EP1141287A1 - Pancreatic polypeptide zsig66 - Google Patents

Pancreatic polypeptide zsig66

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Publication number
EP1141287A1
EP1141287A1 EP99966234A EP99966234A EP1141287A1 EP 1141287 A1 EP1141287 A1 EP 1141287A1 EP 99966234 A EP99966234 A EP 99966234A EP 99966234 A EP99966234 A EP 99966234A EP 1141287 A1 EP1141287 A1 EP 1141287A1
Authority
EP
European Patent Office
Prior art keywords
amino acid
seq
zsigόό
polypeptide
acid number
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP99966234A
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German (de)
French (fr)
Inventor
Paul O. Sheppard
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Zymogenetics Inc
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Zymogenetics Inc
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Filing date
Publication date
Application filed by Zymogenetics Inc filed Critical Zymogenetics Inc
Publication of EP1141287A1 publication Critical patent/EP1141287A1/en
Withdrawn legal-status Critical Current

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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/12—Drugs for genital or sexual disorders; Contraceptives for climacteric disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • hormones and polypeptide growth factors are controlled by hormones and polypeptide growth factors. These diffusible molecules allow cells to communicate with each other and act in concert to regulate cells and form organs, and to repair and regenerate damaged tissue.
  • hormones and growth factors include the steroid hormones (e.g. estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, the interleukins, platelet derived growth factor (PDGF), epidermal growth factor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin.
  • the present invention provides an isolated polynucleotide encoding a zsig ⁇ polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID NO:2, wherein the amino acid percent identity is determined using
  • the isolated polynucleotide disclosed above is selected from the group consisting of: (a) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 431 to nucleotide 472; (b) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 425 to nucleotide 472; (c) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 425 to nucleotide 475; (d) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 305 to nucleotide 475; (e) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 224 to nucleotide 475; and (f) a polynucleotide sequence complementary to (a) or (b).
  • the isolated polynucleotide disclosed above comprises nucleotide 1 to nucleotide 252 of SEQ ID NO:l l .
  • the isolated polynucleotide disclosed above comprises a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ
  • the present invention provides a cultured cell into which has been introduced an expression vector as disclosed above, wherein the cell expresses a polypeptide encoded by the DNA segment.
  • the present invention provides a DNA construct encoding a fusion protein, the DNA construct comprising: a first DNA segment encoding a polypeptide comprising a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO: 2 from residue number 28 (Ala) to amino acid number 84 (Gly); and at least one other DNA segment encoding an additional polypeptide, wherein the first and other DNA segments are connected in-frame; and encode the fusion protein.
  • the present invention provides a fusion protein produced by a method comprising: culturing a host cell into which has been introduced a vector comprising the following operably linked elements: (a) a transcriptional promoter; (b) a DNA construct encoding a fusion protein as disclosed above; and (c) a transcriptional terminator; and recovering the protein encoded by the DNA segment.
  • the present invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number
  • amino acid number 84 Gly
  • the isolated polypeptide disclosed above further comprises motifs 1 through 4 spaced apart from N-terminus to C-terminus in a configuration Ala 28 - ⁇ 7 ⁇ - Ml- ⁇ 3 ⁇ -M2- ⁇ 5 ⁇ -M3- ⁇ 6 ⁇ -M4- ⁇ 10 ⁇ - Gly 84 , wherein Ml is "motif 1,” a sequence of amino acids as shown in amino acids 36 to 41 of SEQ ID NO:2, M2 is “motif 2,” a sequence of amino acids as shown in amino acids 45 to 50 of SEQ ID NO:2, M3 is “motif 3,” a sequence of amino acids as shown in amino acids 56 to 61 of SEQ ID NO:2, M4 is "motif 4," a sequence of amino acids as shown in amino acids 68 to 73 of SEQ ID NO:2, and Ala 28 is the Alanine residue at amino acid number 28 in SEQ ID NO:2, Gly g4 is the Glycine residue at amino acid number 84 in SEQ ID NO:2, and ⁇ # ⁇ denotes the number of amino amino acids
  • the isolated polypeptide disclosed above comprises a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID NO:2.
  • the isolated polypeptide disclosed above further comprises an amidated C-terminal Serine residue.
  • the present invention provides a method of producing a zsig ⁇ polypeptide comprising: culturing a cell as disclosed above; and isolating the zsig ⁇ polypeptide produced by the cell.
  • the present invention provides a method of detecting, in a test sample, the presence of a modulator of zsig ⁇ protein activity, comprising: transfecting a zsig ⁇ -responsive cell, with a reporter gene construct that is responsive to a zsig ⁇ -stimulated cellular pathway; and producing a zsig ⁇ polypeptide by the method as disclosed above; and adding the zsig ⁇ polypeptide to the cell, in the presence and absence of a test sample; and comparing levels of response to the zsig ⁇ polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and determining from the comparison, the presence of the modulator of zsig ⁇ activity in the test sample.
  • the present invention provides a method of producing an antibody to zsig ⁇ polypeptide comprising the following steps in order: inoculating an animal with a polypeptide selected from the group consisting of: (a) a polypeptide consisting of 9 to 57 amino acids, wherein the polypeptide has a contiguous sequence of amino acids as shown within SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); (b) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 from residue number 28 (Ala) to amino acid number 84 (Gly); (c) a polypeptide as disclosed above; (d) a polypeptide consisting of amino acid number 1 (Met) to amino acid number 6 (Glu) of SEQ ID NO:2; (e) a polypeptide consisting of amino acid number 7 (Val) to amino acid number 12 (lie) of SEQ ID NO:2; (f) a polypeptide consisting of amino acid number 26 (Ser) to amino acid
  • the present invention provides an antibody produced by the method as disclosed above, which binds to a zsig ⁇ polypeptide.
  • the antibody disclosed above is a monoclonal antibody.
  • the present invention provides an antibody which specifically binds to a polypeptide as disclosed above.
  • the present invention provides a method for detecting a genetic abnormality in a patient, comprising: obtaining a genetic sample from a patient; producing a first reaction product by incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO:l or the complement of SEQ ID NO:l, under conditions wherein said polynucleotide will hybridize to complementary polynucleotide sequence; visualizing the first reaction product; and comparing said first reaction product to a control reaction product from a wild type patient, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an isolated polypeptide as disclosed above, wherein the polypeptide is in combination with a pharmaceutically acceptable vehicle.
  • the figure is a hydrophobicity plot of zsig ⁇ , determined from a Hopp/Woods hydrophilicity profile based on a sliding six-residue window, with buried G, S, and T residues and exposed H, Y, and W residues ignored.
  • 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 tag any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.
  • Affinity tags include a poly- histidine tract, protein A (Nilsson et al., EMBO J. 4: 1075, 1985; Nilsson et al., Methods Enzymol.
  • 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.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • amino-terminal and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • 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 ⁇ 10 ⁇ M ⁇ l .
  • polynucleotide molecule denotes a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • contig denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5'-ATGGAGCTT-3' are 5'- AGCTTgagt-3' and 3'-tcgacTACC-5'.
  • degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and
  • expression vector is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
  • Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985).
  • An "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • isolated does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • operably linked when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • 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.
  • polynucleotide is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.
  • bp base pairs
  • nt nt
  • kb kilobases
  • promoter is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
  • a "protein” is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • receptor denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell.
  • a bioactive molecule i.e., a ligand
  • Membrane-bound receptors are characterized by a multi-peptide structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell.
  • Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.
  • 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 growth hormone receptor
  • GM-CSF receptor growth hormone receptor
  • G-CSF receptor erythropoietin receptor
  • IL-6 receptor growth hormone receptor
  • secretory signal sequence denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • splice variant is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
  • the present invention is based in part upon the discovery of a novel
  • polypeptide DNA sequence that encodes a novel polypeptide.
  • the polypeptide and corresponding polynucleotide were isolated from a human pituitary library.
  • the polypeptide has been designated zsig ⁇ .
  • novel zsig ⁇ polypeptides of the present invention were initially identified by querying an EST database for proteins homologous to proteins having a secretory signal sequence. These proteins are characterized by an upstream methionine start site and a hydrophobic region of approximately 13 amino acids, followed by a peptide signal peptidase cleavage site. An EST database was queried for novel DNA sequences whose translations would meet these search criteria. An EST was found and its corresponding cDNA was sequenced. The zsig ⁇ nucleotide sequence is believed to encode the entire coding sequence of the predicted protein.
  • Zsig ⁇ may be a novel pituitary hormone, pituitary hormone activating hormone, cell-cell signaling molecule, growth factor, cytokine, nerve cell modulating protein, secreted extracellular matrix associated protein with growth factor hormone activity, or the like, and is a member a novel protein family.
  • the sequence of the zsig ⁇ polypeptide was obtained from a single clone believed to contain its corresponding polynucleotide sequence.
  • the clone was obtained from a pituitary library.
  • Other libraries that might also be searched for such sequences include fetal libraries, pancreas, liver, ovary, placenta, and the like.
  • the nucleotide sequence of a representative zsig ⁇ -encoding DNA is described in SEQ ID NO:l
  • its deduced 84 amino acid sequence is described in SEQ ID NO:2.
  • the zsig ⁇ polypeptide represents a full- length polypeptide segment (residue 1 (Met) to residue 84 (Gly) of SEQ ID NO:2).
  • the domains and structural features of zsig ⁇ are further described below.
  • SEQ ID NO:2 Analysis of the zsig ⁇ polypeptide encoded by the DNA sequence of SEQ ID NO:l revealed an open reading frame encoding 84 amino acids (SEQ ID NO:2) comprising a predicted signal peptide of 27 amino acid residues (residue 1 (Met) to residue 27 (Ser) of SEQ ID NO:2), and a mature polypeptide of 57 amino acids (residue 28 (Ala) to residue 84 (Gly) of SEQ ID NO:2). Further, post-translational processing may truncate zsig ⁇ , for example at a cysteine bond, and still provide a short active polypeptide.
  • the Serine residue of the short active polypeptide amino acid residues 69 (Phe) to 83 (Ser) of SEQ ID NO:2 or amino acid residues 70 (Cys) to 83 (Ser) of SEQ ID NO:2 can further be amidated at the C- terminal Serine residue.
  • zsig ⁇ can have cardiovascular or metabolic vasodilatation capability.
  • vasopressin and oxytocin see Rehbein, M. et al., Biol. Chem. 367:695-704, 1986; Sausville, E. et al., J. Biol. Chem. 260:10236-10241, 1985; Ivell, R. et al., Endocrinol. 127:2990-2996, 1990; Mohr, E. et al., FEBS Lett.
  • the zsig ⁇ polypeptide contains several regions of low variance or low degeneracy (see, Sheppard, P. et al., Gene 150:163-167, 1994). These regions that exhibit low degeneracy are described in the following zsig ⁇ motifs:
  • Motif 4" corresponding to amino acids 68 (Lys) to 73 (Asn) of SEQ ID NO:2; and Motifs 1 through 4 are spaced apart from N-terminus to C-terminus in a configuration represented by the following:
  • Ala 28 is the starting residue of the mature polypeptide (as shown in SEQ ID NO:2)
  • Gly 84 is the ending residue of the mature polypeptide(as shown in SEQ ID NO:2)
  • M# denotes the specific motif disclosed above (e.g., Ml is motif 1, etc.), and
  • ⁇ # ⁇ denotes the approximate number of amino acid residues between the motifs, up to plus or minus 2 residues.
  • conserved and low variance motifs generally correlates with or defines important structural regions in proteins. Regions of low variance (e.g., hydrophobic clusters) are generally present in regions of structural importance (Sheppard, P. et a .Gene 150:163-167, 1994). Such regions of low variance often contain rare or infrequent amino acids, such as Tryptophan. The regions flanking and between such conserved and low variance motifs may be more variable, but are often functionally significant because they may relate to or define important structures and activities such as binding domains, biological and enzymatic activity, signal transduction, tissue localization domains and the like.
  • Cysteine residues that form a disulfide bond located in SEQ ID NO:2 at amino acid number 70 and 74 that can be important for structure or activity of zsig ⁇ polypeptide.
  • PKC protein kinase C
  • CK2 casein kinase II
  • genomic structure of zsig ⁇ is readily determined by one of skill in the art by comparing the cDNA sequence of SEQ ID NO: 1 and the translated amino acid of SEQ ID NO:2 with the genomic DNA in which the gene is contained (Genbank Accession No. ACO 15783). For example, such analysis can be readily done using FASTA as described herein. As such, the intron and exon junctions in this region of genomic DNA can be determined for the zsig ⁇ gene.
  • the region of human genomic DNA where zsig ⁇ is located is shown in SEQ ID NO:24. The entire coding sequence appears to be contained in a single exon, as there are no putative introns within SEQ ID NO:24.
  • the present invention includes the zsig ⁇ gene as located in human genomic DNA.
  • the low degeneracy amino acids in zsig ⁇ can be used as a tool to identify new family members.
  • reverse transcription-polymerase chain reaction RT-PCR
  • RT-PCR reverse transcription-polymerase chain reaction
  • highly degenerate primers designed from the zsig ⁇ sequences are useful for this purpose.
  • degenerate oligonucleotide primers designed from the following zsig ⁇ amino acid sequences of motifs 1 through 5 are useful for this purpose: a) WIQGMG (motif 1; SEQ ID NO:3), corresponding to degenerate polynucleotides of SEQ ID NO:7 and their complement; b) TDFNPF (motif 2; SEQ ID NO:4), corresponding to degenerate polynucleotides of SEQ ID NO:8 and their complement; c) KFCVVN (motif 3; SEQ ID NO:5), corresponding to degenerate polynucleotides of SEQ ID NO:9 and their complement; d) EIELFV (motif 4; SEQ ID NO:6), corresponding to degenerate polynucleotides of SEQ ID NO: 10 and their complement.
  • the present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the zsig ⁇ polypeptides disclosed herein.
  • polynucleotide molecules including DNA and RNA molecules, that encode the zsig ⁇ polypeptides disclosed herein.
  • SEQ ID NO:l 1 is a degenerate DNA sequence that encompasses all DNAs that encode the zsig ⁇ polypeptide of SEQ ID NO:2.
  • the degenerate sequence of SEQ ID NO: 11 also provides all RNA sequences encoding SEQ ID NO:2 by substituting U for T.
  • zsig ⁇ polypeptide- encoding polynucleotides comprising nucleotide 1 to nucleotide 252 of SEQ ID NO:l 1 and their RNA equivalents are contemplated by the present invention.
  • Table 1 sets forth the one-letter codes used within SEQ ID NO: 11 to denote degenerate nucleotide positions. "Resolutions” are the nucleotides denoted by a code letter. "Complement” indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.
  • degenerate codons used in SEQ ID NO: 11, encompassing all possible codons for a given amino acid, are set forth in Table 2.
  • any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid.
  • the degenerate codon for serine can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY).
  • WSN can, in some circumstances, encode arginine
  • MGN degenerate codon for arginine
  • AGY serine
  • some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO:2. Variant sequences can be readily tested for functionality as described herein.
  • 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 mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art.
  • the degenerate codon sequence disclosed in SEQ ID NO:l 1 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
  • the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:l, 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 defmed 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.
  • Suitable stringent hybridization conditions are equivalent to about a 5 h to overnight incubation at about 42°C in a solution comprising: about 40-50% formamide, up to about 5X SSC, about 5X Denhardt's solution, up to about 10% dextran sulfate, and about 10-20 ⁇ g/ml denatured commercially-available carrier DNA; hybridization is then followed by washing filters in up to about 2X SSC.
  • a suitable wash stringency is equivalent to 0.1X SSC to 2X SSC, 0.1% SDS, at 55°C to 65 °C.
  • Stringent hybridization and wash conditions depend on the length of the probe, reflected in the Tm, hybridization and wash solutions used, and are routinely determined empirically by one of skill in the art.
  • the isolated polynucleotides of the present invention include DNA and RNA.
  • Methods for preparing DNA and RNA are well known in the art.
  • RNA is isolated from a tissue or cell that produces large amounts of zsig ⁇ RNA.
  • Such tissues and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include pituitary, pancreas, tissues of endocrine origin, and the like.
  • Total RNA can be prepared using guanidinium isothiocyanate extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al, Biochemistry 18:52-94, 1979).
  • Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).
  • Complementary DNA cDNA is prepared from poly(A)+ RNA using known methods.
  • genomic DNA can be isolated. Polynucleotides encoding zsig ⁇ polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • a full-length clone encoding zsig ⁇ can be obtained by conventional cloning procedures.
  • Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron.
  • Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library.
  • Expression libraries can be probed with antibodies to zsig ⁇ , receptor fragments, or other specific binding partners.
  • the polynucleotides of the present invention can also be synthesized using DNA synthesis machines.
  • the method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately.
  • the production of short polynucleotides 60 to 80 bp is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them.
  • special strategies are usually employed, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%.
  • synthetic genes double-stranded
  • synthetic genes 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. Each internal section of the gene has complementary 3' and 5' terminal extensions designed to base pair precisely with an adjacent section. Thus, after the gene is assembled, process is completed by sealing the nicks along the backbones of the two strands with T4 DNA ligase.
  • synthetic genes can be designed with terminal sequences that facilitate insertion into a restriction endonuclease site of a cloning vector.
  • An alternative way to prepare a full- length gene is to synthesize a specified set of overlapping oligonucleotides (40 to 100 nucleotides).
  • Zsig ⁇ polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a zsig ⁇ gene.
  • this gene region is expected to provide for ovary and pancreas-specific expression.
  • Promoter elements from a zsig ⁇ gene could thus be used to direct the tissue-specific expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy. Cloning of 5' flanking sequences also facilitates production of zsig ⁇ proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670.
  • an endogenous zsig ⁇ gene in a cell is altered by introducing into the zsig ⁇ locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site.
  • the targeting sequence is a zsig ⁇ 5' non- coding sequence that permits homologous recombination of the construct with the endogenous zsig ⁇ locus, whereby the sequences within the construct become operably linked with the endogenous zsig ⁇ coding sequence.
  • an endogenous zsig ⁇ promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.
  • the present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are zsig ⁇ polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human zsig ⁇ 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 zsig ⁇ 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 zsig ⁇ -encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction (PCR) (Mullis, U.S. Patent No.
  • PCR polymerase chain reaction
  • 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 zsig ⁇ polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
  • SEQ ID NO:l represents a single allele of human zsig ⁇ and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO:l, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO:2.
  • cDNAs generated from alternatively spliced mRNAs, which retain the properties of the zsig ⁇ polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
  • Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.
  • the present invention also provides isolated zsig ⁇ polypeptides that are substantially similar to the polypeptides of SEQ ID NO:2 and their orthologs.
  • substantially similar is used herein to denote polypeptides having 70%, preferably 75%, more preferably at least 80%, sequence identity to the sequences shown in SEQ ID NO:2 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO:2 or its orthologs.) Percent sequence identity is determined by conventional methods. See, for example,
  • the "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant zsig ⁇ .
  • the FASTA algorithm is described by Pearson and Lipman, Proc. NatT 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 rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score.
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions.
  • 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 default.
  • the present invention includes nucleic acid molecules that encode a polypeptide having one or more conservative amino acid changes, compared with the amino acid sequence of SEQ ID NO:2.
  • the BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. NatT Acad. 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.
  • the language "conservative amino acid substitution” refers to a substitution represented by a BLOSUM62 value of greater than -1.
  • an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
  • Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • Variant zsig ⁇ polypeptides or substantially homologous zsig ⁇ polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
  • the present invention thus includes polypeptides of from about 35 to about 90 amino acid residues, or in some embodiments, 15 to 16 amino acid residues (e.g., residues 69-83 of SEQ ID NO:2 with or without an amide group), that comprise a sequence that is at least 80%, preferably at least 90%, and more preferably 95% or more identical to the corresponding region of SEQ ID NO:2.
  • Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zsig ⁇ polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.
  • Aromatic phenylalanine tryptophan tyrosine Small : glycine alanine serine threonine methionine
  • the present invention further provides a variety of other polypeptide fusions and related multimeric proteins comprising one or more polypeptide fusions.
  • a zsig ⁇ polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584.
  • Preferred dimerizing proteins in this regard include immunoglobulin constant region domains.
  • Immunoglobulin-zsig ⁇ polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric zsig ⁇ analogs.
  • Auxiliary domains can be fused to zsig ⁇ polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen).
  • a zsig ⁇ polypeptide or protein could be targeted to a predetermined cell type by fusing a zsig ⁇ polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell.
  • polypeptides and proteins can be targeted for therapeutic or diagnostic purposes.
