, in order to link the antibody to an insoluble support such as a polymer-coated bead, a plate or a tube.
  • polymer such as aminodextran
  • anti-Zdsc4 antibodies can be used to detect Zdsc4 in tissue sections prepared from a biopsy specimen. Such immunochemical detection can be used to determine the relative abundance of Zdsc4 and to determine the distribution of Zdsc4 in the examined tissue.
  • General immunochemistry techniques are well established [see, for example, Ponder, "Cell Marking Techniques and Their Application,” in Mammalian Development: A Practical Approach, Monk (ed.), pages 115-38 (IRL Press 1987), Coligan at pages 5.8.1-5.8.8, Ausubel (1995) at pages 14.6.1 to 14.6.13 (Wiley Interscience 1990), and Manson (ed.), Methods In Molecular Biology, Vol 10: Immunochemical Protocols (The Humana Press, Inc. 1992)].
  • Immunochemical detection can be performed by contacting a biological sample with an anti-Zdsc4 antibody, and then contacting the biological sample with a detectably labeled molecule that binds to the antibody.
  • the detectably labeled molecule can comprise an antibody moiety that binds to anti-Zdsc4 antibody.
  • the anti-Zdsc4 antibody can be conjugated with avidin/streptavidin (or biotin) and the detectably labeled molecule can comprise biotin (or avidin/streptavidin). Numerous variations of this basic technique are well known to those of skill in the art.
  • an anti-Zdsc4 antibody can be conjugated with a detectable label to form an anti-Zdsc4 immunoconjugate.
  • Suitable detectable labels include, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label or colloidal gold. Methods of making and detecting such detectably labeled immunoconjugates are well-known to those of ordinary skill in the art, and are described in more detail below.
  • the detectable label can be a radioisotope that is detected by autoradiography. Isotopes that are particularly useful for the purpose of the present invention are 3 H, 125 1, 131 1, 35 S and 14 C.
  • Anti-Zdsc4 immunoconjugates can also be labeled with a fluorescent compound.
  • the presence of a fluorescently labeled antibody is determined by exposing the immunoconjugate to light of the proper wavelength and detecting the resultant fluorescence.
  • Fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • anti-Zdsc4 immunoconjugates can be detectably labeled by coupling an antibody component to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged immunoconjugate is determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.
  • a bioluminescent compound can be used to label anti-Zdsc4 immunoconjugates of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.
  • anti-Zdsc4 immunoconjugates can be detectably labeled by linking an anti-Zdsc4 antibody component to an enzyme.
  • the enzyme moiety reacts with the substrate to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorometric or visual means.
  • enzymes that can be used to detectably label polyspecific immunoconjugates include ⁇ -galactosidase, glucose oxidase, peroxidase and alkaline phosphatase.
  • anti-Zdsc4 antibodies that have been conjugated with avidin, streptavidin, and biotin [see, for example, Wilchek et al. (eds.), "Avidin-Biotin "
  • kits for performing an immunological diagnostic assay for Zdsc4 gene expression comprise at least one container comprising an anti-Zdsc4 antibody, or antibody fragment.
  • a kit may also comprise a second container comprising one or more reagents capable of indicating the presence of Zdsc4 antibody or antibody fragments.
  • indicator reagents include detectable labels such as a radioactive label, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label, colloidal gold, and the like.
  • a kit may also comprise a means for conveying to the user that Zdsc4 antibodies or antibody fragments are used to detect Zdsc4 protein.
  • written instructions may state that the enclosed antibody or antibody fragment can be used to detect Zdsc4.
  • the written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert.
  • the dosage of administered Zdsc4 will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. Typically, it is desirable to provide the recipient with a dosage of Zdsc4 that is in the range of from about 1 pg/kg to 10 mg kg (amount of agent/body weight of patient), although a lower or higher dosage also may be administered as circumstances dictate.
  • Administration of a molecule having Zdsc4 activity to a subject can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection.
  • Zdsc4 can be administered as a controlled release formulation.
  • Additional routes of administration include oral, dermal, mucosal-membrane, pulmonary, and transcutaneous. Oral delivery is suitable for polyester microspheres, zeiii microspheres, proteinoid microspheres, polycyanoacrylate microspheres, and lipid- based systems [see, for example, DiBase and Morrel, "Oral Delivery of Microencapsulated Proteins," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)]!
  • a pharmaceutical composition comprising a protein, polypeptide, or peptide having Zdsc4 activity can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic proteins are combined in a mixture with a pharmaceutically acceptable carrier.
  • a composition is said to be a "pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient.
  • Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier.
  • Other suitable carriers are well known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
  • molecules having Zdsc4 activity and a pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective amount.
  • a combination of a protein, polypeptide, or peptide having Zdsc4 activity and a pharmaceutically acceptable carrier is said to be administered in * a