  • a zsig ⁇ polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain.
  • Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34:1-9, 1996.
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, tr ⁇ y-3-methylproline, 2,4-methanoproline, cw-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, ⁇ t7 ⁇ -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.
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
  • the non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for zsig ⁇ amino acid residues.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al, Proc. Natl. Acad. Sci. USA 88:4498-502, 1991).
  • site-directed mutagenesis or alanine-scanning mutagenesis Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al, Proc. Natl. Acad. Sci. USA 88:4498-502, 1991.
  • single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699-708, 1996.
  • Sites of ligand-receptor or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al, J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related polypeptides.
  • Determination of amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can determine specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule.
  • Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity and computer analysis using available software (e.g., the Insight II® viewer and homology modeling tools; MSI, San Diego, CA), secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al., Current Opin. Struct. Biol. 6:3-10, 1996).
  • available software e.g., the Insight II® viewer and homology modeling tools; MSI, San Diego, CA
  • secondary structure propensities e.g., binary patterns, complementary packing and buried polar interactions
  • Amino acid sequence changes are made in zsig ⁇ polypeptides so as to minimize disruption of higher order structure essential to biological activity.
  • changes in amino acid residues will be made so as not to disrupt the helix geometry and other components of the molecule where changes in conformation abate some critical function, for example, binding of the molecule to its binding partners.
  • the effects of amino acid sequence changes can be predicted by, for example, computer modeling as disclosed above or determined by analysis of crystal structure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995).
  • CD circular dichrosism
  • NMR nuclear magnetic resonance
  • digestive peptide mapping and epitope mapping are also known methods for analyzing folding and structural similarities between proteins and polypeptides (Schaanan et al conflict Science 257:961-964. 1992).
  • a Hopp/Woods hydrophilicity profile of the zsig ⁇ protein sequence as shown in SEQ ID NO:2 can be generated (Hopp et al., Proc. Natl. Acad. Sci.78:3824- 3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquier et al., Protein Engineering I L 153- 169, 1998).
  • the profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored (See, Figure).
  • hydrophilic regions include: (1) amino acid number 1 (Met) to amino acid number 6 (Glu) of SEQ ID NO:2; (2) amino acid number 7 (Val) to amino acid number 12 (He) of SEQ ID NO:2; (3) amino acid number 26 (Ser) to amino acid number 32 (Ser) of SEQ ID NO:2; (4) amino acid number 29 (Asp) to amino acid number 34 (Ser) of SEQ ID NO:2; and (5) amino acid number 51 (Leu) to amino acid number 56 (Glu) of SEQ ID NO:2.
  • hydrophilicity or hydrophobicity will be taken into account when designing modifications in the amino acid sequence of a zsig ⁇ polypeptide, so as not to disrupt the overall structural and biological profile.
  • hydrophobic residues selected from the group consisting of Val, Leu and He or the group consisting of Met, Gly, Ser, Ala, Tyr and Trp.
  • hydrophobic residues tolerant of substitution could include such residues as shown in SEQ ID NO: 2. Cysteine residues are relatively intolerant of substitution.
  • the identities of essential amino acids can also be inferred from analysis of sequence similarity between other proteins with zsig ⁇ . Using methods such as "FASTA" analysis described previously, regions of high similarity are identified within a family of proteins and used to analyze amino acid sequence for conserved regions.
  • An alternative approach to identifying a variant zsig ⁇ polynucleotide on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant zsig ⁇ polynucleotide can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, as discussed above.
  • the present invention also includes functional fragments of zsig ⁇ polypeptides and nucleic acid molecules encoding such functional fragments.
  • a "functional" zsig ⁇ or fragment thereof defined herein is characterized by its proliferative or differentiating activity, by its ability to induce or inhibit specialized cell functions, or by its ability to bind specifically to an anti-zsig ⁇ antibody or zsig ⁇ receptor (either soluble or immobilized).
  • the present invention further provides fusion proteins encompassing: (a) polypeptide molecules comprising one or more of the motifs described above; and (b) functional fragments comprising one or more of these motifs.
  • the other polypeptide portion of the fusion protein may be contributed by a related, non-native and/or an unrelated secretory signal peptide that facilitates secretion of the fusion protein.
  • Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a zsig ⁇ polypeptide.
  • DNA molecules having the nucleotide sequence of SEQ ID NO: 1 or fragments thereof can be digested with Bal 1 nuclease to obtain a series of nested deletions. These DNA fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for zsig ⁇ activity, or for the ability to bind anti-zsig ⁇ antibodies or zsig ⁇ receptor.
  • exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired zsig ⁇ fragment.
  • particular fragments of a zsig ⁇ polynucleotide can be synthesized using the polymerase chain reaction.
  • Standard methods for identifying functional domains are well-known to those of skill in the art. For example, studies on the truncation at either or both termini of interferons have been summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507 (1995).
  • standard techniques for functional analysis of proteins are described by, for example, Treuter et al., Molec. Gen. Genet.
  • phage display e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204
  • region- directed mutagenesis e.g., region- directed mutagenesis
  • variants of the disclosed zsig ⁇ DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994 and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
  • Mutagenesis methods as disclosed herein can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
  • Mutagenized DNA molecules that encode active polypeptides e.g., secreted and detected by antibodies; or measured by a signal transduction type assay
  • 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.
  • proteins of the present invention can be joined to other bioactive molecules, particularly other cytokines, to provide multi-functional molecules.
  • bioactive molecules particularly other cytokines
  • one or more domains, hydrophilic regions, or regions containing motifs 1-4 from zsig ⁇ can be joined to other proteins to enhance their biological properties or efficiency of production.
  • the present invention thus provides a series of novel, hybrid molecules in which a segment comprising one or more of the domains, hydrophilic regions, or regions containing motifs 1-4 of zsig ⁇ is fused to another polypeptide. Fusion is preferably done by splicing at the DNA level to allow expression of chimeric molecules in recombinant production systems. The resultant molecules are then assayed for such properties as improved solubility, improved stability, prolonged clearance half-life, improved expression and secretion levels, and pharmacodynamics.
  • Such hybrid molecules may further comprise additional amino acid residues (e.g. a polypeptide linker) between the component proteins or polypeptides.
  • polypeptides that are substantially similar to SEQ ID NO:2 or allelic variants thereof and retain the properties of the wild-type protein. For example, using the methods described above, one could identify a receptor binding domain on zsig ⁇ ; an extracellular ligand-binding domain of a receptor for zsig ⁇ ; heterodimeric and homodimeric binding domains; other functional or structural domains; affinity tags; or other domains important for protein-protein interactions or signal transduction.
  • polypeptides may also include additional polypeptide segments as generally disclosed above.
  • zsig ⁇ polypeptide including variants and fusion proteins
  • the zsig ⁇ polypeptides of the present invention can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred.
  • a DNA sequence encoding a zsig ⁇ polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
  • the secretory signal sequence may be that of zsig ⁇ , or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo.
  • the secretory signal sequence is operably linked to the zsig ⁇ DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al, U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).
  • the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway.
  • the present invention provides for such fusion polypeptides.
  • a signal fusion polypeptide can be made wherein a secretory signal sequence derived from residue 1 (Met) to residue 27 (Ser) of SEQ ID 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 fusions may be used in vivo or in vitro to direct peptides through the secretory pathway.
  • Cultured mammalian cells are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981 : Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al, EMBO J.
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-Kl; ATCC No. CCL 61) cell lines.
  • COS-1 ATCC No. CRL 1650
  • COS-7 ATCC No. CRL 1651
  • BHK ATCC No. CRL 1632
  • BHK 570 ATCC No. CRL 10314
  • 293 ATCC No. CRL 1573
  • Graham et al. J. Gen. Virol. 36:59-72, 1977
  • Chinese hamster ovary e.g. CHO-Kl; ATCC No. CCL 61
  • Suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, VA.
  • strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288.
  • Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants".
  • stable transfectants Cells that have been cultured, for example in the presence of the selective agent, and are able to pass the gene of interest to their progeny are referred to as "stable transfectants.”
  • a preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • alternative markers that introduce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
  • eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells.
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) H:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa calif ornica nuclear polyhedrosis virus (AcNPV). See, King, L.A. and Possee, R.D., The Baculovirus Expression
  • the second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, V.A, et al., J Virol 67:4566-79, 1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD).
  • This system utilizes a transfer vector, pFastBaclTM (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zsig ⁇ polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.”
  • the pFastBaclTM transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case zsig ⁇ .
  • pFastBaclTM 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, p ⁇ .9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins.
  • the baculovirus basic protein promoter also known as Pcor, p ⁇ .9 or MP promoter
  • Pcor baculovirus basic protein promoter
  • MP promoter baculovirus basic protein promoter
  • transfer vectors can be constructed which replace the native zsig ⁇ secretory signal sequences with secretory signal sequences derived from insect proteins.
  • a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen, Carlsbad, CA), or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native zsig ⁇ secretory signal sequence.
  • transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed zsig ⁇ polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985).
  • a transfer vector containing zsig ⁇ is transformed into E. coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus that expresses zsig ⁇ is subsequently produced. Recombinant viral stocks are made by methods commonly used in the art.
  • the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C, 1994.
  • Another suitable cell line is the High FiveOTM cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No. 5,300,435).
  • Commercially available serum-free media are used to grow and maintain the cells.
  • Suitable media are Sf900 IITM (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 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
  • Fungal cells including yeast cells, can also be used within the present invention.
  • Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
  • Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
  • a preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No.
  • Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533.
  • Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.
  • DNA molecules for use in transforming R. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
  • 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).
  • DHAS dihydroxyacetone synthase
  • FMD formate dehydrogenase
  • CAT catalase
  • a preferred selectable marker for use in Pichia methanolica is a R. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine.
  • 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 (A UG1 and A UG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P.
  • methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
  • Prokaryotic host cells including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.).
  • the polypeptide When expressing a zsig ⁇ 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.
  • 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.
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
  • suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
  • the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell.
  • R. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25 °C to 35°C.
  • Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors.
  • a preferred culture medium for R. methanolica is YEPD (2% D-glucose, 2% BactoTM Peptone (Difco Laboratories, Detroit, MI), 1% BactoTM yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).
  • polypeptides of the present invention it is preferred to purify the polypeptides of the present invention to >80% purity, more preferably to >90% purity, even more preferably >95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
  • a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • Expressed recombinant zsig ⁇ polypeptides can be purified using fractionation and/or conventional purification methods and media.
  • Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography.
  • Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross- linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used.
  • These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
  • Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries.
  • These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support.
  • the polypeptides of the present invention can be isolated by exploitation of their biological and structural properties.
  • immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • IMAC immobilized metal ion adsorption
  • polypeptide fusions, or hybrid zsig ⁇ proteins are constructed using regions or domains of zsig ⁇ in combination with those of paralogs, orthologs, or heterologous proteins (Sambrook et al., ibid., Altschul et al., ibid., Picard. D., Cur. Opin. Biology. 5:511-515, 1994, and references therein). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest.
  • Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure.
  • Fusion polypeptides can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them.
  • a polynucleotide encoding one or more components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein.
  • part or all of a domain(s) conferring a biological function may be swapped between zsig ⁇ of the present invention with the functionally equivalent domain(s) from another protein.
  • domains include, but are not limited to, the secretory signal sequence, and motifs 1 through 4.
  • Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other similar proteins (e.g. paralogs, or orthologs) or heterologous proteins, depending on the fusion constructed.
  • such fusion proteins may exhibit other properties as disclosed herein.
  • Standard molecular biological and cloning techniques can be used to swap the equivalent domains between the zsig ⁇ polypeptide and those polypeptides to which they are fused.
  • a DNA segment that encodes a domain of interest e.g., a zsig ⁇ domain described herein
  • DNA constructs are made such that the several DNA segments that encode the corresponding regions of a polypeptide are operably linked in frame to make a single construct that encodes the entire fusion protein, or a functional portion thereof.
  • a DNA construct would encode from N-terminus to C- terminus a fusion protein comprising a signal polypeptide followed by a mature polypeptide; or a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by a mature protein.
  • fusion proteins can be expressed, isolated, and assayed for activity as described herein.
  • zsig ⁇ polypeptides or fragments thereof may also be prepared through chemical synthesis, zsig ⁇ 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.
  • Polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. Methods for synthesizing polypeptides are well known in the art. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Kaiser et al., Anal. Biochem. 34:595, 1970. After the entire synthesis of the desired peptide on a solid support, the peptide-resin is with a reagent which cleaves the polypeptide from the resin and removes most of the side-chain protecting groups. Such methods are well established in the art.
  • the activity of molecules of the present invention can be measured using a variety of assays that measure for example, signal transduction, cell motility, steroidogenesis, mitogenesis or binding. Such assays are well known in the art.
  • the zsig ⁇ polypeptides of the present invention can be used to study pancreatic cell proliferation or differentiation. Such methods of the present invention generally comprise incubating cells, ⁇ cells, ⁇ cells, F cells and acinar cells in the presence and absence of zsig ⁇ polypeptide, monoclonal antibody, agonist or antagonist thereof and observing changes in islet cell proliferation or differentiation. Similarly, the zsig ⁇ polypeptides of the present invention can be used to study pituitary cell proliferation or differentiation.
  • a further aspect of the invention provides a method for studying insulin.
  • Such methods of the present invention comprise incubating adipocytes in a culture medium comprising zsig ⁇ polypeptide, monoclonal antibody, agonist or antagonist thereof ⁇ insulin and observing changes in adipocyte protein secretion or differentiation.
  • the present invention also provides methods of studying mammalian cellular metabolism.
  • Such methods of the present invention comprise incubating cells to be studied, for example, an appropriate human cell line, ⁇ zsig ⁇ polypeptide, monoclonal antibody, agonist or antagonist thereof, and observing changes in adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, or the like.
  • zsig ⁇ polypeptides, agonists or antagonists thereof may be therapeutically useful for promoting wound healing, for example, in the pancreas.
  • agonists or antagonists of the present invention such zsig ⁇ polypeptides, agonists or antagonists are evaluated with respect to their ability to facilitate wound healing according to procedures known in the art.
  • zsig ⁇ polypeptide performance in this regard can be compared to growth factors, such as EGF, NGF, TGF- ⁇ , TGF- ⁇ , insulin, IGF-I, IGF-II, fibroblast growth factor (FGF) and the like.
  • zsig ⁇ polypeptides or agonists or antagonists thereof may be evaluated in combination with one or more growth factors to identify synergistic effects.
  • zsig ⁇ polypeptides, agonists or antagonists thereof may be therapeutically useful for anti-microbial applications.
  • agonists or antagonists of the present invention are evaluated with respect to their antimicrobial properties according to procedures known in the art. See, for example, Barsum et al., Eur. Respir. J. 8(5): 709-14, 1995; Sandovsky-Losica et al., J. Med. Vet. Mvcol (England) 28(4): 279-87, 1990; Mehentee et al, J. Gen. Microbiol (England) 135 (Pt.
  • zsig ⁇ polypeptide performance in this regard can be compared to proteins known to be functional in this regard, such as proline-rich proteins, lysozyme, histatins, lactoperoxidase or the like.
  • zsig ⁇ polypeptides or agonists or antagonists thereof may be evaluated in combination with one or more antimicrobial agents to identify synergistic effects.
  • Anti-microbial protective agents may be directly acting or indirectly acting. Such agents operating via membrane association or pore forming mechanisms of action directly attach to the offending microbe. Anti-microbial agents can also act via an enzymatic mechanism, breaking down microbial protective substances or the cell wall membrane thereof. Anti-microbial agents, capable of inhibiting microorganism proliferation or action or of disrupting microorganism integrity by either mechanism set forth above, are useful in methods for preventing contamination in cell culture by microbes susceptible to that anti-microbial activity. Such techniques involve culturing cells in the presence of an effective amount of said zsig ⁇ polypeptide, or an agonist or antagonist thereof.
  • zsig ⁇ polypeptides or agonists thereof may be used as cell culture reagents in in vitro studies of exogenous microorganism infection, such as bacterial, viral or fungal infection. Such moieties may also be used in in vivo animal models of infection. Also, the microorganism-adherence properties of zsig ⁇ polypeptides or agonists thereof can be studied under a variety of conditions in binding assays and the like.
  • Proteins of the present invention are useful for example, in treating ovarian, pancreatic, ocular, blood or bone disorders, can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to the appropriate animal model.
  • host cells expressing a secreted form of zsig ⁇ polypeptide may be embedded in an alginate environment and injected (implanted) into recipient animals.
  • Alginate-poly-L-lysine microencapsulation, permselective membrane encapsulation and diffusion chambers are a means to entrap transfected mammalian cells or primary mammalian cells to permit the diffusion of proteins and other macromolecules secreted or released by the captured cells to the recipient animal.
  • the capsules mask and shield the foreign, embedded cells from the recipient animal's immune response.
  • Such encapsulations can extend the life of the injected cells from a few hours or days (naked cells) to several weeks (embedded cells).
  • Alginate threads provide a simple and quick means for generating embedded cells and testing, in vivo, the proteins secreted therefrom.
  • alginate threads The materials needed to generate the alginate threads are known in the art.
  • 3% alginate is prepared in sterile H2O, and sterile filtered. Just prior to preparation of alginate threads, the alginate solution is again filtered.
  • An approximately 50% cell suspension (containing about 5 x 10 ⁇ to about 5 x
  • a large bore needle is then attached to the syringe, and the thread is intraperitoneally injected into a recipient in a minimal volume of the Lactated Ringer's Solution.
  • An in vivo approach for assaying proteins of the present invention involves viral delivery systems.
  • Exemplary viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV).
  • Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for review, see T.C. Becker et al., Meth. Cell Biol. 43:161-89, 1994; and J.T. Douglas and D.T.
  • adenovirus can accommodate relatively large DNA inserts; (ii) can be grown to high- titer; (iii) infect a broad range of mammalian cell types; and (iv) can be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
  • 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 has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary).
  • adenovirus When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adeno viral delivery system has an El gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.
  • tissue e.g., liver
  • adenoviral vectors containing various deletions of viral genes can be used in an attempt to reduce or eliminate immune responses to the vector.
  • Such adenoviruses are El deleted, and in addition contain deletions of E2A or E4 (Lusky, M. et al., J. Virol. 72:2022-2032, 1998; Raper, S.E. et al., Human Gene Therapy 9:671- 679, 1998).
  • deletion of E2b is reported to reduce immune responses (Amalfitano, A. et al., J. Virol. 72:926-933, 1998).
  • by deleting the entire adenovirus genome very large inserts of heterologous DNA can be accommodated.
  • the adenovirus system can also be used for protein production in vitro.
  • the cells By culturing adenovirus-infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division.
  • adenovirus vector infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (See Gamier et al., Cytotechnol. 15:145-55, 1994).
  • an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the disposition of the expressed protein in the cell. Within the infected 293 cell production protocol, non-secreted proteins may also be effectively obtained.
  • agonists including the natural ligand/ substrate/ cofactor/ etc.
  • antagonists have enormous potential in both in vitro and in vivo applications.
  • Compounds identified as zsig ⁇ agonists are useful for stimulating cell growth or signal transduction in vitro and in vivo.
  • zsig ⁇ and agonist compounds are useful as components of defined cell culture media, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture. Agonists are thus useful in specifically promoting the growth and/or development of cells in culture.
  • zsig ⁇ polypeptides and zsig ⁇ agonists may be particularly useful as research reagents, particularly for the growth of pituitary and endocrine cell types, ovarian cell lines, human eggs, cells from animal embryos or primary cultures derived from these tissues.
  • zsig ⁇ polypeptide can be provided as a supplement in cell culture medium.
  • Antagonists are also useful as research reagents for characterizing sites of ligand-receptor interaction.
  • Inhibitors of zsig ⁇ activity include anti-zsig ⁇ antibodies and soluble proteins which bind zsig ⁇ polypeptide.
  • Inhibitors of zsig ⁇ activity include anti-zsig66 antibodies and soluble zsig ⁇ receptors, as well as other peptidic and non-peptidic agents (including ribozymes).
  • Zsig ⁇ polypeptide can be used to identify inhibitors (antagonists) of its activity.
  • Test compounds are added to the biological or biochemical assays disclosed herein to identify compounds that inhibit the activity of zsig ⁇ .
  • samples can be tested for inhibition of zsig ⁇ activity within a variety of assays designed to measure receptor binding or the stimulation inhibition of zsig ⁇ -dependent cellular responses.
  • zsig ⁇ -responsive cell lines can be transfected with a reporter gene construct that is responsive to a zsig ⁇ -stimulated cellular pathway.
  • Reporter gene constructs of this type are known in the art, and will generally comprise a zsig66-DNA response element operably linked to a gene encoding an assay detectable protein, such as luciferase.
  • DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE) insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) and serum response elements (SRE) (Shaw et al., Cell 56: 563- 72, 1989). Cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem.
  • test samples including solutions, mixtures or extracts, are tested for the level of response to the zsig ⁇ polypeptide.
  • Assays of this type will detect compounds that directly block zsig ⁇ binding to cell-surface receptors, e.g., dimerization, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding.
  • compounds or other samples can be tested for direct blocking of zsig ⁇ binding to receptor using zsig ⁇ tagged with a detectable label (e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like).
  • a detectable label e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like.
  • Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.
  • the above methodology may be used to identify agonists of zsig ⁇ activity.
  • Candidate compounds serving as test samples including solutions, mixtures or extracts, are tested for the ability to mimic the activity of zsig ⁇ polypeptide on the target cells as evidenced by stimulation of reporter gene expression in the presence of a test sample and the absence of zsig ⁇ , relative to a control (cultured in the absence of a test sample and the absence of zsig ⁇ polypeptide), using assays as described above.
  • a zsig ⁇ polypeptide can be expressed as a fusion with an immunoglobulin heavy chain constant region, typically an F c fragment, which contains two constant region domains and lacks the variable region. Methods for preparing such fusions are disclosed in U.S. Patents Nos.
  • Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfide bonded to each other and two non-Ig polypeptides are arrayed in closed proximity to each other. Fusions of this type can be used to affinity purify ligand, as an in vitro assay tool, or an antagonist of zsig ⁇ . For use in assays, the chimeras are bound to a support via the F c region and used in an ELISA format.
  • a zsig ⁇ polypeptide can also be used for purification of receptor or polypeptides to which it binds.
  • the zsig ⁇ polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica- based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • a solid support such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica- based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N- hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation.
  • the resulting medium will generally be configured in the form of a column, and membrane fractions containing receptors are passed through the column one or more times to allow the receptor to bind to the ligand zsig ⁇ polypeptide.
  • the receptor is then eluted using changes in salt concentration, chaotropic agents (guanidine HCI), or pH to disrupt ligand-receptor binding.
  • An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/ anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed.
  • Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip.
  • Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993.
  • a receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell.
  • a test sample is passed through the cell.
  • a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
  • This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
  • Ligand-binding receptor polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 5J_: 660-72, 1949) and calorimetric assays (Cunningham et al., Science 253:545-48, 1991 ; Cunningham et al.. Science 245:821-25. 1991).
  • the activity of zsig ⁇ polypeptide can be measured by a silicon-based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses.
  • An exemplary device is the CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
  • CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
  • a variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell, H.M. et al., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol. 228:84-108, 1997; Arimilli, S.
  • the microphysiometer can be used for assaying adherent or non- adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including zsig ⁇ polypeptide, its agonists, or antagonists. Preferably, the microphysiometer is used to measure responses of a zsig ⁇ -responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to zsig ⁇ polypeptide.
  • Zsig ⁇ -responsive eukaryotic cells comprise cells into which a receptor for zsig ⁇ has been transfected creating a cell that is responsive to zsig ⁇ ; or cells naturally responsive to zsig ⁇ such as cells derived from pituitary tissue. Differences, measured by a change, for example, an increase or diminution in extracellular acidification, in the response of cells exposed to zsig ⁇ polypeptide, relative to a control not exposed to zsig ⁇ , are a direct measurement of zsig ⁇ -modulated cellular responses. Moreover, such zsig ⁇ -modulated responses can be assayed under a variety of stimuli.
  • a method of identifying agonists of zsig ⁇ polypeptide comprising providing cells responsive to a zsig ⁇ polypeptide, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test compound, and detecting a change, for example, an increase or diminution, in a cellular response of the second portion of the cells as compared to the first portion of the cells.
  • the change in cellular response is shown as a measurable change extracellular acidification rate.
  • culturing a third portion of the cells in the presence of zsig ⁇ polypeptide and the absence of a test compound can be used as a positive control for the zsig ⁇ -responsive cells, and as a control to compare the agonist activity of a test compound with that of the zsig ⁇ polypeptide.
  • a method of identifying antagonists of zsig ⁇ polypeptide comprising providing cells responsive to a zsig ⁇ polypeptide, culturing a first portion of the cells in the presence of zsig ⁇ and the absence of a test compound, culturing a second portion of the cells in the presence of zsig ⁇ and the presence of a test compound, and detecting a change, for example, an increase or a diminution in a cellular response of the second portion of the cells as compared to the first portion of the cells.
  • the change in cellular response is shown as a measurable change extracellular acidification rate.
  • Antagonists and agonists, for zsig ⁇ polypeptide can be rapidly identified using this method.
  • zsig ⁇ can be used to identify cells, tissues, or cell lines which respond to a zsig ⁇ -stimulated pathway.
  • the microphysiometer, described above can be used to rapidly identify ligand-responsive cells, such as cells responsive to zsig ⁇ of the present invention.
  • Cells can be cultured in the presence or absence of zsig ⁇ polypeptide. Those cells which elicit a measurable change in extracellular acidification in the presence of zsig ⁇ are responsive to zsig ⁇ .
  • Such cell lines can be used to identify antagonists and agonists of zsig ⁇ polypeptide as described above.
  • the polypeptides, nucleic acid and/or antibodies of the present invention may be used in treatment of disorders associated with gonadal development, pregnancy, pubertal changes, menopause, ovarian cancer, fertility, ovarian function, polycystic ovarian syndrome, pancreas, diabetes, eye disease, pituitary function, blood pressure regulation, water balance, osteoporosis, and other bone diseases.
  • the molecules of the present invention may be used to modulate or to treat or prevent development of pathological conditions in such diverse tissue as pancreas and ovary.
  • certain syndromes or diseases may be amenable to such diagnosis, treatment or prevention.
  • natural functions, such as ovulation may be suppressed or controlled for use in birth control by molecules of the present invention.
  • the zsig ⁇ polypeptide of the present invention may act in the neuroendocrine/exocrine cell fate decision pathway in organ systems in the body, and is therefore capable of regulating the expansion of neuroendocrine and exocrine cells in the pancreas.
  • One such regulatory use is that of islet cell regeneration.
  • zsig ⁇ polypeptide is a developmental gene involved in cell partitioning.
  • Assays and animal models are known in the art for monitoring the exocrine/neuroendocrine cell lineage decision, for observing pancreatic cell balance and for evaluating zsig ⁇ polypeptide, fragment, fusion protein, antibody, agonist or antagonist in the prevention or treatment of the conditions set forth above.
  • the molecules of the present invention will be useful for treating growth disorders, adrenal disorders, hormone and pubertal disorders, reproductive disorders and cancers, menopause, disorders associated with production of breast milk in humans and animals, enhancing production of breast milk in humans and animals, and pituitary disorders and cancers.
  • the polypeptides, nucleic acid and/or antibodies of the present invention can be used in treatment of disorders and complications associated with these diseases.
  • the molecules of the present invention can be used to modulate pituitary function, pituitary hormones, reproductive organs, enhance fertility, birth control, and the like, or to treat or prevent development of pathological conditions in such diverse tissue as pancreas, other endocrine tissues and pituitary.
  • certain syndromes and diseases may be amenable to such diagnosis, treatment or prevention.
  • Zsig ⁇ polypeptide is expressed in the pituitary and may have additional biological activity as described below.
  • Oogenesis is the process by which a diploid stem cell proceeds through multiple stages of differentiation, culminating in the formation of a terminally differentiated cell with a unique function, an oocyte.
  • oogenesis begins during fetal development and by birth, a female's entire supply of primary oocytes are stored in the ovaries in primordial follicles and await maturation and release.
  • folliculogenesis starts when the follicles enter the growth phase.
  • Early growing follicles undergo a dramatic process of cellular proliferation and differentiation.
  • the ovarian cycle in mammals includes the growth and maturation of follicles, followed by ovulation and transformation of follicles into corpea lutea.
  • the physiological events in the ovarian cycle are dependent on interactions between hormones and cells within the hypothalamic-pituitary-ovarian axis, including gonadotropin releasing hormone (GnRH), LH, and FSH.
  • GnRH gonadotropin releasing hormone
  • estradiol synthesized in the follicle, primes the hypothalamic-pituitary axis and is required for the mid-cycle surge of gonadotropin that stimulates the resumption of oocyte meiosis and leads to ovulation and subsequent extrusion of an oocyte from the follicle.
  • This gonadotropin surge also promotes the differentiation of the follicular cells from secreting estradiol to secreting progesterone.
  • Progesterone secreted by the corpus luteum, is needed for uterine development required for the implantation of fertilized oocytes.
  • Such applications include treatments for precocious puberty, endometriosis, uterine leiomyomata, hirsutism, infertility, pre menstrual syndrome (PMS), amenorrhea, and as contraceptive agents.
  • Zsig ⁇ polypeptides, agonists and antagonists which modulate the actions of such hormones can be of therapeutic value.
  • zsig ⁇ may act as a hormone itself, and as such zsig ⁇ polypeptides, agonists and antagonists can be of therapeutic value.
  • Such molecules can also be useful for modulating steroidogenesis, both in vivo and in vitro, and modulating aspects of the ovarian cycle such as oocyte maturation, ovarian cell-cell interactions, follicular development and rupture, luteal function, and promoting uterine implantation of fertilized oocytes.
  • Molecules which modulate hormone action can be beneficial therapeutics for use prior to or at onset of puberty.
  • puberty in females is marked by an establishment of feed-back loops to control hormone levels and hormone production. Abnormalities resulting from hormone imbalances during puberty have been observed and include precocious puberty, where pubertal changes occur in females prior to the age of 8. Hormone- modulating molecules, can be used, in this case, to suppress hormone secretion and delay onset of puberty.
  • the level and ratio of gonadotropin and steroid hormones can be used to assess the existence of hormonal imbalances associated with diseases, as well as determine whether normal hormonal balance has been restored after administration of a therapeutic agent.
  • the level of zsig ⁇ can also be measured in its relationship to hormonal imbalances.
  • estradiol, progesterone, LH, and FSH for example, from serum is known by one of skill in the art.
  • Such assays can be used to monitor the hormone levels after administration of zsig ⁇ in vivo, or in a transgenic mouse model where the zsig ⁇ gene is expressed or the murine ortholog is deleted.
  • zsig ⁇ polypeptide can have therapeutic application for treating, for example, breakthrough menopausal bleeding, as part of a therapeutic regime for pregnancy support, or for treating symptoms associated with polycystic ovarian syndrome (PCOS), PMS and menopause.
  • PCOS polycystic ovarian syndrome
  • Proteins of the present invention may also be used in applications for enhancing fertilization during assisted reproduction in humans and in animals.
  • assisted reproduction methods are known in the art and include artificial insemination, in vitro fertilization, embryo transfer, and gamete intrafallopian transfer. Such methods are useful for assisting those who may have physiological or metabolic disorders that prevent or impede natural conception.
  • animal breeding programs e.g., for livestock, racehorses, domestic and wild animals, and could be used as methods for the creation of transgenic animals.
  • Zsig ⁇ polypeptides could be used in the induction of ovulation, either independently or in conjunction with a regimen of gonadotropins or agents such as clomiphene citrate or bromocriptine (Speroff et al., Induction of ovulation, Clinical Gynecologic Endocrinology and Infertility, 5 th ed., Baltimore, Williams & Wilkins, 1994).
  • proteins of the present invention can be administered to the recipient prior to fertilization or combined with the sperm, an egg or an egg-sperm mixture prior to in vitro or in vivo fertilization.
  • Such proteins can also be mixed with oocytes prior to cryopreservation to enhance viability of the preserved oocytes for use in assisted reproduction.
  • the zsig ⁇ polypeptides, agonists and antagonists of the present invention may be directly used as or incorporated into therapies for treating reproductive disorders. Disorders such as luteal phase deficiency would benefit from such therapy (Soules, "Luteal phase deficiency: A subtle abnormality of ovulation" in, Infertility: Evaluation and Treatment, Keye et al., eds., Philadelphia, WB Saunders, 1995). Moreover, administration of gonadotropin-releasing hormone is shown to stimulate reproductive behavior (Riskin and Moss, Res. Bull. 11:481-5, 1983; Kadar et al., Phvsiol. Behav. 51:601-5, 1992 and Silver et al., J.
  • the zsig ⁇ polypeptides of the present invention can be used to study ovarian and other reproductive tissue cell proliferation, maturation, and differentiation, i.e., by acting as a luteinizing agent that converts granulosa cells from estradiol to progesterone-producing cells.
  • such methods of the present invention generally comprise incubating granulosa cells, theca cells, oocytes or a combination thereof, in the presence and absence of zsig ⁇ polypeptide, monoclonal antibody, agonist or antagonist thereof and observing changes in cell proliferation, maturation and differentiation. See for example, Basini et al..(J. Rep. Immunol. 37:139-53, 1998);
  • the molecules of the present invention are useful as components of defined cell culture media, as described herein, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture.
  • Molecules of the present invention are particularly useful in specifically promoting the growth, development, differentiation, and/or maturation of ovarian cells in culture, and may also prove useful in the study of the ovarian cycle, reproductive function, ovarian cell-cell interactions, and fertilization.
  • zsig ⁇ may affect other hormones aside from those in reproductive tissues.
  • the present invention also provides methods for studying steroidogenesis and steroid hormone secretion.
  • Such methods generally comprise incubating ovarian or other hormone-producing cells in culture medium comprising zsig ⁇ polypeptides, monoclonal antibodies, agonists or antagonists thereof with and without gonadotropins and/or steroid hormones, and subsequently observing protein and steroid secretion.
  • gonadotropin hormones include luteinizing hormone and follicle stimulating hormone (Rouillier et al., Mol. Reprod. Dev. 50:170- 7, 1998).
  • Exemplary steroid hormones include estradiol, androstenedione, and progesterone.
  • Effects of zsig ⁇ on steroidogenesis or steroid secretion can be determined by methods known in the art, such as radioimmunoassay (to detect levels of estradiol, androstenedione, progesterone, and the like), and immunoradiometric assay (IRMA).
  • radioimmunoassay to detect levels of estradiol, androstenedione, progesterone, and the like
  • IRMA immunoradiometric assay
  • Molecules expressed in the pituitary such as zsig ⁇ polypeptide, and which may modulate hormones, hormone receptors, growth factors, or cell-cell interactions, of the reproductive cascade, other organ systems or the cardiovascular system, or are involved in oocyte or ovarian development or other development, would be useful as markers for cancer of reproductive organs and other organ systems and as therapeutic agents for hormone-dependent cancers, by inhibiting hormone-dependent growth and/or development of tumor cells.
  • Human reproductive system cancers such as ovarian, uterine, cervical, testicular and prostate cancers are common.
  • Diagnostic methods of the present invention involve the detection of zsig ⁇ polypeptides in the serum or tissue biopsy of a patient undergoing analysis of reproductive function or evaluation for possible reproductive or other cancer or disease.
  • Such polypeptides can be detected using immunoassay techniques and antibodies, described herein, that are capable of recognizing polypeptide epitopes.
  • the present invention contemplates methods for detecting zsig ⁇ polypeptides comprising: exposing a test sample potentially containing zsig ⁇ polypeptides to an antibody attached to a solid support, wherein said antibody binds to a first epitope of a zsig ⁇ polypeptide; washing the immobilized antibody-polypeptide to remove unbound contaminants; exposing the immobilized antibody-polypeptide to a second antibody directed to a second epitope of a zsig ⁇ polypeptide, wherein the second antibody is associated with a detectable label; and detecting the detectable label.
  • Altered levels of zsig ⁇ polypeptides in a test sample can be monitored as an indication of reproductive function or of cancer or disease, when compared against a normal control.
  • Additional methods using probes or primers derived, for example, from the nucleotide sequences disclosed herein can also be used to detect zsig ⁇ expression in a patient sample, such as a blood, saliva, sweat, tissue sample, or the like.
  • probes can be hybridized to tumor tissues and the hybridized complex detected by in situ hybridization.
  • Zsig ⁇ sequences can also be detected by PCR amplification using cDNA generated by reverse translation of sample mRNA as a template (PCR Primer A Laboratory Manual.
  • zsig ⁇ may regulate blood pressure and water
  • zsig ⁇ , agonists, and antagonists have great therapeutic potential in regulating blood pressure.
  • zsig ⁇ , agonists, and antagonists have therapeutic potential in enhancing blood pressure, for example in the case of traumatic shock; or decreasing blood pressure, for example in the case of hypertension.
  • water balance is important in maintaining electrolytes as well as affecting overall health.
  • zsig ⁇ , agonists, and antagonists have great therapeutic potential in regulating water balance, for example after major organ surgeries, trauma nutritional or disease states that alter such balance.
  • zsig ⁇ polypeptides may modulate mammalian energy balance.
  • the pituitary expression of zsig ⁇ suggests that zsig ⁇ may exhibit effects on glucose uptake, e.g. through GLUT-1, and thermogenesis (thermoregulation).
  • zsig ⁇ polypeptides may find utility in modulating nutrient uptake, as demonstrated, for example, by 2-deoxy-glucose uptake in the brain or the like.
  • mammalian energy balance may be evaluated by monitoring one or more of the following metabolic functions: adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, protein synthesis, thermogenesis, oxygen utilization or the like.
  • glucoregulatory effects of insulin are predominantly exerted in the liver, skeletal muscle and adipose tissue.
  • Insulin binds to its cellular receptor in these three tissues and initiates tissue-specific actions that result in, for example, the inhibition of glucose production and the stimulation of glucose utilization.
  • insulin stimulates glucose uptake and inhibits gluconeogenesis and glycogenolysis.
  • skeletal muscle and adipose tissue insulin acts to stimulate the uptake, storage and utilization of glucose.
  • Zsig ⁇ polypeptide is expressed in pituitary but may exhibit extra- pituitary activity in organs that affect metabolic functions.
  • pharmaceutical compositions of the present invention may be useful in prevention or treatment of pancreatic and other endocrine, exocrine or neurocrine disorders.
  • zsig ⁇ may be associated with pathological regulation of the expansion of neurocrine and exocrine cells in the pancreas, as evident in IDDM, pancreatic cancer or the like, or zsig ⁇ may be associated with pathological regulation of reproductive organs such as the ovaries.
  • Pharmaceutical compositions of the present invention may also be involved in prevention or treatment of pancreatic conditions characterized by dysfunction associated with pathological regulation of blood glucose levels, insulin resistance or digestive function.
  • Adipogenesis, gluconeogenesis and glycogenolysis are interrelated components of mammalian energy balance, which may be evaluated by known techniques using, for example, ob/ob mice or db/db mice.
  • the ob/ob mice are inbred mice that are homozygous for an inactivating mutation at the ob (obese) locus.
  • Such ob/ob mice are hyperphagic and hypometabolic, and are believed to be deficient in production of circulating OB protein.
  • the db/db mice are inbred mice that are homozygous for an inactivating mutation at the db (diabetes) locus.
  • the db/db mice display a phenotype similar to that of ob/ob mice, except db/db mice display a more severe diabetic phenotype.
  • Such db/db mice are believed to be resistant to the effects of circulating OB protein.
  • various in vitro methods of assessing these parameters are known in the art.
  • Insulin-stimulated lipogenesis may be monitored by measuring the incorporation of ⁇ C-acetate into triglyceride (Mackall et al. J. Biol. Chem. 25J,:6462-6464, 1976) or triglyceride accumulation (Kletzien et al., Mol. Pharmacol. 41 :393-398. 1992).
  • Glucose uptake may be evaluated, for example, in an assay for insulin- stimulated glucose transport.
  • Non-transfected, differentiated L6 myotubes (maintained in the absence of G418) are placed in DMEM containing 1 g/l glucose, 0.5 or 1.0% BSA, 20 mM Hepes, and 2 mM glutamine.
  • the medium is replaced with fresh, glucose-free DMEM containing 0.5 or 1.0% BSA, 20 mM Hepes, 1 mM pyruvate, and 2 mM glutamine.
  • Appropriate concentrations of insulin or IGF-1, or a dilution series of the test substance, are added, and the cells are incubated for 20-30 minutes.
  • -1H or 14c-labeled deoxyglucose is added to «50 1 M final concentration, and the cells are incubated for approximately 10-30 minutes.
  • the cells are then quickly rinsed with cold buffer (e.g. PBS), then lysed with a suitable lysing agent (e.g. 1% SDS or 1 N NaOH).