  • tumorapeutically effective amount if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
  • An inhibition of tumor growth may be indicated, for example, by a decrease in the number of tumor cells, decreased metastasis, a decrease in the size of a solid tumor, or increased necrosis of a tumor.
  • Indicators of viral infection inhibition include decreased viral titer, a decrease in detectable viral antigen, or an increase in anti- viral antibody titer.
  • a pharmaceutical composition comprising molecules having Zdsc4activity can be furnished in liquid form, in an aerosol, or in solid form.
  • Proteins having Zdsc4 activity such as human or murine Zdsc4
  • Liquid forms, including liposome-encapsulated formulations, are illustrated by injectable solutions and oral suspensions.
  • Exemplary solid forms include capsules, tablets, and controlled-release forms, such as a miniosmotic pump or an implant.
  • Zdsc4 pharmaceutical compositions may be supplied as a kit comprising a container that comprises Zdsc4, a Zdsc4 agonist, or an Zdsc4 antagonist (e.g., an anti-Zdsc4 antibody or antibody fragment).
  • Zdsc4 can be provided in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection.
  • a kit can include a dry- powder disperser, liquid aerosol generator, or nebulizer for administration of a therapeutic polypeptide.
  • Such a kit may further comprise written information on indications and usage of the pharmaceutical composition.
  • such information may include a statement that the Zdsc4 composition is contraindicated in patients with known hypersensitivity to Zdsc4.
  • Immunomodulator genes can be introduced into a subject to enhance immunological responses.
  • immunomodulator gene therapy has been examined in model systems using vectors that express IL-2, IL-3, JL-4, TL-6, JJL- .10, IL-12, JL-15, Zdsc4 ⁇ , tumor necrosis factor- ⁇ , or granulocyte-macrophage colony- stimulating factor (see, for example, Cao et al, J. Gastroenterol Hepatol. 11:1053 (1996), Tahara et al, Ann. N. Y. Acad. Sci. 795:275 (1996), Rakhmilevich ⁇ t al, Hum. Gene Ther.
  • a therapeutic expression vector can be provided that inhibits Zdsc4 gene expression, such as an anti-sense molecule, a ribozyme, or an external guide sequence molecule.
  • Zdsc4 gene expression such as an anti-sense molecule, a ribozyme, or an external guide sequence molecule.
  • murine Zdsc4 nucleotide sequences can be used for these methods, compositions comprising human Zdsc4 nucleotide sequences are preferred for treatment of human subjects.
  • an expression vector is constracted in which a nucleotide sequence encoding a Zdsc4 gene is operably linked to a core promoter, and optionally a regulatory element, to control gene transcription.
  • a core promoter and optionally a regulatory element
  • a Zdsc4gene can be delivered using recombinant viral vectors, including for example, adenoviral vectors [e.g., Kass-Eisler et ⁇ l, Proc. Nat 'I Acad. Sci. USA 90: 11498 (1993), Kolls et al, Proc. Nat'l Acad. Sci. USA 91 :215
  • adenoviral vectors e.g., Kass-Eisler et ⁇ l, Proc. Nat 'I Acad. Sci. USA 90: 11498 (1993), Kolls et al, Proc. Nat'l Acad. Sci. USA 91 :215
  • adenovirus a double-stranded DNA virus
  • the adenovirus system offers several advantages including: (i) the ability to accommodate relatively large DNA inserts, (ii) the ability to be grown to high-titer, (iii) the ability to infect a broad range of mammalian cell types, and (iv) the ability to be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters.
  • adenoviruses can be administered by intravenous injection, because the viruses are stable in the bloodstream.
  • adenovirus vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
  • 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.
  • adenovirus When intravenously administered to intact animals, adenovirus primarily targets the liver. Although an adenoviral delivery system with an El gene deletion cannot replicate in the host cells, the host's tissue will express and process an encoded heterologous protein. Host cells will also secrete the heterologous protein if the corresponding gene includes a secretory signal sequence. Secreted proteins will enter the circulation from tissue that expresses the heterologous gene (e.g., the highly vascularized liver).
  • adenoviral vectors containing various deletions of viral genes can be used to reduce or eliminate immune responses to the vector.
  • Such adenoviruses are El -deleted, and in addition, contain deletions of E2A or E4 [Lusky ⁇ t al, J. Virol 72:2022 (1998); Raper et al, Human Gene Therapy 9:671 (1998)].
  • the deletion of E2b has also been reported to reduce immune responses [Amalfitano et al, J. Virol 72:926 (1998)]. By deleting the entire adenovirus genome, very large inserts of heterologous DNA can be accommodated.
  • High titer stocks of recombinant viruses capable of expressing a therapeutic gene can be obtained from infected mammalian cells using standard methods.
  • recombinant HS V can be prepared in Vero cells, as described by Brandt et al, J. Gen. Virol 72:2043 (1991), Herald et ⁇ /., J. Gen. Virol. 75:1211 (1994), Visalli and Brandt, Virology 185:419 (1991), Grau et al, Invest. Ophthalmol Vis. Sci. 30:2474 (1989), Brandt etal, J. Virol. Meth. 36:209 (1992), and by Brown and MacLean (eds.), HSV Virus Protocols (Humana Press 1997).
  • an expression vector comprising a Zdsc4 gene can be introduced into a subject's cells by lipofection in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker [Feigner ⁇ t al, Proc. Nat'lAcad. Sci. USA 84:7413 (1987); Mackey et al, Proc. Nat'lAcad. Sci. USA 85:8027 (1988)].
  • the use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages.