  • cold buffer e.g. PBS
  • a suitable lysing agent e.g. 1% SDS or 1 N NaOH.
  • the cell lysate is then evaluated by counting in a scintillation counter.
  • Cell-associated radioactivity is taken as a measure of glucose transport after subtracting non-specific binding as determined by incubating cells in the presence of cytochalasin b, an inhibitor of glucose transport.
  • Other methods include those described by, for example, Manchester et al., Am. J. Phvsiol. 266 (Endocrinol. Metab. 29):E326-E333, 1994 (insulin-stimulated glucose transport).
  • Protein synthesis may be evaluated, for example, by comparing precipitation of 35 S-methionine-labeled proteins following incubation of the test cells with 35 S-methionine and 35 S-methionine and a putative modulator of protein synthesis.
  • Thermogenesis may be evaluated as described by B. Stanley in The Biology of Neuropeptide Y and Related Peptides. W. Colmers and C. Wahlestedt (eds.), Humana Press, Ottawa, 1993, pp. 457-509; C. Billington et al., Am. J. Phvsiol. 260:R321, 1991 ; N. Zarjevski et al., Endocrinology 133:1753, 1993; C. Billington et al.. Am. J. Phvsiol. 266:R1765. 1994; Heller et al.. Am. J. Phvsiol. 252(4 Pt 2): R661-7, 1987; and Heller et al.. Am. J. Phvsiol. 245(3): R321-8, 1983.
  • metabolic rate which may be measured by a variety of techniques, is an indirect measurement of thermogenesis.
  • Oxygen utilization may be evaluated as described by Heller et al., Pflugers Arch 369(1): 55-9, 1977. This method also involved an analysis of hypothalmic temperature and metabolic heat production. Oxygen utilization and thermoregulation have also been evaluated in humans as described by Haskell et al., J. Appl. Phvsiol. 51(4): 948-54, 1981.
  • the zsig ⁇ polypeptides of the present invention may act in the neuroendocrine/exocrine cell fate decision pathway and is therefore capable of regulating the expansion of neuroendocrine and exocrine cells in the pancreas.
  • One such regulatory use is that of islet cell regeneration.
  • zsig ⁇ polypeptide is a developmental gene involved in cell partitioning.
  • Assays and animal models are known in the art for monitoring the exocrine/neuroendocrine cell lineage decision, for observing pancreatic cell balance and for evaluating zsig ⁇ polypeptide, fragment, fusion protein, antibody, agonist or antagonist in the prevention or treatment of the conditions set forth above.
  • zsig ⁇ polypeptides can also be used to prepare antibodies that bind to zsig ⁇ epitopes, peptides or polypeptides.
  • the zsig ⁇ polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response.
  • antigenic, epitope-bearing polypeptides contain a sequence of at least 6, preferably at least 9, and more preferably at least 15 to about 30 contiguous amino acid residues of a zsig ⁇ polypeptide (e.g., SEQ ID NO:2).
  • Polypeptides comprising a larger portion of a zsig ⁇ polypeptide, i.e., from 30 to 10 residues up to the entire length of the amino acid sequence are included.
  • Antigens or immunogenic epitopes can also include attached tags, adjuvants and carriers, as described herein.
  • Suitable antigens include the zsig ⁇ polypeptide encoded by SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly), or a contiguous 9 to 57 amino acid fragment thereof.
  • Other suitable antigens include motifs 1 through 4, as disclosed herein.
  • Preferred peptides to use as antigens are hydrophilic peptides such as those predicted by one of skill in the art from a hydrophobicity plot (See Figure).
  • Zsig ⁇ hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: (1) amino acid number 1 (Met) to amino acid number 6 (Glu) of SEQ ID NO:2; (2) amino acid number 7 (Val) to amino acid number 12 (He) of SEQ ID NO:2; (3) amino acid number 26 (Ser) to amino acid number 32 (Ser) of SEQ ID NO:2; (4) amino acid number 29 (Asp) to amino acid number 34 (Ser) of SEQ ID NO:2; and (5) amino acid number 51 (Leu) to amino acid number 56 (Glu) of SEQ ID NO:2.
  • Antibodies from an immune response generated from inoculation with these antigens can be isolated and purified as described herein.
  • polyclonal antibodies can be generated by inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a zsig ⁇ polypeptide or a fragment thereof.
  • the immunogenicity of a zsig ⁇ polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of zsig ⁇ or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • the polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • tetanus toxoid tetanus toxoid
  • antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments.
  • Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non- human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody).
  • humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics.
  • human antibodies can be produced in transgenic, non-human animals that have been engineered to contain human immunoglobulin genes as disclosed in WIPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination.
  • Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zsig ⁇ protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zsig ⁇ protein or peptide).
  • Genes encoding polypeptides having potential zsig ⁇ 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 which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US Patent NO. 5,223,409; Ladner et al., US Patent NO. 4,946,778; Ladner et al., US Patent NO. 5,403,484 and Ladner et al., US Patent NO.
  • Random peptide display libraries can be screened using the sequences disclosed herein to identify proteins which bind to zsig ⁇ . These "binding polypeptides" which interact with zsig ⁇ polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like.
  • binding polypeptides can also be used in analytical methods such as for screening expression libraries and neutralizing activity, e.g., for blocking interaction between ligand and receptor, or viral binding to a receptor.
  • the binding polypeptides can also be used for diagnostic assays for determining circulating levels of zsig ⁇ sequences disclosed herein to identify proteins which bind to zsig ⁇ .
  • binding polypeptides which interact with zsig ⁇ polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like.
  • binding polypeptides can also be used in analytical methods such as for screening expression libraries and neutralizing activity, e.g., for blocking interaction between ligand and receptor, or viral binding to a receptor.
  • the binding polypeptides can also be used for diagnostic assays for determining circulating levels of zsig ⁇ polypeptides; for detecting or quantitating soluble zsig ⁇ polypeptides as marker of underlying pathology or disease.
  • These binding polypeptides can also act as zsig ⁇ "antagonists" to block zsig ⁇ binding and signal transduction in vitro and in vivo. These anti-zsig ⁇ binding polypeptides would be useful for inhibiting zsig ⁇ activity or protein-binding.
  • Antibodies are considered to be specifically binding if: 1) they exhibit a threshold level of binding activity, and 2) they do not significantly cross-react with known related polypeptide molecules.
  • a threshold level of binding is determined if anti-zsig ⁇ antibodies herein bind to a zsig ⁇ polypeptide, peptide or epitope with an affinity at least 10-fold greater than the binding affinity to control (non-zsig66) polypeptide. It is preferred that the antibodies exhibit a binding affinity (K a ) of 10 M "
  • binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis
  • anti-zsig ⁇ antibodies do not significantly cross-react with known related polypeptide molecules is shown, for example, by the antibody detecting zsig ⁇ polypeptide but not known related polypeptides using a standard Western blot analysis (Ausubel et al., ibid.).
  • known related polypeptides are those disclosed in the prior art, such as known orthologs, and paralogs, and similar known members of a protein family, Screening can also be done using non-human zsig ⁇ , and zsig ⁇ mutant polypeptides.
  • antibodies can be "screened against" known related polypeptides, to isolate a population that specifically binds to the zsig ⁇ polypeptides.
  • antibodies raised to zsig ⁇ are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to zsig ⁇ will flow through the matrix under the proper buffer conditions. Screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to known closely related polypeptides (Antibodies: A Laboratory Manual. Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology. Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995). Screening and isolation of specific antibodies is well known in the art. See, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol.
  • Specifically binding anti-zsig ⁇ antibodies can be detected by a number of methods in the art, and disclosed below.
  • assays known to those skilled in the art can be utilized to detect antibodies which bind to zsig ⁇ proteins or polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual. Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant zsig ⁇ protein or polypeptide.
  • Antibodies to zsig ⁇ and zsig ⁇ binding polypeptides described herein may be used for tagging cells that express zsig ⁇ ; for isolating zsig ⁇ by affinity purification; for diagnostic assays for determining circulating levels of zsig ⁇ polypeptides; for detecting or quantitating soluble zsig ⁇ as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; for detecting or quantitating soluble zsig ⁇ polypeptides as marker of underlying pathology or disease.
  • These antibodies and binding polypeptides can also act as zsig ⁇ "antagonists" to block zsig ⁇ binding and signal transduction in vitro and in vivo. These anti-zsig ⁇ binding polypeptides would be useful for inhibiting zsig ⁇ activity or protein-binding.
  • Antibodies to zsig ⁇ may be used for tagging cells that express zsig ⁇ ; for isolating zsig ⁇ by affinity purification; for diagnostic assays for determining circulating levels of zsig ⁇ polypeptides; for detecting or quantitating soluble zsig ⁇ as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zsig ⁇ activity in vitro and in vivo.
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti- complement pairs as intermediates.
  • Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • antibodies to zsig ⁇ or fragments thereof may be used in vitro to detect denatured zsig ⁇ or fragments thereof in assays, for example, Western Blots or other assays known in the art.
  • Antibodies or polypeptides herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance).
  • zsig ⁇ polypeptides or anti-zsig ⁇ antibodies, or bioactive fragments or portions thereof can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti- complementary molecule.
  • Suitable detectable molecules may be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like.
  • Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium- 188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance).
  • Polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin.
  • cytotoxic drugs such as adriamycin.
  • the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion.
  • biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
  • polypeptide-toxin fusion proteins or antibody- toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues).
  • a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest.
  • the anti-complementary molecule can be conjugated to a detectable or cytotoxic molecule.
  • Such domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue- specific delivery of generic anti-complementary-detectable/ cytotoxic molecule conjugates.
  • zsig ⁇ -cytokine fusion proteins or antibody- cytokine fusion proteins can be used for enhancing in vivo killing of target tissues (for example, blood and bone marrow cancers), if the zsig ⁇ polypeptide or anti-zsig66 antibody targets the hyperproliferative blood or bone marrow cell (See, generally, Hornick et al., Blood 89:4437-47, 1997).
  • target tissues for example, blood and bone marrow cancers
  • zsig ⁇ polypeptide or anti-zsig66 antibody targets the hyperproliferative blood or bone marrow cell
  • Suitable zsig ⁇ polypeptides or anti-zsig ⁇ antibodies target an undesirable cell or tissue (i.e., a tumor or a leukemia), and the fused cytokine mediated improved target cell lysis by effector cells.
  • Suitable cytokines for this purpose include interleukin 2 and granulocyte-macrophage colony-stimulating factor (GM- CSF), for instance.
  • GM- CSF granulocyte-macrophage colony-stimulating factor
  • Differentiation is a progressive and dynamic process, beginning with pluripotent stem cells and ending with terminally differentiated cells. Pluripotent stem cells that can regenerate without commitment to a lineage express a set of differentiation markers that are lost when commitment to a cell lineage is made.
  • Progenitor cells express a set of differentiation markers that may or may not continue to be expressed as the cells progress down the cell lineage pathway toward maturation. Differentiation markers that are expressed exclusively by mature cells are usually functional properties such as cell products, enzymes to produce cell products, and receptors. The stage of a cell population's differentiation is monitored by identification of markers present in the cell population. Myocytes, osteoblasts, adipocytes, chrondrocytes, fibroblasts and reticular cells are believed to originate from a common mesenchymal stem cell (Owen et al., Ciba Fdn. Svmp. 136:42-46, 1988). Markers for mesenchymal stem cells have not been well identified (Owen et al., J.
  • novel polypeptides of the present invention may be useful for studies to isolate mesenchymal stem cells and myocyte or other progenitor cells, both in vivo and ex vivo.
  • the present invention includes stimulating or inhibiting the proliferation of myocytes, smooth muscle cells, osteoblasts, adipocytes, chrondrocytes, neuronal and endothelial cells.
  • Molecules of the present invention may while stimulating proliferation or differentiation of cardiac myocytes, inhibit proliferation or differentiation of adipocytes, by virtue of the affect on their common precursor/stem cells.
  • molecules of the present invention may have use in inhibiting chondrosarcomas, atherosclerosis, restenosis and obesity.
  • Assays measuring differentiation include, for example, measuring cell markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses. 161-171, 1989; all incorporated herein by reference).
  • ZSIG ⁇ polypeptide itself can serve as an additional cell-surface or secreted marker associated with stage-specific expression of a tissue.
  • direct measurement of ZSIG66 polypeptide, or its loss of expression in a tissue as it differentiates can serve as a marker for differentiation of tissues.
  • direct measurement of ZSIG66 polypeptide, or its loss of expression in a tissue can be determined in a tissue or cells as they undergo tumor progression.
  • Increases in invasiveness and motility of cells, or the gain or loss of expression of ZSIG ⁇ in a pre-cancerous or cancerous condition, in comparison to normal tissue can serve as a diagnostic for transformation, invasion and metastasis in tumor progression.
  • knowledge of a tumor's stage of progression or metastasis will aid the physician in choosing the most proper therapy, or aggressiveness of treatment, for a given individual cancer patient.
  • Methods of measuring gain and loss of expression are well known in the art and described herein and can be applied to ZSIG66 expression.
  • polypeptides that regulate cell motility can be used to aid diagnosis and prognosis of prostate cancer (Banyard, J. and Zetter, B.R., Cancer and Metast. Rev. 17:449-458, 1999).
  • ZSIG66 gain or loss of expression may serve as a diagnostic for prostate and other cancers.
  • tumor cells passaged in culture are implanted into mice of the same strain as the tumor donor.
  • the cells will develop into tumors having similar characteristics in the recipient mice, and metastasis will also occur in some of the models.
  • Appropriate tumor models for our studies include the Lewis lung carcinoma (ATCC No. CRL- 1642) and B 16 melanoma (ATCC No. CRL-6323), amongst others. These are both commonly used tumor lines, syngeneic to the C57BL6 mouse, that are readily cultured and manipulated in vitro.
  • Tumors resulting from implantation of either of these cell lines are capable of metastasis to the lung in C57BL6 mice.
  • the Lewis lung carcinoma model has recently been used in mice to identify an inhibitor of angiogenesis (O'Reilly MS, et al. C l 79: 315-328,1994).
  • C57BL6/J mice are treated with an experimental agent either through daily injection of recombinant protein, agonist or antagonist or a one time injection of recombinant adenovirus.
  • an experimental agent either through daily injection of recombinant protein, agonist or antagonist or a one time injection of recombinant adenovirus.
  • 10 5 to 10 cells are implanted under the dorsal skin.
  • the cells themselves may be infected with recombinant adenovirus, such as one expressing zsig ⁇ , before implantation so that the protein is synthesized at the tumor site or intracellularly, rather than systemically.
  • the mice normally develop visible tumors within 5 days.
  • the tumors are allowed to grow for a period of up to 3 weeks, during which time they may reach a size of 1500 - 1800 mm in the control treated group. Tumor size and body weight are carefully monitored throughout the experiment.
  • the tumor is removed and weighed along with the lungs and the liver.
  • the lung weight has been shown to correlate well with metastatic tumor burden.
  • lung surface metastases are counted.
  • the resected tumor, lungs and liver are prepared for histopathological examination, immunohistochemistry, and in situ hybridization, using methods known in the art and described herein.
  • the influence of the expressed polypeptide in question, e.g., zsig ⁇ , on the ability of the tumor to recruit vasculature and undergo metastasis can thus be assessed.
  • the implanted cells can be transiently transfected with zsig ⁇ .
  • the zsig ⁇ polypeptide or anti- zsig ⁇ antibody targets vascular cells or tissues
  • such polypeptide or antibody may be conjugated with a radionuclide, and particularly with a beta-emitting radionuclide, to reduce restenosis.
  • a radionuclide and particularly with a beta-emitting radionuclide
  • Such therapeutic approach poses less danger to clinicians who administer the radioactive therapy.
  • iridium-192 impregnated ribbons placed into stented vessels of patients until the required radiation dose was delivered showed decreased tissue growth in the vessel and greater luminal diameter than the control group, which received placebo ribbons. Further, revascularisation and stent thrombosis were significantly lower in the treatment group. Similar results are predicted with targeting of a bioactive conjugate containing a radionuclide, as described herein.
  • the bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action.
  • Molecules of the present invention can be used to identify and isolate receptors that bind zsig ⁇ .
  • proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column (Immobilized Affinity Ligand Techniques. Hermanson et al., eds., Academic Press, San Diego, CA, 1992, pp.195-202).
  • Proteins and peptides can also be radiolabeled (Methods in Enzymol.. vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990, 721-37) or photoaffinity labeled (Brunner et al., Ann- Rev. Biochem. 62:483-514, 1993 and Fedan et al., Biochem. Pharmacol. 33:1167-80, 1984) and specific cell-surface proteins can be identified.
  • the molecules of the present invention although expressed in pituitary, may exert their effects elsewhere and hence may be useful for treating diabetes, and pancreatic cancer.
  • the polypeptides, nucleic acid and/or antibodies of the present invention can be used in treatment of disorders and complications associated with these diseases.
  • the molecules of the present invention can be used to modulate insulin, glucagon, and the like, or to treat or prevent development of pathological conditions in such diverse tissue as pancreas and other endocrine tissues. In particular, certain syndromes and diseases may be amenable to such diagnosis, treatment or prevention.
  • zsig ⁇ appears to have structural similarity to vasopressin and oxytocin
  • zsig ⁇ could be useful as modulator blood pressure, muscle tension or and osmotic balance.
  • blood pressure modification is important in situations such as heart attack, stroke, traumatic shock, surgery, and any number of bleeding complications.
  • zsig ⁇ may modulate contractility in the organ systems and tissues that it effects.
  • the activity of molecules of the present invention can be measured using a variety of assays that measure cell contractility. Such assays are well known in the art.
  • As zsig ⁇ is structurally related to vasopressin, it can be used to modualte tissues that contract.
  • contractile tissues upon which zsig ⁇ may act include skeletal muscle, uterus; tissues in testis, e.g., vas deferens; prostate tissues; gastrointestinal tissues, e.g., colon and small intestine; and heart.
  • tissue contractility can be measured in vitro using a tensiometer with or without electrical field stimulation.
  • Such assays are known in the art and can be applied to tissue samples, such as aortic rings, vas deferens, ilium, uterine and other contractile tissue samples, as well as to organ systems, such as atria, and can be used to determine whether zsig ⁇ polypeptide, its agomsts or antagonists, enhance or depress contractility.
  • Molecules of the present invention are hence useful for treating dysfunction associated with contractile tissues or can be used to suppress or enhance contractility in vivo.
  • molecules of the present invention have utility in treating cardiovascular disease, infertility, in vitro fertilization, birth control, treating impotence or other male reproductive dysfunction, as well as inducing birth.
  • the effect of the zsig ⁇ polypeptides, antagonists and agonists of the present invention on contractility of tissues including uterus, prostate, testis, gastrointestinal tissues, and heart can be measured in a tensiometer that measures contractility and relaxation in tissues. See, Dainty et al., J. Pharmacol. 100:767. 1990; Rhee et al, Neurotox. 16: 179, 1995; Anderson, M.B., Endocrinol. 114:364-368, 1984; and Downing, S.J. and Sherwood, O.D, Endocrinol. 116:1206-1214, 1985. For example, measuring vasodilatation of aortic rings is well known in the art.
  • aortic rings are taken from 4 month old Sprague Dawley rats and placed in a buffer solution, such as modified Krebs solution (118.5 mM NaCI, 4.6 mM KCI, 1.2 mM MgSO 4 .7H 2 O, 1.2 mM KH 2 PO 4 , 2.5 mM CaCl 2 .2H 2 O, 24.8 mM NaHCO 3 and 10 mM glucose).
  • a buffer solution such as modified Krebs solution (118.5 mM NaCI, 4.6 mM KCI, 1.2 mM MgSO 4 .7H 2 O, 1.2 mM KH 2 PO 4 , 2.5 mM CaCl 2 .2H 2 O, 24.8 mM NaHCO 3 and 10 mM glucose).
  • modified Krebs solution 118.5 mM NaCI, 4.6 mM KCI, 1.2 mM MgSO 4 .7H 2 O, 1.2 mM KH 2 PO 4 , 2.5 mM CaCl 2 .
  • the rings are then attached to an isometric force transducer (Radnoti Inc., Monrovia, CA) and the data recorded with a Ponemah physiology platform (Gould Instrument systems, Inc., Valley View, OH) and placed in an oxygenated (95% O 2 , 5% CO 2 ) tissue bath containing the buffer solution.
  • the tissues are adjusted to 1 gram resting tension and allowed to stabilize for about one hour before testing.
  • the integrity of the rings can be tested with norepinepherin (Sigma Co., St. Louis, MO) and Carbachol, a muscarinic acetylcholine agonist (Sigma Co.). After integrity is checked, the rings are washed three times with fresh buffer and allowed to rest for about one hour.
  • the rings are contracted to two grams tension and allowed to stabilize for fifteen minutes.
  • a zsig ⁇ polypeptide sample is then added to 1 , 2 or 3 of the 4 baths, without flushing, and tension on the rings recorded and compared to the control rings containing buffer only. Enhancement or relaxation of contractility by zsig ⁇ polypeptides, their agonists and antagonists is directly measured by this method, and it can be applied to other contractile tissues such as uterus, prostate, and testis.
  • the activity of molecules of the present invention can be measured using a variety of assays that measure stimulation of gastrointestinal cell contractility, modulation of nutrient uptake and/or secretion of digestive enzymes.
  • assays that measure stimulation of gastrointestinal cell contractility, modulation of nutrient uptake and/or secretion of digestive enzymes.
  • changes in contractility of smooth muscle cells For example, the contractile response of segments of mammalian duodenum or other gastrointestinal smooth muscles tissue (Depoortere et al., J. Gastrointestinal Motility 1:150-159, 1989, incorporated herein by reference).
  • An exemplary in vivo assay uses an ultrasonic micrometer to measure the dimensional changes radially between commissures and longiturdinally to the plane of the valve base (Hansen et al., Society of Thoracic Surgeons 60:S384-390. 1995).
  • Gastric motility is generally measured in the clinical setting as the time required for gastric emptying and subsequent transit time through the gastrointestinal tract.
  • Gastric emptying scans are well known to those skilled in the art, and briefly, comprise use of an oral contrast agent, such as barium, or a radiolabeled meal. Solids and liquids can be measured independently.
  • a test food or liquid is radiolabeled with an isotope (e.g. 99m Tc), and after ingestion or administration, transit time through the gastrointestinal tract and gastric emptying are measured by visualization using gamma cameras (Mever et al.. Am. J. Dig. Pis. 21 :296. 1976; Collins et al, Gut 24:1117, 1983; Maughan et al, Diabet. Med.
  • polypeptides, antagonists, agonists, nucleic acid and/or antibodies of the present invention can also be used in treatment of disorders associated with gastrointestinal cell contractility, secretion of digestive enzymes and acids, gastrointestinal motility, recruitment of digestive enzymes; inflammation, particularly as it affects the gastrointestinal system; reflux disease and regulation of nutrient absorption.
  • Specific conditions that will benefit from treatment with molecules of the present invention include, but are not limited to, diabetic gastroparesis, post-surgical gastroparesis, vagotomy, chronic idiopathic intestinal pseudo-obstruction and gastroesophageal reflux disease. Additional uses include, gastric emptying for radiological studies, stimulating gallbladder contraction and antrectomy.
  • the motor and neurological affects of molecules of the present invention make it useful for treatment of obesity and other metabolic disorders where neurological feedback modulates nutritional absorption.
  • the molecules of the present invention are useful for regulating satiety, glucose absorption and metabolism, and neuropathy-associated gastrointestinal disorders.
  • Molecules of the present invention are also useful as additives to anti-hypoglycemic preparations containing glucose and as adsorption enhancers for oral drugs which require fast nutrient action. Additionally, molecules of the present invention can be used to stimulate glucose-induced insulin release.
  • Polynucleotides encoding zsig ⁇ polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zsig ⁇ activity. If a mammal has a mutated or absent zsig ⁇ gene, the zsig ⁇ gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zsig ⁇ polypeptide is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like.
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred.
  • a defective virus is not infectious after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30, 1992; and a defective adeno-associated virus vector (Samulski et al., J. Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).
  • HSV1 herpes simplex virus 1
  • a zsig ⁇ gene can be introduced in a retroviral vector, e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No.
  • the vector can be introduced by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., Proc. Natl. Acad. Sci.
  • lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages.
  • Molecular targeting of liposomes to specific cells represents one area of benefit. More particularly, directing transfection to particular cells represents one area of benefit. For instance, directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters
  • proteins such as antibodies, or non- peptide molecules
  • liposomes chemically. It is possible to remove the target cells from the body; to introduce the vector as a naked DNA plasmid; and then to re-implant the transformed cells into the body. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem. 267:963-7,
  • Antisense methodology can be used to inhibit zsig ⁇ gene transcription, such as to inhibit cell proliferation in vivo.
  • Polynucleotides that are complementary to a segment of a zsig ⁇ -encoding polynucleotide e.g., a polynucleotide as set froth in SEQ ID NO:l
  • Such antisense polynucleotides are used to inhibit expression of zsig ⁇ polypeptide-encoding genes in cell culture or in a subject.
  • the present invention also provides reagents which will find use in diagnostic applications.
  • the zsig ⁇ gene, a probe comprising zsig ⁇ DNA or RNA or a subsequence thereof can be used to determine if the zsig ⁇ gene is present on a chromosome or if a mutation has occurred.
  • Detectable chromosomal aberrations at the zsig ⁇ gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
  • Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid. ; Ausubel et. al., ibid. ; Marian, Chest 108:255-65, 1995).
  • molecular genetic techniques such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid. ; Ausubel et. al., ibid. ; Marian, Chest 108:255-65, 1995).
  • Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes (Cox et al., Science 250:245-50, 1990). Partial or full knowledge of a gene's sequence allows one to design PCR primers suitable for use with chromosomal radiation hybrid mapping panels. Radiation hybrid mapping panels are commercially available which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL). These panels enable rapid, PCR-based chromosomal localizations and ordering of genes, sequence-tagged sites (STSs), and other nonpolymorphic and polymorphic markers within a region of interest. This includes establishing directly proportional physical distances between newly discovered genes of interest and previously mapped markers.
  • the precise knowledge of a gene's position can be useful for a number of purposes, including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have.
  • Sequence tagged sites can also be used independently for chromosomal localization.
  • An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome.
  • An STS is defined by a pair of oligonucleotide primers that are used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences.
  • STSs are based solely on DNA sequence they can be completely described within an electronic database, for example, Database of Sequence Tagged Sites (dbSTS), GenBank, (National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), and can be searched with a gene sequence of interest for the mapping data contained within these short genomic landmark STS sequences.
  • dbSTS Database of Sequence Tagged Sites
  • GenBank National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov
  • the zsig ⁇ gene can be used to detect genes of known function map to the same region of the human chromosome.
  • zsig ⁇ polynucleotide probes can be used to detect abnormalities or genotypes associated with human disease states as well as map to the same region of the human genome. See the Online Mendellian Inheritance of Man (OMIM) gene map, on a publicly available WWW server (http://www3.ncbi.nlm.nih.gov/htbin-post/Omim).
  • OMIM Online Mendellian Inheritance of Man
  • the zsig ⁇ gene is located at the 4q28-q31 region of chromosome 4 (Example 3).
  • the Fibrinogin alpha, beta, and gamma gene cluster is located at 4q28, and mutations therein are associated with diseases such as dysfibrinogenemia, hypofibrinogenemia, and fibrinogenemia which result in blood clotting malfunction and other complications (See, for example, Berg, K. and Keirulf, P. Clin. Genet. 36: 229- 235, 1989).
  • Zsig ⁇ polynucleotide probes can be used to detect abnormalities or genotypes associated with these fibrinogen associated markers.
  • zsig ⁇ polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 4q28-q31 deletions and translocations associated with human diseases, such as Reiger syndrome (deletion in 4q); loss of heterozygosity, or translocation between 4q28-q31 and another chromosome; translocations involved with malignant progression of tumors; or mutations, which are expected to be involved in chromosome rearrangements in malignancy; or deletions and translocations in other cancers.
  • diseases such as Reiger syndrome (deletion in 4q); loss of heterozygosity, or translocation between 4q28-q31 and another chromosome; translocations involved with malignant progression of tumors; or mutations, which are expected to be involved in chromosome rearrangements in malignancy; or deletions and translocations in other cancers.
  • zsig ⁇ polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 4q28-31 trisomy and chromosome loss associated with human diseases such as Reiger syndrome (above).
  • human diseases such as Reiger syndrome (above).
  • PHA autosomal dominant pseudohypoaldosteroidism
  • anterior segment mesenchymal dysgenesis and Reiger syndrome (4q28- q31)
  • Hepatitis B virus associated hepatocellular carcinoma susceptability (4q32.1), and others, all manifest themselves in human disease states as well as map to this region of the human genome.
  • zsig ⁇ polynucleotide probes can be used to detect abnormalities or genotypes associated with these defects.
  • defects in the zsig ⁇ locus itself may result in a heritable human disease state.
  • defects the zsig ⁇ molecules of the present invention can have broad reaching effects in the human body.
  • Molecules of the present invention such as the polypeptides, antagonists, agonists, polynucleotides and antibodies of the present invention would aid in the detection, diagnosis prevention, and treatment associated with a zsig ⁇ genetic defect.
  • a diagnostic could assist physicians in determining the type of genetic disease and appropriate associated therapy, or assistance in genetic counseling.
  • inventive anti-zsig ⁇ antibodies, polynucleotides, and polypeptides can be used for the detection of zsig ⁇ polypeptide, mRNA or anti-zsig ⁇ antibodies, thus serving as markers and be directly used for detecting or diagnosing known or yet to be discovered diseases, or cancers, as described herein, using methods known in the art and described herein.
  • zsig ⁇ polynucleotide probes can be used to detect allelic differences between diseased or non-diseased individuals at the zsig ⁇ chromosomal locus.
  • the zsig ⁇ sequences can be used as diagnostics in forensic DNA profiling. Such profiling can be applied to commercial animals as well for use in breeding programs.
  • diagnostic methods used in genetic linkage analysis, to detect a genetic abnormality or aberration in a patient, are known in the art.
  • Most diagnostic methods comprise the steps of (a) obtaining a genetic sample from a potentially diseased patient, diseased patient or potential non-diseased carrier of a recessive disease allele; (b) producing a first reaction product by incubating the genetic sample with a ZSMF16 polynucleotide probe wherein the polynucleotide will hybridize to complementary polynucleotide sequence, such as in RFLP analysis or by incubating the genetic sample with sense and antisense primers in a PCR reaction under appropriate PCR reaction conditions; (iii) Visualizing the first reaction product by gel electrophoresis and/or other known method such as visualizing the first reaction product with a ZSMF16 polynucleotide probe wherein the polynucleotide will hybridize to the complementary polynucleotide sequence of the first reaction; and (iv) comparing
  • a difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the diseased or potentially diseased patient, or the presence of a heterozygous recessive carrier phenotype for a non-diseased patient, or the presence of a genetic defect in a tumor from a diseased patient, or the presence of a genetic abnormality in a fetus or pre- implantation embryo.
  • a difference in restriction fragment pattern, length of PCR products, length of repetitive sequences at the ZSIG66 genetic locus, and the like are indicative of a genetic abnormality, genetic aberration, or allelic difference in comparison to the normal wild type control. Controls can be from unaffected family members, or unrelated individuals, depending on the test and availability of samples.
  • Genetic samples for use within the present invention include genomic DNA, mRNA, and cDNA isolated form any tissue or other biological sample from a patient, such as but not limited to, blood, saliva, semen, embryonic cells, amniotic fluid, and the like.
  • the polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO:l, the complement of SEQ ID NO: 1, or an RNA equivalent thereof.
  • Such methods of showing genetic linkage analysis to human disease phenotypes are well known in the art. For reference to PCR based methods in diagnostics see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc.
  • Abe ⁇ ations associated with the ZSIG66 locus can be detected using nucleic acid molecules of the present invention by employing standard methods for direct mutation analysis, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc.
  • standard methods for direct mutation analysis such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-a
  • mice engineered to express the zsig ⁇ gene referred to as "transgenic mice,” and mice that exhibit a complete absence of zsig ⁇ gene function, referred to as “knockout mice,” may also be generated (Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature 366:740-42, 1993; Capecchi, M.R., Science 244: 1288-1292, 1989; Palmiter, R.D. et al. Annu Rev Genet. 20: 465-499, 1986).
  • transgenic mice that over-express zsig ⁇ either ubiquitously or under a tissue-specific or tissue-restricted promoter can be used to ask whether over-expression causes a phenotype.
  • over-expression of a wild-type zsig ⁇ polypeptide, polypeptide fragment or a mutant thereof may alter normal cellular processes, resulting in a phenotype that identifies a tissue in which zsig ⁇ expression is functionally relevant and may indicate a therapeutic target for the zsig ⁇ , its agonists or antagonists.
  • a preferred transgenic mouse to engineer is one that over-expresses the zsig ⁇ mature polypeptide (approximately amino acids 28 (Ala) to 84 (Gly) of SEQ ID NO:2).
  • over-expression may result in a phenotype that shows similarity with human diseases.
  • knockout zsig ⁇ mice can be used to determine where zsig ⁇ is absolutely required in vivo.
  • the phenotype of knockout mice is predictive of the in vivo effects of that a zsig ⁇ antagonist, such as those described herein, may have.
  • the human zsig ⁇ cDNA can be used to isolate murine zsig ⁇ mRNA, cDNA and genomic DNA, which are subsequently used to generate knockout mice. These mice may be employed to study the zsig ⁇ gene and the protein encoded thereby in an in vivo system, and can be used as in vivo models for corresponding human diseases.
  • transgenic mice expression of zsig ⁇ antisense polynucleotides or ribozymes directed against zsig ⁇ , described herein can be used analogously to transgenic mice described above.
  • the proteins of the present invention are formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods.
  • Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours.
  • pharmaceutical formulations will include a zsig ⁇ protein in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like.
  • Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
  • Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy.
  • Therapeutic doses will generally be in the range of 0.1 to 100 ⁇ g/kg of patient weight per day, preferably 0.5-20 _g/kg per day, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art.
  • the proteins may be administered for acute treatment, over one week or less, often over a period of one to three days or may be used in chronic treatment, over several months or years.
  • This cDNA was contained in a plasmid from a human pituitary library, and was sequenced using the following primers to generate complete double stranded sequence of this clone: ZC6,768 (SEQ ID NO:12), ZC20,137 (SEQ ID NO:13), ZC20,136 (SEQ ID NO: 14), ZC20,132 (SEQ ID NO:15), ZC20,134 (SEQ ID NO: 16), ZC19,964 (SEQ ID NO: 17), ZC19,965 (SEQ ID NO:18), ZC694 (SEQ ID NO: 19).
  • Example 1 was used in a PCR reaction using oligos ZC21,336 (SEQ ID NO:20) and
  • ZC21.338 (SEQ ID NO:21) as primers.
  • PCR conditions were as follows: 94°C for 1.5 minutes; 35 cycles at 94°C for 15 seconds , 66°C for 30 seconds; 72°C for 30 seconds, one cycle at 72°C for 5 minutes, followed by a 4°C hold.
  • a sample of the PCR reaction product was run on a 4% agarose gel. A band of the expected size of about 220 bp was seen.
  • the 220 bp PCR fragment was gel purified using a commercially available kit (QiaexIITM; Qiagen) and then radioactively labeled with 32 P-dCTP using Rediprime IITM (Amersham), a random prime labeling system, according to the manufacturer's specifications.
  • the probe was then purified using a Nuc-TrapTM column (Stratagene) according to the manufacturer's instructions. ExpressHybTM (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 55°C using 1-2 x 10 6 cpm/ml of labeled probe. The blots were then washed for 30 minutes in 2X SSC/0.1% SDS at room temperature, followed by a wash in 2X SSC/0.1% SDS at 65°C , followed by a wash in 0.1X SSC/0.1% SDS at 65°C. The blots were exposed 7 days. No transcript signals were observed in the tissues represented on the blots.
  • Dot Blots were also performed using Human RNA Master BlotsTM (Clontech). The methods and conditions for the Dot Blots are the same as for the Multiple Tissue Blots described above. A signal was observed in genomic DNA.
  • Each of the 85 PCR reactions consisted of 2 ⁇ l 10X KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, CA), 1.6 ⁇ l dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, CA), 1 ⁇ l sense primer, ZC21,450 (SEQ ID NO:22), 1 ⁇ l antisense primer, ZC21,451 (SEQ ID NO:23), 2 ⁇ l "RediLoad” (Research Genetics, Inc., Huntsville, AL), 0.4 ⁇ l 50X Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and ddH2O for a total volume of 20 ⁇ l.
  • the reactions were overlaid with an equal amount of mineral oil and sealed.
  • the PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 94°C, 35 cycles of a 45 seconds denaturation at 94°C, 45 seconds annealing at 60°C and 1 minute AND

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Abstract

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

Description

Description PANCREATIC POLYPEPTIDE ZSIG66
BACKGROUND OF THE INVENTION
Proliferation and differentiation of cells of multicellular organisms are controlled by hormones and polypeptide growth factors. These diffusible molecules allow cells to communicate with each other and act in concert to regulate cells and form organs, and to repair and regenerate damaged tissue. Examples of hormones and growth factors include the steroid hormones (e.g. estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, the interleukins, platelet derived growth factor (PDGF), epidermal growth factor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin.
Hormones and growth factors influence cellular metabolism by binding to proteins. These proteins may be integral membrane proteins that are linked to signaling pathways within the cell, such as second messenger systems. Other classes of proteins that hormones and growth factors influence are soluble molecules, such as the transcription factors.
Thus, there is a continuing need to discover new hormones, growth factors and the like. The in vivo activities of these cytokines illustrates the enormous clinical potential of, and need for, other cytokines, cytokine agonists, and cytokine antagonists. The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
SUMMARY OF THE INVENTION
Within one aspect, the present invention provides an isolated polynucleotide encoding a zsigόό polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID NO:2, wherein the amino acid percent identity is determined using a FASTA program with ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution with other parameters set as default. Within one embodiment, the isolated polynucleotide disclosed above is selected from the group consisting of: (a) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 431 to nucleotide 472; (b) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 425 to nucleotide 472; (c) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 425 to nucleotide 475; (d) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 305 to nucleotide 475; (e) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 224 to nucleotide 475; and (f) a polynucleotide sequence complementary to (a) or (b). Within another embodiment, the isolated polynucleotide disclosed above comprises nucleotide 1 to nucleotide 252 of SEQ ID NO:l l . Within another embodiment, the isolated polynucleotide disclosed above comprises a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID Within a second aspect, the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a zsigόό polypeptide comprising an amino acid sequence as shown in SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and a transcription terminator. Within one embodiment, the expression vector disclosed above further comprising a secretory signal sequence operably linked to the DNA segment.
Within a third aspect, the present invention provides a cultured cell into which has been introduced an expression vector as disclosed above, wherein the cell expresses a polypeptide encoded by the DNA segment.
Within a fourth aspect, the present invention provides a DNA construct encoding a fusion protein, the DNA construct comprising: a first DNA segment encoding a polypeptide comprising a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO: 2 from residue number 28 (Ala) to amino acid number 84 (Gly); and at least one other DNA segment encoding an additional polypeptide, wherein the first and other DNA segments are connected in-frame; and encode the fusion protein.
Within another aspect, the present invention provides a fusion protein produced by a method comprising: culturing a host cell into which has been introduced a vector comprising the following operably linked elements: (a) a transcriptional promoter; (b) a DNA construct encoding a fusion protein as disclosed above; and (c) a transcriptional terminator; and recovering the protein encoded by the DNA segment.
Within another aspect, the present invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number
28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID NO:2, wherein the amino acid percent identity is determined using a FASTA program with ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62, with other parameters set as default. Within one embodiment, the isolated polypeptide disclosed above further comprises motifs 1 through 4 spaced apart from N-terminus to C-terminus in a configuration Ala28-{7}- Ml-{3}-M2-{5}-M3-{6}-M4-{ 10}- Gly84, wherein Ml is "motif 1," a sequence of amino acids as shown in amino acids 36 to 41 of SEQ ID NO:2, M2 is "motif 2," a sequence of amino acids as shown in amino acids 45 to 50 of SEQ ID NO:2, M3 is "motif 3," a sequence of amino acids as shown in amino acids 56 to 61 of SEQ ID NO:2, M4 is "motif 4," a sequence of amino acids as shown in amino acids 68 to 73 of SEQ ID NO:2, and Ala28 is the Alanine residue at amino acid number 28 in SEQ ID NO:2, Glyg4 is the Glycine residue at amino acid number 84 in SEQ ID NO:2, and {#} denotes the number of amino acids between the motifs. Within another embodiment, the isolated polypeptide disclosed above comprises a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser); (b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser); (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 84 (Gly); (d) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID NO:2. Within another embodiment, the isolated polypeptide disclosed above further comprises an amidated C-terminal Serine residue.