  • Liposomes can be used to direct transfection to particular cell types, which is particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters
  • proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.
  • Electroporation is another alternative mode of administration.
  • Aihara and Miyazaki Nature Biotechnology 16:867 (1998), have demonstrated the use of in vivo electroporation for gene transfer into muscle.
  • a therapeutic gene may encode a Zdsc4 anti-sense RNA that inhibits the expression of Zdsc4.
  • Suitable sequences for anti-sense molecules can be derived from the nucleotide sequences of Zdsc4 ⁇ disclosed herein.
  • an expression vector can be constructed in which a regulatory element is operably linked to a nucleotide sequence that encodes a ribozyme.
  • 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).
  • ribozymes include nucleotide sequences that bind with Zdsc4 mRNA.
  • 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 an Zdsc4 gene.
  • an external guide sequence can be 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, Airman ⁇ t al, U.S. Patent No. 5,168,053, Yuan etal, Science 263:1269 (1994), Pace et al, international publication No. WO 96/18733, George et al, international publication No.
  • the external guide sequence comprises a ten to fifteen nucleotide sequence complementary ' to Zdsc4 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 dosage of a composition comprising a therapeutic vector having a Zdsc4 nucleotide acid sequence, such as a recombinant virus will vary depending upon such factors as the subject's age, weight, height, sex, general medical condition and previous medical history.
  • Suitable routes of administration of therapeutic vectors include intravenous injection, intraarterial injection, intraperitoneal injection, intramuscular injection, intratumoral injection, and injection into a cavity that contains a tumor.
  • a composition comprising viral vectors, non-viral vectors, or a combination of viral and non- viral vectors of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby vectors or viruses are combined in a mixture with a pharmaceutically acceptable carrier.
  • a composition such as phosphate-buffered saline is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient subject.
  • suitable carriers are well-known to those in the art [see, for example, Remington 's Pharmaceutical Sciences, 19th Ed. (Mack
  • a therapeutic gene expression vector, or a recombinant virus comprising such a vector, and a pharmaceutically acceptable carrier are administered to a subject in a therapeutically effective amount.
  • a combination of an expression vector (or virus) and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient subject.
  • the therapy is preferably somatic cell gene therapy.
  • the preferred treatment of a human with a therapeutic gene expression vector or a recombinant virus does not entail introducing into cells a nucleic acid molecule that can form part of a human germ line and be passed onto successive generations (i.e., human germ line gene therapy).
  • Transgenic mice can be engineered to over-express the human or murine Zdsc4gene in all tissues or under the control of a tissue-specific or tissue- preferred regulatory element. These over-producers of Zdsc4 can be used to characterize the phenotype that results from over-expression, and the transgenic animals can serve as models for human disease caused by excess Zdsc4. Transgenic mice that over-express Zdsc4 also provide model bioreactors for production of Zdsc4 in the milk or blood of larger animals.
  • a method for producing a transgenic mouse that expresses a Zdsc4 gene can begin with adult, fertile males (studs) [B6C3fl, 2-8 months of age (Taconic Farms, Germantown, NY)), vasectomized males (duds) (B6D2fl, 2-8 months, (Taconic Farms)], prepubescent fertile females (donors) [B6C3fl, 4-5 weeks, (Taconic Farms)] and adult fertile females (recipients) [B6D2f 1, 2-4 months, (Taconic
  • the donors are acclimated for one week and then injected with approximately 8 IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma Chemical Company; St. Louis, MO) IP., and 46-47 hours later, 8 IU/mouse of human Chorionic Gonadotropin [hCG (Sigma)] I.P. to induce superovulation.
  • Donors are mated with studs subsequent to hormone injections. Ovulation generally occurs within 13 hours of hCG injection. Copulation is confirmed by the presence of a vaginal plug the morning following mating.
  • Fertilized eggs are collected under a surgical scope.
  • the oviducts are collected and eggs are released into urinanalysis slides containing hyaluronidase (Sigma).
  • Eggs are washed once in hyaluronidase, and twice in Whitten's W640 medium [described, for example, by Menino and O'Claray, Biol. Reprod. 77:159 (1986), and Dienhart and Downs, Zygote 4:129 (1996)] that has been incubated with 5% CO 2 , 5% O 2 , and 90% N 2 at 37°C.
  • the eggs are then stored in a 37°C/5% CO 2 incubator until microinjection.
  • Ten to twenty micrograms of plasmid DNA containing a Zdsc4 encoding sequence is linearized, gel-purified, and resuspended in 10 mM Tris-HCl (pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of 5-10 nanograms per microliter for microinjection.
  • Plasmid DNA is microinjected into harvested eggs contained in a drop of W640 medium overlaid by warm, C ⁇ 2-equilibrated mineral oil.
  • the DNA is drawn into an injection needle (pulled from a 0.75mm ED, 1mm OD borosilicate glass capillary), and injected into individual eggs. Each egg is penetrated with the injection needle, into one or both of the haploid pronuclei.