Within another aspect, the present invention provides a method of producing a zsigόό polypeptide comprising: culturing a cell as disclosed above; and isolating the zsigόό polypeptide produced by the cell.
Within another aspect, the present invention provides a method of detecting, in a test sample, the presence of a modulator of zsigόό protein activity, comprising: transfecting a zsigόό-responsive cell, with a reporter gene construct that is responsive to a zsigόό-stimulated cellular pathway; and producing a zsigόό polypeptide by the method as disclosed above; and adding the zsigόό polypeptide to the cell, in the presence and absence of a test sample; and comparing levels of response to the zsigόό polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and determining from the comparison, the presence of the modulator of zsigόό activity in the test sample.
Within another aspect, the present invention provides a method of producing an antibody to zsigόό polypeptide comprising the following steps in order: inoculating an animal with a polypeptide selected from the group consisting of: (a) a polypeptide consisting of 9 to 57 amino acids, wherein the polypeptide has a contiguous sequence of amino acids as shown within SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); (b) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 from residue number 28 (Ala) to amino acid number 84 (Gly); (c) a polypeptide as disclosed above; (d) a polypeptide consisting of amino acid number 1 (Met) to amino acid number 6 (Glu) of SEQ ID NO:2; (e) a polypeptide consisting of amino acid number 7 (Val) to amino acid number 12 (lie) of SEQ ID NO:2; (f) a polypeptide consisting of amino acid number 26 (Ser) to amino acid number 32 (Ser) of SEQ ID NO:2; (g) a polypeptide consisting of amino acid number 29 (Asp) to amino acid number 34 (Ser) of SEQ ID NO:2; and (h) a polypeptide consisting of amino acid number 51 (Leu) to amino acid number 56 (Glu) of SEQ ID NO:2; and wherein the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal.
Within another aspect, the present invention provides an antibody produced by the method as disclosed above, which binds to a zsigόό polypeptide. Within one embodiment, the antibody disclosed above is a monoclonal antibody.
Within another aspect, the present invention provides an antibody which specifically binds to a polypeptide as disclosed above.
Within another aspect, the present invention provides a method for detecting a genetic abnormality in a patient, comprising: obtaining a genetic sample from a patient; producing a first reaction product by incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO:l or the complement of SEQ ID NO:l, under conditions wherein said polynucleotide will hybridize to complementary polynucleotide sequence; visualizing the first reaction product; and comparing said first reaction product to a control reaction product from a wild type patient, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient.
Within another aspect, the present invention provides a pharmaceutical composition comprising an isolated polypeptide as disclosed above, wherein the polypeptide is in combination with a pharmaceutically acceptable vehicle.
These and other aspects of the invention will become evident upon reference to the following detailed description of the invention and attached drawings.
BRIEF DESCRIPTION OF THE DRAWING
The figure is a hydrophobicity plot of zsigόό, determined from a Hopp/Woods hydrophilicity profile based on a sliding six-residue window, with buried G, S, and T residues and exposed H, Y, and W residues ignored.
DETAILED DESCRIPTION OF THE INVENTION
Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms: The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly- histidine tract, protein A (Nilsson et al., EMBO J. 4: 1075, 1985; Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952- 4, 1985), substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107, 1991. DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ).
The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
The terms "amino-terminal" and "carboxyl-terminal" are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
The term "complement/anti-complement pair" denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair. Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of <10^ M~l .
The term "complements of a polynucleotide molecule" denotes a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
The term "contig" denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5'-ATGGAGCTT-3' are 5'- AGCTTgagt-3' and 3'-tcgacTACC-5'.
The term "degenerate nucleotide sequence" denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
The term "expression vector" is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985). An "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
The term "operably linked", when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
The term "ortholog" 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, α- globin, β-globin, and myoglobin are paralogs of each other.
A "polynucleotide" is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.
Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides
("nt"), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides".
The term "promoter" is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes. A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
The term "receptor" denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell. Membrane-bound receptors are characterized by a multi-peptide structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. In general, receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g.,
PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).
The term "secretory signal sequence" denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
The term "splice variant" is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to ±10%.
All references cited herein are incorporated by reference in their entirety.
The present invention is based in part upon the discovery of a novel
DNA sequence that encodes a novel polypeptide. The polypeptide and corresponding polynucleotide were isolated from a human pituitary library. The polypeptide has been designated zsigόό.
The novel zsigόό polypeptides of the present invention were initially identified by querying an EST database for proteins homologous to proteins having a secretory signal sequence. These proteins are characterized by an upstream methionine start site and a hydrophobic region of approximately 13 amino acids, followed by a peptide signal peptidase cleavage site. An EST database was queried for novel DNA sequences whose translations would meet these search criteria. An EST was found and its corresponding cDNA was sequenced. The zsigόό nucleotide sequence is believed to encode the entire coding sequence of the predicted protein. Zsigόό may be a novel pituitary hormone, pituitary hormone activating hormone, cell-cell signaling molecule, growth factor, cytokine, nerve cell modulating protein, secreted extracellular matrix associated protein with growth factor hormone activity, or the like, and is a member a novel protein family.
The sequence of the zsigόό polypeptide was obtained from a single clone believed to contain its corresponding polynucleotide sequence. The clone was obtained from a pituitary library. Other libraries that might also be searched for such sequences include fetal libraries, pancreas, liver, ovary, placenta, and the like. The nucleotide sequence of a representative zsigόό-encoding DNA is described in SEQ ID NO:l, and its deduced 84 amino acid sequence is described in SEQ ID NO:2. In its entirety, the zsigόό polypeptide (SEQ ID NO:2) represents a full- length polypeptide segment (residue 1 (Met) to residue 84 (Gly) of SEQ ID NO:2). The domains and structural features of zsigόό are further described below.
Analysis of the zsigόό polypeptide encoded by the DNA sequence of SEQ ID NO:l revealed an open reading frame encoding 84 amino acids (SEQ ID NO:2) comprising a predicted signal peptide of 27 amino acid residues (residue 1 (Met) to residue 27 (Ser) of SEQ ID NO:2), and a mature polypeptide of 57 amino acids (residue 28 (Ala) to residue 84 (Gly) of SEQ ID NO:2). Further, post-translational processing may truncate zsigόό, for example at a cysteine bond, and still provide a short active polypeptide. As such, amino acids residues 69 (Phe) to 84 (Gly) of SEQ ID NO:2, preferably amino acids residues 69 (Phe) to 83 (Ser) of SEQ ID NO:2, and more preferably amino acids residues 70 (Cys) to 83 (Ser) of SEQ ID NO:2, comprise an active zsigόό polypeptide. In another embodiment, the Serine residue of the short active polypeptide amino acid residues 69 (Phe) to 83 (Ser) of SEQ ID NO:2 or amino acid residues 70 (Cys) to 83 (Ser) of SEQ ID NO:2 can further be amidated at the C- terminal Serine residue. The structure of zsigόό surrounding the C-C bond (see below), shows some similarity with vasopressin and oxytocin. As such, zsigόό can have cardiovascular or metabolic vasodilatation capability. For general references on vasopressin and oxytocin, see Rehbein, M. et al., Biol. Chem. 367:695-704, 1986; Sausville, E. et al., J. Biol. Chem. 260:10236-10241, 1985; Ivell, R. et al., Endocrinol. 127:2990-2996, 1990; Mohr, E. et al., FEBS Lett. 193:12-16, 1985; Chauvet, M.-T. et al., Proc. NatT. Acad. Sci. USA 80:2839-2843, 1983; Schlesinger, D.H. and Audhya, T.K., FEBS Lett. 128:325-328, 1981; and Light, A. and DuVigneaud, V., Proc. Soc. Exp. Biol. Med. 98:692-696. 1958. Moreover, the zsigόό polypeptide contains several regions of low variance or low degeneracy (see, Sheppard, P. et al., Gene 150:163-167, 1994). These regions that exhibit low degeneracy are described in the following zsigόό motifs:
1) "motif 1" (corresponding to amino acids 36 (Trp) to 41 (Gly) of SEQ ID NO:2); 2) "motif 2" (corresponding to amino acids 45 (Thr) to 50 (Phe) of SEQ
ID NO:2); 3) "motif 3" (corresponding to amino acids 56 (Glu) to 61 (Val) of SEQ ID NO:2); and
4) "motif 4" (corresponding to amino acids 68 (Lys) to 73 (Asn) of SEQ ID NO:2); and Motifs 1 through 4 are spaced apart from N-terminus to C-terminus in a configuration represented by the following:
Ala28-{7}-Ml-{3}-M2-{5}-M3-{6}-M4-{ 10}- Gly84, where Ala28is the starting residue of the mature polypeptide (as shown in SEQ ID NO:2), Gly84 is the ending residue of the mature polypeptide(as shown in SEQ
ID NO:2),
M# denotes the specific motif disclosed above (e.g., Ml is motif 1, etc.), and
{#} denotes the approximate number of amino acid residues between the motifs, up to plus or minus 2 residues.
The presence of conserved and low variance motifs generally correlates with or defines important structural regions in proteins. Regions of low variance (e.g., hydrophobic clusters) are generally present in regions of structural importance (Sheppard, P. et a .Gene 150:163-167, 1994). Such regions of low variance often contain rare or infrequent amino acids, such as Tryptophan. The regions flanking and between such conserved and low variance motifs may be more variable, but are often functionally significant because they may relate to or define important structures and activities such as binding domains, biological and enzymatic activity, signal transduction, tissue localization domains and the like. In addition, there are two Cysteine residues that form a disulfide bond located in SEQ ID NO:2 at amino acid number 70 and 74 that can be important for structure or activity of zsigόό polypeptide. Moreover, there are 2 consensus phosphorylation sites in zsigόό polypeptide: a protein kinase C (PKC) site at amino acid 53 (Ser) of SEQ ID NO:2; and a casein kinase II (CK2) site at amino acid 43 (Thr) ofSEQ ID NO:2. Moreover the genomic structure of zsigόό is readily determined by one of skill in the art by comparing the cDNA sequence of SEQ ID NO: 1 and the translated amino acid of SEQ ID NO:2 with the genomic DNA in which the gene is contained (Genbank Accession No. ACO 15783). For example, such analysis can be readily done using FASTA as described herein. As such, the intron and exon junctions in this region of genomic DNA can be determined for the zsigόό gene. The region of human genomic DNA where zsigόό is located is shown in SEQ ID NO:24. The entire coding sequence appears to be contained in a single exon, as there are no putative introns within SEQ ID NO:24. Thus, the present invention includes the zsigόό gene as located in human genomic DNA.
The corresponding polynucleotides encoding the zsigόό polypeptide regions, domains, motifs, residues and sequences described above are as shown in SEQ ID NO:l.
The low degeneracy amino acids in zsigόό can be used as a tool to identify new family members. For instance, reverse transcription-polymerase chain reaction (RT-PCR) can be used to amplify sequences, encoding the low degeneracy motifs, from RNA obtained from a variety of tissue sources or cell lines. In particular, highly degenerate primers designed from the zsigόό sequences are useful for this purpose. In particular, degenerate oligonucleotide primers designed from the following zsigόό amino acid sequences of motifs 1 through 5 are useful for this purpose: a) WIQGMG (motif 1; SEQ ID NO:3), corresponding to degenerate polynucleotides of SEQ ID NO:7 and their complement; b) TDFNPF (motif 2; SEQ ID NO:4), corresponding to degenerate polynucleotides of SEQ ID NO:8 and their complement; c) KFCVVN (motif 3; SEQ ID NO:5), corresponding to degenerate polynucleotides of SEQ ID NO:9 and their complement; d) EIELFV (motif 4; SEQ ID NO:6), corresponding to degenerate polynucleotides of SEQ ID NO: 10 and their complement.
The present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the zsigόό polypeptides disclosed herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. SEQ ID NO:l 1 is a degenerate DNA sequence that encompasses all DNAs that encode the zsigόό polypeptide of SEQ ID NO:2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO: 11 also provides all RNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus, zsigόό polypeptide- encoding polynucleotides comprising nucleotide 1 to nucleotide 252 of SEQ ID NO:l 1 and their RNA equivalents are contemplated by the present invention. Table 1 sets forth the one-letter codes used within SEQ ID NO: 11 to denote degenerate nucleotide positions. "Resolutions" are the nucleotides denoted by a code letter. "Complement" indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.
TABLE 1
Nucleotide Nucleotide
Base Code Resolution Base Code Complement
A A T T
C C G G
G G C C
T T A A
R A|G Y C|T
Y C|T R A|G
M A|C K G|T
K G|T M A|C
S C|G S C|G w A|T w A|T
H A|C|T D A|G|T
B C|G|T V A|C|G
V A|C|G B C|G|T
D A|G|T H A|C|T
N A|C|G|T N A|C|G|T
The degenerate codons used in SEQ ID NO: 11, encompassing all possible codons for a given amino acid, are set forth in Table 2.
TABLE 2
Three One
Letter Letter Degenerate
Code Code Synonymous Codons Codon
Cys C TGC TGT TGY
Ser S AGC AGT TCA TCC TCG TCT WSN
Thr T ACA ACC ACG ACT ACN
Pro P CCA CCC CCG CCT CCN
Ala A GCA GCC GCG GCT GCN
Gly G GGA GGC GGG GGT GGN
Asn N AAC AAT AAY
Asp D GAC GAT GAY
Glu E GAA GAG GAR
Gin Q CAA CAG CAR
His H CAC CAT CAY
Arg R AGA AGG CGA CGC CGG CGT MGN
Lys K AAA AAG AAR
Met M ATG ATG
He I ATA ATC ATT ATH
Leu L CTA CTC CTG CTT TTA TTG YTN
Val V GTA GTC GTG GTT GTN
Phe F TTC TTT TTY
Tyr Y TAC TAT TAY
Tip W TGG TGG
Ter TAA TAG TGA TRR
Asn|Asp B RAY
Glu|Gln Z SAR
Any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY). A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO:2. Variant sequences can be readily tested for functionality as described herein.
One of ordinary skill in the art will also appreciate that different species can exhibit "preferential codon usage." In general, see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64. 1981; Grosjean and Fiers. Gene 18:199-209. 1982; Holm, Nuc. Acids Res. 14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As used herein, the term "preferential codon usage" or "preferential codons" is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2). For example, the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential. Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NO:l 1 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein. Within preferred embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:l, or a sequence complementary thereto, under stringent conditions. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defmed ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Suitable stringent hybridization conditions are equivalent to about a 5 h to overnight incubation at about 42°C in a solution comprising: about 40-50% formamide, up to about 5X SSC, about 5X Denhardt's solution, up to about 10% dextran sulfate, and about 10-20 μg/ml denatured commercially-available carrier DNA; hybridization is then followed by washing filters in up to about 2X SSC. For example, a suitable wash stringency is equivalent to 0.1X SSC to 2X SSC, 0.1% SDS, at 55°C to 65 °C. Stringent hybridization and wash conditions depend on the length of the probe, reflected in the Tm, hybridization and wash solutions used, and are routinely determined empirically by one of skill in the art.
As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of zsigόό RNA. Such tissues and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include pituitary, pancreas, tissues of endocrine origin, and the like. Total RNA can be prepared using guanidinium isothiocyanate extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al, Biochemistry 18:52-94, 1979). Poly (A)+ RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972). Complementary DNA (cDNA) is prepared from poly(A)+ RNA using known methods. In the alternative, genomic DNA can be isolated. Polynucleotides encoding zsigόό polypeptides are then identified and isolated by, for example, hybridization or PCR.
A full-length clone encoding zsigόό can be obtained by conventional cloning procedures. Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron. Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library. Expression libraries can be probed with antibodies to zsigόό, receptor fragments, or other specific binding partners.
The polynucleotides of the present invention can also be synthesized using DNA synthesis machines. Currently the method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short polynucleotides (60 to 80 bp) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. However, for producing longer polynucleotides (longer than about 300 bp), special strategies are usually employed, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%. To overcome this problem, synthetic genes (double-stranded) are assembled in modular form from single- stranded fragments that are from 20 to 100 nucleotides in length.
One method for building a synthetic gene requires the initial production of a set of overlapping, complementary oligonucleotides, each of which is between 20 to 60 nucleotides long. Each internal section of the gene has complementary 3' and 5' terminal extensions designed to base pair precisely with an adjacent section. Thus, after the gene is assembled, process is completed by sealing the nicks along the backbones of the two strands with T4 DNA ligase. In addition to the protein coding sequence, synthetic genes can be designed with terminal sequences that facilitate insertion into a restriction endonuclease site of a cloning vector. An alternative way to prepare a full- length gene is to synthesize a specified set of overlapping oligonucleotides (40 to 100 nucleotides). After the 3' and 5' short overlapping complementary regions are annealed, large gaps still remain, but the short base-paired regions are both long enough and stable enough to hold the structure together. The gaps are filled and the DNA duplex is completed via enzymatic DNA synthesis by E. coli DNA polymerase I. After the enzymatic synthesis is completed, the nicks are sealed. Double-stranded constructs are sequentially linked to one another to form the entire gene sequence which is verified by DNA sequence analysis. See Glick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA. (ASM Press, Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984 and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-7. 1990.
Zsigόό polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a zsigόό gene. In view of the tissue-specific expression observed for zsigόό by Northern blotting, this gene region is expected to provide for ovary and pancreas-specific expression. Promoter elements from a zsigόό gene could thus be used to direct the tissue-specific expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy. Cloning of 5' flanking sequences also facilitates production of zsigόό proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous zsigόό gene in a cell is altered by introducing into the zsigόό locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site. The targeting sequence is a zsigόό 5' non- coding sequence that permits homologous recombination of the construct with the endogenous zsigόό locus, whereby the sequences within the construct become operably linked with the endogenous zsigόό coding sequence. In this way, an endogenous zsigόό promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.
The present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are zsigόό polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human zsigόό can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zsigόό 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 zsigόό-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction (PCR) (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human zsigόό sequence disclosed herein. Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to zsigόό polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
Those skilled in the art will recognize that the sequence disclosed in SEQ ID NO:l represents a single allele of human zsigόό and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO:l, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO:2. cDNAs generated from alternatively spliced mRNAs, which retain the properties of the zsigόό polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.
The present invention also provides isolated zsigόό polypeptides that are substantially similar to the polypeptides of SEQ ID NO:2 and their orthologs. The term
"substantially similar" is used herein to denote polypeptides having 70%, preferably 75%, more preferably at least 80%, sequence identity to the sequences shown in SEQ ID NO:2 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO:2 or its orthologs.) Percent sequence identity is determined by conventional methods. See, for example,
Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9. 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by the standard one-letter codes). The percent identity is then calculated as:
Total number of identical matches x lOO
[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences]
> •* H r H 1
!2 M ro
1 en <# H ro CM CM
1 1 1 fc o CN CM H ro H
1 1 1 1 s tn o CM H rH H rH
1 1 1 1 1
« tn rH ro H O H ro CN CN 1 1 1 1 1 1 1
S£> CM CM ro ro CM o CM CM ro ro
1 1 1 1 1 1 1 1 1 1 1 ω UO CM O ro ro H CM ro H o H ro CM CM
1 1 1 1 1 1 1 1 1 1 α tn CN CM O ro CM H o ro H o H CM H CM
1 1 1 1 1 1 1 1 1 u σs ro ro ro rH H ro H CM ro H H CM CM H
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Q vo ro O CM H H ro ^r1 H ro ro H O ro ro
1 1 1 1 1 1 1 1 1 1 1 1 1
S3 SD H ro o O o H ro ro o CM ro CM H O CM ro
1 1 1 1 1 1 1 1 1
P, tn O ro H O CM O ro CM CM H ro CN rH H ro CM ro
1 1 1 1 1 1 1 1 1 1 1 1 : «* H CM o H H o CM H H rH M H H O ro CM O
1 1 1 1 1 1 1 1 1 1 1 1 1 c P, S3 Q U σ W O ffi M J « 2 Ot CΛ E-i 5 !* >
LΠ LΠ o Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above.
Those skilled in the art appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant zsigόό. The FASTA algorithm is described by Pearson and Lipman, Proc. NatT Acad. Sci. USA 85:2444 (1988), and by Pearson. Meth. Enzymol. 183:63 (1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO:2) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions. Preferred parameters for FASTA analysis are: ktup=T, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).
FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.