  • Picoliters of DNA are injected into the pronuclei, and the injection needle withdrawn without coming into contact with the nucleoli. The procedure is repeated until all the eggs are injected. Successfully microinjected eggs are transferred into an organ tissue-culture dish with pre-gassed W640 medium for storage overnight in a 37°C/5% CO incubator.
  • two-cell embryos are transferred into pseudopregnant recipients.
  • the recipients are identified by the presence of copulation plugs, after copulating with vasectomized duds.
  • Recipients are anesthetized and shaved on the dorsal left side and transferred to a surgical microscope.
  • a small incision is made in the skin and through the muscle wall in the middle of the abdominal area outlined by the ribcage, the saddle, and the hind leg, midway between knee and spleen.
  • the reproductive organs are exteriorized onto a small surgical drape.
  • the fat pad is stretched out over the surgical drape, and a baby serrefine (Roboz,
  • the recipients are returned to cages in pairs, and allowed 19-21 days gestation. After birth, 19-21 days postpartum is allowed before weaning.
  • the weanlings are sexed and placed into separate sex cages, and a 0.5 cm biopsy (used for genotyping) is snipped off the tail with clean scissors.
  • Genomic DNA is prepared from the tail snips using, for example, a QIAGEN DNEASY kit following the manufacturer's instructions. Genomic DNA is analyzed by PCR using primers designed to amplify a Zdsc4 gene or a selectable marker gene that was introduced in the same plasmid. After animals are confirmed to be transgenic, they are backcrossed into an inbred strain by placing a transgenic female with a wild-type male, or a transgenic male with one or two wild-type female(s). As pups are born and weaned, the sexes are separated, and their tails snipped for genotyping. To check for expression of a transgene in a live animal, a partial hepatectomy is performed.
  • a surgical prep is made of the upper abdomen directly below the zyphoid process. Using sterile technique, a small 1.5-2 cm incision is made below the sternum and the left lateral lobe of the liver exteriorized. Using 4-0 silk, a tie is made around the lower lobe securing it outside the body cavity. An atraumatic clamp is used to hold the tie while a second loop of absorbable Dexon (American Cyanamid; Wayne, NJ.) is placed proximal to the first tie. A distal cut is made from the Dexon tie and approximately 100 mg of the excised liver tissue is placed in a sterile petri dish. The excised liver section is transferred to a 14 ml polypropylene round bottom tube and snap frozen in liquid nitrogen and then stored on dry ice. The surgical site is closed with suture and wound clips, and the animal's cage placed on a
  • Zdsc4 mRNA is examined for each transgenic mouse using an RNA solution hybridization assay or polymerase chain reaction.
  • Zdsc4 gene expression can be inhibited using anti- sense genes, ribozyme genes, or external guide sequence genes.
  • transgenic mice that under-express the Zdsc4 gene such inhibitory sequences are targeted to murine Zdsc4 mRNA.
  • Methods for producing transgenic mice that have abnormally low expression of a particular gene are known to those in the art [see, for example, Wu et al, "Gene Underexpression in Cultured Cells and Animals by Antisense DNA and RNA Strategies," in Methods in Gene Biotechnology, pages 205- 224 (CRC Press 1997)].
  • An alternative approach to producing transgenic mice that have little or no Zdsc4 gene expression is to generate mice having at least one normal Zdsc4 allele replaced by a nonfunctional Zdsc4 gene.
  • One method of designing a nonfunctional Zdsc4 gene is to insert another gene, such as a selectable marker gene, within a nucleic acid molecule that encodes murine Zdsc4.
  • Standard methods for producing these so- called “knockout mice” are known to those skilled in the art [see, for example, Jacob, "Expression and Knockout of Interferons in Transgenic Mice,” in Overexpression and Knockout of Cytokines in Transgenic Mice, Jacob (ed.), pages 111-124 (Academic Press, Ltd. 1994), and Wu ⁇ t al, "New Strategies for Gene Knockout,” in Methods in Gene Biotechnology, pages 339-365 (CRC Press 1997)].
  • 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
  • Zdsc4 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 Zdsc4 is the gene to be expressed; for determining the restriction endonuclease cleavage sites of the polynucleotides; determining mRNA and DNA localization of Zdsc4 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.
  • Zdsc4 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 Zdsc4 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.
  • native and tagged protein i.e., receptor binding, signal transduction, proliferation, and differentiation
  • Zdsc4 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.
  • 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.
  • a kit containing the Zdsc4 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 Zdsc4 would be unique unto itself.
  • the antibodies which bind specifically to Zdsc4 can be used as a teaching aid to instruct students how to prepare affinity chromatography columns to purify Zdsc4, cloning and sequencing the polynucleotide that encodes an ' antibody and thus as a practicum for teaching a student how to design humanized antibodies.
  • the Zdsc4 gene, polypeptide or antibody would then be packaged by reagent companies and sold to universities so that the students gain skill in art of molecular biology. Because each gene and protein is unique, each gene and protein creates unique challenges and learning experiences for students in a lab practicum.
  • Such educational kits containing the Zdsc4 gene, polypeptide or antibody are considered within the scope of the present invention.

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Abstract

L'invention concerne des polynucléotides et de polypeptides de protéine-4 à noyau disulfure mammifère (Zdsc4) codant pour les polypeptides, des anticorps se liant spécifiquement aux polypeptides, des vecteurs d'expression comprenant ces polynucléotides, et des cellules hôtes transformées avec les vecteurs d'expression.
PCT/US2001/011506 2000-04-10 2001-04-09 Proteine 4 a noyau disulfure mammifere WO2001077152A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001251471A AU2001251471A1 (en) 2000-04-10 2001-04-09 Mammalian disulfide core protein-4

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US54644600A 2000-04-10 2000-04-10
US09/546,446 2000-04-10

Publications (2)

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WO2001077152A2 true WO2001077152A2 (fr) 2001-10-18
WO2001077152A3 WO2001077152A3 (fr) 2002-01-24

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PCT/US2001/011506 WO2001077152A2 (fr) 2000-04-10 2001-04-09 Proteine 4 a noyau disulfure mammifere

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AU (1) AU2001251471A1 (fr)
WO (1) WO2001077152A2 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999006549A2 (fr) * 1997-08-01 1999-02-11 Genset Est 5' pour proteines exprimees dans les testicules et autres tissus
WO1999031236A2 (fr) * 1997-12-17 1999-06-24 Genset ADNc PROLONGES POUR PROTEINES SECRETEES

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999006549A2 (fr) * 1997-08-01 1999-02-11 Genset Est 5' pour proteines exprimees dans les testicules et autres tissus
WO1999031236A2 (fr) * 1997-12-17 1999-06-24 Genset ADNc PROLONGES POUR PROTEINES SECRETEES

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [Online] ACCESSION NO: AI673291, 19 May 1999 (1999-05-19) "tw69g07.x1 NCI_CGAP_Ut3 Homo sapiens cDNA clone IMAGE:2264988 3', mRNA sequence." XP002179484 *
DATABASE EMBL [Online] ACCESSION NO: AL031671, 22 September 1998 (1998-09-22) SKUCE C.;: " Human DNA sequence from clone RP4-688G8 on chromosome 20q11.2-12. Contains the gene for a novel protein similar to ribosomal protein S2 (RPS2), a gene encoding a protein similar to basic protease inhibitor chelonianin, a novel gene, the 3' end of a novel gene, ESTs, STSs, GSSs and a CpG island." XP002179485 *

Also Published As

Publication number Publication date
AU2001251471A1 (en) 2001-10-23
WO2001077152A3 (fr) 2002-01-24

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