The present invention includes nucleic acid molecules that encode a polypeptide having one or more conservative amino acid changes, compared with the amino acid sequence of SEQ ID NO:2. The BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. NatT Acad. 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. As used herein, the language "conservative amino acid substitution" refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
Variant zsigόό polypeptides or substantially homologous zsigόό polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. The present invention thus includes polypeptides of from about 35 to about 90 amino acid residues, or in some embodiments, 15 to 16 amino acid residues (e.g., residues 69-83 of SEQ ID NO:2 with or without an amide group), that comprise a sequence that is at least 80%, preferably at least 90%, and more preferably 95% or more identical to the corresponding region of SEQ ID NO:2. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zsigόό polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.
Table 4
Conservative a ino acid substitutions
Basic: arginine lysine histidine Acidic: glutamic acid aspartic acid
Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine
Aromatic: phenylalanine tryptophan tyrosine Small : glycine alanine serine threonine methionine
The present invention further provides a variety of other polypeptide fusions and related multimeric proteins comprising one or more polypeptide fusions. For example, a zsigόό polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in this regard include immunoglobulin constant region domains. Immunoglobulin-zsigόό polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric zsigόό analogs. Auxiliary domains can be fused to zsigόό polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen). For example, a zsigόό polypeptide or protein could be targeted to a predetermined cell type by fusing a zsigόό polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell. In this way, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. A zsigόό polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain. Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34:1-9, 1996.
The proteins of the present invention can also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, tr< y-3-methylproline, 2,4-methanoproline, cw-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, αt7ø-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722. 1991; Εllman et al.. Methods Enzymol. 202:301. 1991; Chung et al., Science 259:806-9, 1993; and Chung et al.. Proc. Natl. Acad. Sci. USA 90:10145-9. 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993). A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for zsigόό amino acid residues.
Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al, Proc. Natl. Acad. Sci. USA 88:4498-502, 1991). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699-708, 1996. Sites of ligand-receptor or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al, J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related polypeptides.
Determination of amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can determine specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule. Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity and computer analysis using available software (e.g., the Insight II® viewer and homology modeling tools; MSI, San Diego, CA), secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). In general, when designing modifications to molecules or identifying specific fragments determination of structure will be accompanied by evaluating activity of modified molecules.
Amino acid sequence changes are made in zsigόό polypeptides so as to minimize disruption of higher order structure essential to biological activity. For example, when the zsigόό polypeptide comprises one or more helices, changes in amino acid residues will be made so as not to disrupt the helix geometry and other components of the molecule where changes in conformation abate some critical function, for example, binding of the molecule to its binding partners. The effects of amino acid sequence changes can be predicted by, for example, computer modeling as disclosed above or determined by analysis of crystal structure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995). Other techniques that are well known in the art compare folding of a variant protein to a standard molecule (e.g., the native protein). For example, comparison of the cysteine pattern in a variant and standard molecules can be made. Mass spectrometry and chemical modification using reduction and alkylation provide methods for determining cysteine residues which are associated with disulfide bonds or are free of such associations (Bean et al., Anal. Biochem. 201:216- 226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Patterson et al., Anal. Chem. 66:3727-3732, 1994). It is generally believed that if a modified molecule does not have the same disulfide bonding pattern as the standard molecule folding would be affected. Another well known and accepted method for measuring folding is circular dichrosism (CD). Measuring and comparing the CD spectra generated by a modified molecule and standard molecule is routine (Johnson, Proteins 7:205-214, 1990). Crystallography is another well known method for analyzing folding and structure. Nuclear magnetic resonance (NMR), digestive peptide mapping and epitope mapping are also known methods for analyzing folding and structural similarities between proteins and polypeptides (Schaanan et al„ Science 257:961-964. 1992).
A Hopp/Woods hydrophilicity profile of the zsigόό protein sequence as shown in SEQ ID NO:2 can be generated (Hopp et al., Proc. Natl. Acad. Sci.78:3824- 3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquier et al., Protein Engineering I L 153- 169, 1998). The profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored (See, Figure). For example, in zsigόό, hydrophilic regions include: (1) amino acid number 1 (Met) to amino acid number 6 (Glu) of SEQ ID NO:2; (2) amino acid number 7 (Val) to amino acid number 12 (He) of SEQ ID NO:2; (3) amino acid number 26 (Ser) to amino acid number 32 (Ser) of SEQ ID NO:2; (4) amino acid number 29 (Asp) to amino acid number 34 (Ser) of SEQ ID NO:2; and (5) amino acid number 51 (Leu) to amino acid number 56 (Glu) of SEQ ID NO:2.
Those skilled in the art will recognize that hydrophilicity or hydrophobicity will be taken into account when designing modifications in the amino acid sequence of a zsigόό polypeptide, so as not to disrupt the overall structural and biological profile. Of particular interest for replacement are hydrophobic residues selected from the group consisting of Val, Leu and He or the group consisting of Met, Gly, Ser, Ala, Tyr and Trp. For example, hydrophobic residues tolerant of substitution could include such residues as shown in SEQ ID NO: 2. Cysteine residues are relatively intolerant of substitution.
The identities of essential amino acids can also be inferred from analysis of sequence similarity between other proteins with zsigόό. Using methods such as "FASTA" analysis described previously, regions of high similarity are identified within a family of proteins and used to analyze amino acid sequence for conserved regions. An alternative approach to identifying a variant zsigόό polynucleotide on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant zsigόό polynucleotide can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, as discussed above.
Other methods of identifying essential amino acids in the polypeptides of the present invention are procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et al., Proc. Natl Acad. Sci. USA 88:4498 (1991), Coombs and Corey, "Site-Directed Mutagenesis and Protein Engineering," in Proteins: Analysis and Design, Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al, J. Biol. Chem. 271:4699 (1996).
The present invention also includes functional fragments of zsigόό polypeptides and nucleic acid molecules encoding such functional fragments. A "functional" zsigόό or fragment thereof defined herein is characterized by its proliferative or differentiating activity, by its ability to induce or inhibit specialized cell functions, or by its ability to bind specifically to an anti-zsigόό antibody or zsigόό receptor (either soluble or immobilized). The present invention further provides fusion proteins encompassing: (a) polypeptide molecules comprising one or more of the motifs described above; and (b) functional fragments comprising one or more of these motifs. The other polypeptide portion of the fusion protein may be contributed by a related, non-native and/or an unrelated secretory signal peptide that facilitates secretion of the fusion protein.
Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a zsigόό polypeptide. As an illustration, DNA molecules having the nucleotide sequence of SEQ ID NO: 1 or fragments thereof, can be digested with Bal 1 nuclease to obtain a series of nested deletions. These DNA fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for zsigόό activity, or for the ability to bind anti-zsigόό antibodies or zsigόό receptor. One alternative to exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired zsigόό fragment. Alternatively, particular fragments of a zsigόό polynucleotide can be synthesized using the polymerase chain reaction. Standard methods for identifying functional domains are well-known to those of skill in the art. For example, studies on the truncation at either or both termini of interferons have been summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover, standard techniques for functional analysis of proteins are described by, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993); Content et al., "Expression and preliminary deletion analysis of the 42 kDa 2-5A synthetase induced by human interferon," in Biological Interferon Systems. Proceedings of ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72 (Nijhoff 1987); Herschman, "The EGF Receptor," in Control of Animal Cell Proliferation _. Boynton et al., (eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol. Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995); and Meisel et al., Plant Molec. Biol. 30:1 (1996). Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer f Science 241:53-7, 1988) or Bowie and Sauer fProc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region- directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).
Variants of the disclosed zsigόό DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994 and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
Mutagenesis methods as disclosed herein can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells. Mutagenized DNA molecules that encode active polypeptides (e.g., secreted and detected by antibodies; or measured by a signal transduction type assay) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
In addition, the proteins of the present invention (or polypeptide fragments thereof) can be joined to other bioactive molecules, particularly other cytokines, to provide multi-functional molecules. For example, one or more domains, hydrophilic regions, or regions containing motifs 1-4 from zsigόό can be joined to other proteins to enhance their biological properties or efficiency of production.
The present invention thus provides a series of novel, hybrid molecules in which a segment comprising one or more of the domains, hydrophilic regions, or regions containing motifs 1-4 of zsigόό is fused to another polypeptide. Fusion is preferably done by splicing at the DNA level to allow expression of chimeric molecules in recombinant production systems. The resultant molecules are then assayed for such properties as improved solubility, improved stability, prolonged clearance half-life, improved expression and secretion levels, and pharmacodynamics. Such hybrid molecules may further comprise additional amino acid residues (e.g. a polypeptide linker) between the component proteins or polypeptides.
Using the methods discussed herein, one of ordinary skill in the art can identify and/or prepare a variety of polypeptides that are substantially similar to SEQ ID NO:2 or allelic variants thereof and retain the properties of the wild-type protein. For example, using the methods described above, one could identify a receptor binding domain on zsigόό; an extracellular ligand-binding domain of a receptor for zsigόό; heterodimeric and homodimeric binding domains; other functional or structural domains; affinity tags; or other domains important for protein-protein interactions or signal transduction. Such polypeptides may also include additional polypeptide segments as generally disclosed above.
For any zsigόό 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 zsigόό polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides, can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., eds., Current Protocols in Molecular Biology. John Wiley and Sons, Inc., NY, 1987.
In general, a DNA sequence encoding a zsigόό polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
To direct a zsigόό polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of zsigόό, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo. The secretory signal sequence is operably linked to the zsigόό DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al, U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).
Alternatively, the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway. The present invention provides for such fusion polypeptides. A signal fusion polypeptide can be made wherein a secretory signal sequence derived from residue 1 (Met) to residue 27 (Ser) of SEQ ID 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. For example, these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein. Such fusions may be used in vivo or in vitro to direct peptides through the secretory pathway. Cultured mammalian cells are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981 : Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al, EMBO J. 1:841-5, 1982), DEAE- dextran mediated transfection (Ausubel et al., ibid.), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90, 1989; Wang and Finer, Nature Med. 2:714-6, 1996). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Patent No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-Kl; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, VA. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter. Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured, for example in the presence of the selective agent, and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. In addition to selection of transfectants by agents, alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) H:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa calif ornica nuclear polyhedrosis virus (AcNPV). See, King, L.A. and Possee, R.D., The Baculovirus Expression
System: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D.R. et al., Baculovirus Expression Vectors: A Laboratory Manual. New York, Oxford University Press., 1994; and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology. Totowa, NJ, Humana Press, 1995. The second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, V.A, et al., J Virol 67:4566-79, 1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBacl™ (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zsigόό polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid." The pFastBacl™ transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case zsigόό. However, pFastBacl™ can be modified to a considerable degree. The polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, pό.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins, M.S. and Possee, R.D., J. Gen. Virol. 71:971-6, 1990; Bonning, B.C. et al., J. Gen. Virol. 75:1551-6, 1994; and, Chazenbalk, G.D., and Rapoport, B., J. Biol. Chem. 270:1543-9. 1995. In such transfer vector constructs, a short or long version of the basic protein promoter can be used. Moreover, transfer vectors can be constructed which replace the native zsigόό secretory signal sequences with secretory signal sequences derived from insect proteins. For example, a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen, Carlsbad, CA), or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native zsigόό secretory signal sequence. In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed zsigόό polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Using a technique known in the art, a transfer vector containing zsigόό is transformed into E. coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus that expresses zsigόό is subsequently produced. Recombinant viral stocks are made by methods commonly used in the art.
The recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C, 1994. Another suitable cell line is the High FiveO™ cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No. 5,300,435). Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa, KS) or Express FiveO™ (Life Technologies) for the T. ni cells. The cells are grown up from an inoculation density of approximately 2-5 x 105 cells to a density of 1-2 x 106 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3. Procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R.D., ibid.; O'Reilly, D.R. et al., ibid.: Richardson, C. D., ibid.). Subsequent purification of the zsigόό polypeptide from the supernatant can be achieved using methods described herein.
Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-65, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533.
The use of Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in transforming R. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. For polypeptide production in R. methanolica, it is preferred that the promoter and terminator in the plasmid be that of a 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), and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid flanked at both ends by host DNA sequences. A preferred selectable marker for use in Pichia methanolica is a R. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine. For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (A UG1 and A UG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
Prokaryotic host cells, including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.). When expressing a zsigόό polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell. R. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25 °C to 35°C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. A preferred culture medium for R. methanolica is YEPD (2% D-glucose, 2% Bacto™ Peptone (Difco Laboratories, Detroit, MI), 1% Bacto™ yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).
It is preferred to purify the polypeptides of the present invention to >80% purity, more preferably to >90% purity, even more preferably >95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
Expressed recombinant zsigόό polypeptides (or chimeric zsigόό polypeptides) can be purified using fractionation and/or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred. Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross- linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles & Methods. Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, R. (ed.) Protein Purification: Principles and practice. Springer- Verlag, New York, 1987.
The polypeptides of the present invention can be isolated by exploitation of their biological and structural properties. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents. Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol., Vol. 182, "Guide to Protein Purification", M. Deutscher, (ed.), Acad. Press, San Diego, 1990, pp.529-39). Within additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (e.g., maltose-binding protein, an immunoglobulin domain) may be constructed to facilitate purification.
Moreover, using methods described in the art, polypeptide fusions, or hybrid zsigόό proteins, are constructed using regions or domains of zsigόό in combination with those of paralogs, orthologs, or heterologous proteins (Sambrook et al., ibid., Altschul et al., ibid., Picard. D., Cur. Opin. Biology. 5:511-515, 1994, and references therein). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest. Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure. Fusion polypeptides can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide encoding one or more components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. For example, part or all of a domain(s) conferring a biological function may be swapped between zsigόό of the present invention with the functionally equivalent domain(s) from another protein. Such domains include, but are not limited to, the secretory signal sequence, and motifs 1 through 4. Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other similar proteins (e.g. paralogs, or orthologs) or heterologous proteins, depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein.
Standard molecular biological and cloning techniques can be used to swap the equivalent domains between the zsigόό polypeptide and those polypeptides to which they are fused. Generally, a DNA segment that encodes a domain of interest, e.g., a zsigόό domain described herein, is operably linked in frame to at least one other DNA segment encoding an additional polypeptide (for instance an analogous domain or region in a similar protein), and inserted into an appropriate expression vector, as described herein. Generally DNA constructs are made such that the several DNA segments that encode the corresponding regions of a polypeptide are operably linked in frame to make a single construct that encodes the entire fusion protein, or a functional portion thereof. For example, a DNA construct would encode from N-terminus to C- terminus a fusion protein comprising a signal polypeptide followed by a mature polypeptide; or a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by a mature protein. Such fusion proteins can be expressed, isolated, and assayed for activity as described herein. zsigόό polypeptides or fragments thereof may also be prepared through chemical synthesis, zsigόό 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. Polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. Methods for synthesizing polypeptides are well known in the art. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Kaiser et al., Anal. Biochem. 34:595, 1970. After the entire synthesis of the desired peptide on a solid support, the peptide-resin is with a reagent which cleaves the polypeptide from the resin and removes most of the side-chain protecting groups. Such methods are well established in the art.
The activity of molecules of the present invention can be measured using a variety of assays that measure for example, signal transduction, cell motility, steroidogenesis, mitogenesis or binding. Such assays are well known in the art. The zsigόό polypeptides of the present invention can be used to study pancreatic cell proliferation or differentiation. Such methods of the present invention generally comprise incubating cells, β cells, δ cells, F cells and acinar cells in the presence and absence of zsigόό polypeptide, monoclonal antibody, agonist or antagonist thereof and observing changes in islet cell proliferation or differentiation. Similarly, the zsigόό polypeptides of the present invention can be used to study pituitary cell proliferation or differentiation.
A further aspect of the invention provides a method for studying insulin. Such methods of the present invention comprise incubating adipocytes in a culture medium comprising zsigόό polypeptide, monoclonal antibody, agonist or antagonist thereof ± insulin and observing changes in adipocyte protein secretion or differentiation.
The present invention also provides methods of studying mammalian cellular metabolism. Such methods of the present invention comprise incubating cells to be studied, for example, an appropriate human cell line, ± zsigόό polypeptide, monoclonal antibody, agonist or antagonist thereof, and observing changes in adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, or the like.
Also, zsigόό polypeptides, agonists or antagonists thereof may be therapeutically useful for promoting wound healing, for example, in the pancreas. To verify the presence of this capability in zsigόό polypeptides, agonists or antagonists of the present invention, such zsigόό polypeptides, agonists or antagonists are evaluated with respect to their ability to facilitate wound healing according to procedures known in the art. If desired, zsigόό polypeptide performance in this regard can be compared to growth factors, such as EGF, NGF, TGF-α, TGF-β, insulin, IGF-I, IGF-II, fibroblast growth factor (FGF) and the like. In addition, zsigόό polypeptides or agonists or antagonists thereof may be evaluated in combination with one or more growth factors to identify synergistic effects.
In addition, zsigόό polypeptides, agonists or antagonists thereof may be therapeutically useful for anti-microbial applications. To verify the presence of this capability in zsigόό polypeptides, agonists or antagonists of the present invention, such zsigόό polypeptides, agonists or antagonists are evaluated with respect to their antimicrobial properties according to procedures known in the art. See, for example, Barsum et al., Eur. Respir. J. 8(5): 709-14, 1995; Sandovsky-Losica et al., J. Med. Vet. Mvcol (England) 28(4): 279-87, 1990; Mehentee et al, J. Gen. Microbiol (England) 135 (Pt. 8 : 2181-8, 1989; Segal and Savage, Journal of Medical and Veterinary Mycology 24: 477-479, 1986 and the like. If desired, zsigόό polypeptide performance in this regard can be compared to proteins known to be functional in this regard, such as proline-rich proteins, lysozyme, histatins, lactoperoxidase or the like. In addition, zsigόό polypeptides or agonists or antagonists thereof may be evaluated in combination with one or more antimicrobial agents to identify synergistic effects.
Anti-microbial protective agents may be directly acting or indirectly acting. Such agents operating via membrane association or pore forming mechanisms of action directly attach to the offending microbe. Anti-microbial agents can also act via an enzymatic mechanism, breaking down microbial protective substances or the cell wall membrane thereof. Anti-microbial agents, capable of inhibiting microorganism proliferation or action or of disrupting microorganism integrity by either mechanism set forth above, are useful in methods for preventing contamination in cell culture by microbes susceptible to that anti-microbial activity. Such techniques involve culturing cells in the presence of an effective amount of said zsigόό polypeptide, or an agonist or antagonist thereof.
Also, zsigόό polypeptides or agonists thereof may be used as cell culture reagents in in vitro studies of exogenous microorganism infection, such as bacterial, viral or fungal infection. Such moieties may also be used in in vivo animal models of infection. Also, the microorganism-adherence properties of zsigόό polypeptides or agonists thereof can be studied under a variety of conditions in binding assays and the like.
Proteins of the present invention are useful for example, in treating ovarian, pancreatic, ocular, blood or bone disorders, can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to the appropriate animal model. For instance, host cells expressing a secreted form of zsigόό polypeptide may be embedded in an alginate environment and injected (implanted) into recipient animals. Alginate-poly-L-lysine microencapsulation, permselective membrane encapsulation and diffusion chambers are a means to entrap transfected mammalian cells or primary mammalian cells to permit the diffusion of proteins and other macromolecules secreted or released by the captured cells to the recipient animal. Most importantly, the capsules mask and shield the foreign, embedded cells from the recipient animal's immune response. Such encapsulations can extend the life of the injected cells from a few hours or days (naked cells) to several weeks (embedded cells). Alginate threads provide a simple and quick means for generating embedded cells and testing, in vivo, the proteins secreted therefrom.
The materials needed to generate the alginate threads are known in the art. In an exemplary procedure, 3% alginate is prepared in sterile H2O, and sterile filtered. Just prior to preparation of alginate threads, the alginate solution is again filtered. An approximately 50% cell suspension (containing about 5 x 10^ to about 5 x
10' cells/ml) is mixed with the 3% alginate solution. One ml of the alginate/cell suspension is extruded into a 100 mM sterile filtered CaCl2 solution over a time period of -15 min, forming a "thread". The extruded thread is then transferred into a solution of 50 mM CaCl2, and then into a solution of 25 mM CaCl2. The thread is then rinsed with deionized water before coating the thread by incubating in a 0.01% solution of poly-L-lysine. Finally, the thread is rinsed with Lactated Ringer's Solution and drawn from solution into a syringe barrel (without needle). A large bore needle is then attached to the syringe, and the thread is intraperitoneally injected into a recipient in a minimal volume of the Lactated Ringer's Solution. An in vivo approach for assaying proteins of the present invention involves viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for review, see T.C. Becker et al., Meth. Cell Biol. 43:161-89, 1994; and J.T. Douglas and D.T. Curiel, Science & Medicine 4:44-53, 1997). The adenovirus system offers several advantages: (i) adenovirus can accommodate relatively large DNA inserts; (ii) can be grown to high- titer; (iii) infect a broad range of mammalian cell types; and (iv) can be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
Using adenovirus vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In an exemplary system, the essential El gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary). When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adeno viral delivery system has an El gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.
Moreover, adenoviral vectors containing various deletions of viral genes can be used in an attempt to reduce or eliminate immune responses to the vector. Such adenoviruses are El deleted, and in addition contain deletions of E2A or E4 (Lusky, M. et al., J. Virol. 72:2022-2032, 1998; Raper, S.E. et al., Human Gene Therapy 9:671- 679, 1998). In addition, deletion of E2b is reported to reduce immune responses (Amalfitano, A. et al., J. Virol. 72:926-933, 1998). Moreover, by deleting the entire adenovirus genome, very large inserts of heterologous DNA can be accommodated.
Generation of so called "gutless" adenoviruses where all viral genes are deleted are particularly advantageous for insertion of large inserts of heterologous DNA. For review, see Yeh, P. and Perricaudet, M., FASEB J. 1 1 :615-623, 1997.
The adenovirus system can also be used for protein production in vitro. By culturing adenovirus-infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division. Alternatively, adenovirus vector infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (See Gamier et al., Cytotechnol. 15:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the disposition of the expressed protein in the cell. Within the infected 293 cell production protocol, non-secreted proteins may also be effectively obtained.
In view of the tissue distribution observed for zsigόό, agonists (including the natural ligand/ substrate/ cofactor/ etc.) and antagonists have enormous potential in both in vitro and in vivo applications. Compounds identified as zsigόό agonists are useful for stimulating cell growth or signal transduction in vitro and in vivo. For example, zsigόό and agonist compounds are useful as components of defined cell culture media, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture. Agonists are thus useful in specifically promoting the growth and/or development of cells in culture. Considering the high expression of zsigόό ovary and pancreas, zsigόό polypeptides and zsigόό agonists may be particularly useful as research reagents, particularly for the growth of pituitary and endocrine cell types, ovarian cell lines, human eggs, cells from animal embryos or primary cultures derived from these tissues. As such, zsigόό polypeptide can be provided as a supplement in cell culture medium.
Antagonists are also useful as research reagents for characterizing sites of ligand-receptor interaction. Inhibitors of zsigόό activity (zsigόό antagonists) include anti-zsigόό antibodies and soluble proteins which bind zsigόό polypeptide. Inhibitors of zsigόό activity (zsigόό antagonists) include anti-zsig66 antibodies and soluble zsigόό receptors, as well as other peptidic and non-peptidic agents (including ribozymes).
Zsigόό polypeptide can be used to identify inhibitors (antagonists) of its activity. Test compounds are added to the biological or biochemical assays disclosed herein to identify compounds that inhibit the activity of zsigόό. In addition to those assays disclosed herein, samples can be tested for inhibition of zsigόό activity within a variety of assays designed to measure receptor binding or the stimulation inhibition of zsigόό-dependent cellular responses. For example, zsigόό-responsive cell lines can be transfected with a reporter gene construct that is responsive to a zsigόό-stimulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a zsig66-DNA response element operably linked to a gene encoding an assay detectable protein, such as luciferase. DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE) insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) and serum response elements (SRE) (Shaw et al., Cell 56: 563- 72, 1989). Cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem. 263 (19):9063-6; 1988 and Habener, Molec. Endocrinol. 4 (8):1087-94; 1990. Hormone response elements are reviewed in Beato, Cell 56:335-44; 1989. Candidate compounds that serve as test samples including solutions, mixtures or extracts, are tested for the level of response to the zsigόό polypeptide. The ability of a test sample to inhibit the activity of zsigόό polypeptide on the target cells as evidenced by a decrease in zsigόό stimulation of reporter gene expression in the presence of a test sample relative to a control which was cultured in the absence of a test sample. Assays of this type will detect compounds that directly block zsigόό binding to cell-surface receptors, e.g., dimerization, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding. Alternatively, compounds or other samples can be tested for direct blocking of zsigόό binding to receptor using zsigόό tagged with a detectable label (e.g., 125I, biotin, horseradish peroxidase, FITC, and the like). Within assays of this type, the ability of a test sample to inhibit the binding of labeled zsigόό to the receptor is indicative of inhibitory activity, which can be confirmed through secondary assays. Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.
Alternatively, the above methodology may be used to identify agonists of zsigόό activity. Candidate compounds serving as test samples including solutions, mixtures or extracts, are tested for the ability to mimic the activity of zsigόό polypeptide on the target cells as evidenced by stimulation of reporter gene expression in the presence of a test sample and the absence of zsigόό, relative to a control (cultured in the absence of a test sample and the absence of zsigόό polypeptide), using assays as described above. A zsigόό polypeptide can be expressed as a fusion with an immunoglobulin heavy chain constant region, typically an Fc fragment, which contains two constant region domains and lacks the variable region. Methods for preparing such fusions are disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584. Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfide bonded to each other and two non-Ig polypeptides are arrayed in closed proximity to each other. Fusions of this type can be used to affinity purify ligand, as an in vitro assay tool, or an antagonist of zsigόό. For use in assays, the chimeras are bound to a support via the Fc region and used in an ELISA format.
A zsigόό polypeptide can also be used for purification of receptor or polypeptides to which it binds. The zsigόό polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica- based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use. Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N- hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation. The resulting medium will generally be configured in the form of a column, and membrane fractions containing receptors are passed through the column one or more times to allow the receptor to bind to the ligand zsigόό polypeptide. The receptor is then eluted using changes in salt concentration, chaotropic agents (guanidine HCI), or pH to disrupt ligand-receptor binding. An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/ anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed. Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993. A receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell. A test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
Ligand-binding receptor polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 5J_: 660-72, 1949) and calorimetric assays (Cunningham et al., Science 253:545-48, 1991 ; Cunningham et al.. Science 245:821-25. 1991).
As a ligand, the activity of zsigόό polypeptide can be measured by a silicon-based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses. An exemplary device is the Cytosensor™ Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA. A variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell, H.M. et al., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol. 228:84-108, 1997; Arimilli, S. et al, J. Immunol. Meth. 212:49-59, 1998; Van Liefde, I. et al., Eur. J. Pharmacol. 346:87-95, 1998. The microphysiometer can be used for assaying adherent or non- adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including zsigόό polypeptide, its agonists, or antagonists. Preferably, the microphysiometer is used to measure responses of a zsigόό-responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to zsigόό polypeptide. Zsigόό-responsive eukaryotic cells comprise cells into which a receptor for zsigόό has been transfected creating a cell that is responsive to zsigόό; or cells naturally responsive to zsigόό such as cells derived from pituitary tissue. Differences, measured by a change, for example, an increase or diminution in extracellular acidification, in the response of cells exposed to zsigόό polypeptide, relative to a control not exposed to zsigόό, are a direct measurement of zsigόό-modulated cellular responses. Moreover, such zsigόό-modulated responses can be assayed under a variety of stimuli. Using the microphysiometer, there is provided a method of identifying agonists of zsigόό polypeptide, comprising providing cells responsive to a zsigόό polypeptide, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test compound, and detecting a change, for example, an increase or diminution, in a cellular response of the second portion of the cells as compared to the first portion of the cells. The change in cellular response is shown as a measurable change extracellular acidification rate. Moreover, culturing a third portion of the cells in the presence of zsigόό polypeptide and the absence of a test compound can be used as a positive control for the zsigόό-responsive cells, and as a control to compare the agonist activity of a test compound with that of the zsigόό polypeptide. Moreover, using the microphysiometer, there is provided a method of identifying antagonists of zsigόό polypeptide, comprising providing cells responsive to a zsigόό polypeptide, culturing a first portion of the cells in the presence of zsigόό and the absence of a test compound, culturing a second portion of the cells in the presence of zsigόό and the presence of a test compound, and detecting a change, for example, an increase or a diminution in a cellular response of the second portion of the cells as compared to the first portion of the cells. The change in cellular response is shown as a measurable change extracellular acidification rate. Antagonists and agonists, for zsigόό polypeptide, can be rapidly identified using this method.
Moreover, zsigόό can be used to identify cells, tissues, or cell lines which respond to a zsigόό-stimulated pathway. The microphysiometer, described above, can be used to rapidly identify ligand-responsive cells, such as cells responsive to zsigόό of the present invention. Cells can be cultured in the presence or absence of zsigόό polypeptide. Those cells which elicit a measurable change in extracellular acidification in the presence of zsigόό are responsive to zsigόό. Such cell lines, can be used to identify antagonists and agonists of zsigόό polypeptide as described above. The polypeptides, nucleic acid and/or antibodies of the present invention may be used in treatment of disorders associated with gonadal development, pregnancy, pubertal changes, menopause, ovarian cancer, fertility, ovarian function, polycystic ovarian syndrome, pancreas, diabetes, eye disease, pituitary function, blood pressure regulation, water balance, osteoporosis, and other bone diseases. The molecules of the present invention may used to modulate or to treat or prevent development of pathological conditions in such diverse tissue as pancreas and ovary. In particular, certain syndromes or diseases may be amenable to such diagnosis, treatment or prevention. Moreover, natural functions, such as ovulation, may be suppressed or controlled for use in birth control by molecules of the present invention. For a review on pituitary function and diseases of which zsigόό may effect or regulate, see Imura, H.
(ed) The Pituitary Gland. Raven Press, New York, 1994.
As a pituitary-expressed polypeptide, the zsigόό polypeptide of the present invention may act in the neuroendocrine/exocrine cell fate decision pathway in organ systems in the body, and is therefore capable of regulating the expansion of neuroendocrine and exocrine cells in the pancreas. One such regulatory use is that of islet cell regeneration. Also, it has been hypothesized that the autoimmunity that triggers IDDM starts in utero, and zsigόό polypeptide is a developmental gene involved in cell partitioning. Assays and animal models are known in the art for monitoring the exocrine/neuroendocrine cell lineage decision, for observing pancreatic cell balance and for evaluating zsigόό polypeptide, fragment, fusion protein, antibody, agonist or antagonist in the prevention or treatment of the conditions set forth above. The molecules of the present invention will be useful for treating growth disorders, adrenal disorders, hormone and pubertal disorders, reproductive disorders and cancers, menopause, disorders associated with production of breast milk in humans and animals, enhancing production of breast milk in humans and animals, and pituitary disorders and cancers. The polypeptides, nucleic acid and/or antibodies of the present invention can be used in treatment of disorders and complications associated with these diseases. The molecules of the present invention can be used to modulate pituitary function, pituitary hormones, reproductive organs, enhance fertility, birth control, and the like, or to treat or prevent development of pathological conditions in such diverse tissue as pancreas, other endocrine tissues and pituitary. In particular, certain syndromes and diseases may be amenable to such diagnosis, treatment or prevention. Zsigόό polypeptide is expressed in the pituitary and may have additional biological activity as described below.
Oogenesis is the process by which a diploid stem cell proceeds through multiple stages of differentiation, culminating in the formation of a terminally differentiated cell with a unique function, an oocyte. Unlike spermatogenesis, which begins at puberty and continues on through the life of a male, oogenesis begins during fetal development and by birth, a female's entire supply of primary oocytes are stored in the ovaries in primordial follicles and await maturation and release. In the adult ovary, folliculogenesis starts when the follicles enter the growth phase. Early growing follicles undergo a dramatic process of cellular proliferation and differentiation. The classic control of ovarian function by luteinizing hormone (LH) and follicle stimulating hormone (FSH) is now thought to include the action of a variety of molecules that act to promote cell-cell interactions between cells of the follicle. For review, see Gougeon, A., Endocrine Rev. 17:121-155, 1996. Hence, the mechanisms for controlling ovarian folliculogenesis and dominant follicle selection are still under investigation. As zsigόό is expressed in the pituitary, it may serve a role in regulating folliculogenesis and dominant follicle selection, by affecting proliferation or differentiation of follicular cells, affecting cell-cell interactions, modulating hormones involved in the process, and the like. The ovarian cycle in mammals includes the growth and maturation of follicles, followed by ovulation and transformation of follicles into corpea lutea. The physiological events in the ovarian cycle are dependent on interactions between hormones and cells within the hypothalamic-pituitary-ovarian axis, including gonadotropin releasing hormone (GnRH), LH, and FSH. In addition, estradiol, synthesized in the follicle, primes the hypothalamic-pituitary axis and is required for the mid-cycle surge of gonadotropin that stimulates the resumption of oocyte meiosis and leads to ovulation and subsequent extrusion of an oocyte from the follicle. This gonadotropin surge also promotes the differentiation of the follicular cells from secreting estradiol to secreting progesterone. Progesterone, secreted by the corpus luteum, is needed for uterine development required for the implantation of fertilized oocytes. The central role of hypothalamic-pituitary-gonadal hormones in the ovarian cycle and reproductive cascade, and the role of sex steroids on target tissues and organs, e.g., uterus, breast, adipose, bones and liver, has made modulators of their activity desirable for therapeutic applications. Such applications include treatments for precocious puberty, endometriosis, uterine leiomyomata, hirsutism, infertility, pre menstrual syndrome (PMS), amenorrhea, and as contraceptive agents.
Zsigόό polypeptides, agonists and antagonists which modulate the actions of such hormones can be of therapeutic value. Moreover, zsigόό may act as a hormone itself, and as such zsigόό polypeptides, agonists and antagonists can be of therapeutic value. Such molecules can also be useful for modulating steroidogenesis, both in vivo and in vitro, and modulating aspects of the ovarian cycle such as oocyte maturation, ovarian cell-cell interactions, follicular development and rupture, luteal function, and promoting uterine implantation of fertilized oocytes. Molecules which modulate hormone action can be beneficial therapeutics for use prior to or at onset of puberty. For example, puberty in females is marked by an establishment of feed-back loops to control hormone levels and hormone production. Abnormalities resulting from hormone imbalances during puberty have been observed and include precocious puberty, where pubertal changes occur in females prior to the age of 8. Hormone- modulating molecules, can be used, in this case, to suppress hormone secretion and delay onset of puberty. The level and ratio of gonadotropin and steroid hormones can be used to assess the existence of hormonal imbalances associated with diseases, as well as determine whether normal hormonal balance has been restored after administration of a therapeutic agent. Similarly, the level of zsigόό can also be measured in its relationship to hormonal imbalances. Determination of estradiol, progesterone, LH, and FSH, for example, from serum is known by one of skill in the art. Such assays can be used to monitor the hormone levels after administration of zsigόό in vivo, or in a transgenic mouse model where the zsigόό gene is expressed or the murine ortholog is deleted. Thus, as a hormone-modulating molecule, zsigόό polypeptide can have therapeutic application for treating, for example, breakthrough menopausal bleeding, as part of a therapeutic regime for pregnancy support, or for treating symptoms associated with polycystic ovarian syndrome (PCOS), PMS and menopause. In addition, other in vivo rodent models are known in the art to assay effects of zsigόό polypeptide on, for example, polycystic ovarian syndrome (PCOS). Proteins of the present invention may also be used in applications for enhancing fertilization during assisted reproduction in humans and in animals. Such assisted reproduction methods are known in the art and include artificial insemination, in vitro fertilization, embryo transfer, and gamete intrafallopian transfer. Such methods are useful for assisting those who may have physiological or metabolic disorders that prevent or impede natural conception. Such methods are also used in animal breeding programs, e.g., for livestock, racehorses, domestic and wild animals, and could be used as methods for the creation of transgenic animals. Zsigόό polypeptides could be used in the induction of ovulation, either independently or in conjunction with a regimen of gonadotropins or agents such as clomiphene citrate or bromocriptine (Speroff et al., Induction of ovulation, Clinical Gynecologic Endocrinology and Infertility, 5th ed., Baltimore, Williams & Wilkins, 1994). As such, proteins of the present invention can be administered to the recipient prior to fertilization or combined with the sperm, an egg or an egg-sperm mixture prior to in vitro or in vivo fertilization. Such proteins can also be mixed with oocytes prior to cryopreservation to enhance viability of the preserved oocytes for use in assisted reproduction. The zsigόό polypeptides, agonists and antagonists of the present invention may be directly used as or incorporated into therapies for treating reproductive disorders. Disorders such as luteal phase deficiency would benefit from such therapy (Soules, "Luteal phase deficiency: A subtle abnormality of ovulation" in, Infertility: Evaluation and Treatment, Keye et al., eds., Philadelphia, WB Saunders, 1995). Moreover, administration of gonadotropin-releasing hormone is shown to stimulate reproductive behavior (Riskin and Moss, Res. Bull. 11:481-5, 1983; Kadar et al., Phvsiol. Behav. 51:601-5, 1992 and Silver et al., J. Neruoendocrin. 4:207-10, 199; King and Millar, Cell. Mol. Neurobiol., 15:5-23, 1995). Given the high prevalence of sexual dysfunction and impotence in humans, molecules, such as zsigόό, which may modulate or enhance gonadotropin activity can find application in developing treatments for these conditions.
The zsigόό polypeptides of the present invention can be used to study ovarian and other reproductive tissue cell proliferation, maturation, and differentiation, i.e., by acting as a luteinizing agent that converts granulosa cells from estradiol to progesterone-producing cells. For example, such methods of the present invention generally comprise incubating granulosa cells, theca cells, oocytes or a combination thereof, in the presence and absence of zsigόό polypeptide, monoclonal antibody, agonist or antagonist thereof and observing changes in cell proliferation, maturation and differentiation. See for example, Basini et al..(J. Rep. Immunol. 37:139-53, 1998);
Duleba et al..(Fert. Ster. 69:335-40, 1998); and Campbell, B.K. et al., J. Reprod. and Fert. 112:69-77. 1998V
The molecules of the present invention are useful as components of defined cell culture media, as described herein, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture. Molecules of the present invention are particularly useful in specifically promoting the growth, development, differentiation, and/or maturation of ovarian cells in culture, and may also prove useful in the study of the ovarian cycle, reproductive function, ovarian cell-cell interactions, and fertilization. In addition, zsigόό may affect other hormones aside from those in reproductive tissues. In addition, the present invention also provides methods for studying steroidogenesis and steroid hormone secretion. Such methods generally comprise incubating ovarian or other hormone-producing cells in culture medium comprising zsigόό polypeptides, monoclonal antibodies, agonists or antagonists thereof with and without gonadotropins and/or steroid hormones, and subsequently observing protein and steroid secretion. Exemplary gonadotropin hormones include luteinizing hormone and follicle stimulating hormone (Rouillier et al., Mol. Reprod. Dev. 50:170- 7, 1998). Exemplary steroid hormones include estradiol, androstenedione, and progesterone. Effects of zsigόό on steroidogenesis or steroid secretion can be determined by methods known in the art, such as radioimmunoassay (to detect levels of estradiol, androstenedione, progesterone, and the like), and immunoradiometric assay (IRMA).
Molecules expressed in the pituitary, such as zsigόό polypeptide, and which may modulate hormones, hormone receptors, growth factors, or cell-cell interactions, of the reproductive cascade, other organ systems or the cardiovascular system, or are involved in oocyte or ovarian development or other development, would be useful as markers for cancer of reproductive organs and other organ systems and as therapeutic agents for hormone-dependent cancers, by inhibiting hormone-dependent growth and/or development of tumor cells. Human reproductive system cancers such as ovarian, uterine, cervical, testicular and prostate cancers are common. Moreover, receptors for steroid hormones involved in the reproductive cascade are found in human tumors and tumor cell lines (breast, prostate, endometrial, ovarian, kidney, and pancreatic tumors) (Kakar et al., Mol. Cell. Endocrinol.. 106:145-49, 1994; Kakar and Jennes, Cancer Letts.. 98:57-62, 1995). Thus, expression of zsigόό in pituitary, with concomitant effects in reproductive tissues and other organ systems suggests that polypeptides of the present invention would be useful in diagnostic methods for the detection and monitoring of reproductive tissue and other cancers.
Diagnostic methods of the present invention involve the detection of zsigόό polypeptides in the serum or tissue biopsy of a patient undergoing analysis of reproductive function or evaluation for possible reproductive or other cancer or disease. Such polypeptides can be detected using immunoassay techniques and antibodies, described herein, that are capable of recognizing polypeptide epitopes. More specifically, the present invention contemplates methods for detecting zsigόό polypeptides comprising: exposing a test sample potentially containing zsigόό polypeptides to an antibody attached to a solid support, wherein said antibody binds to a first epitope of a zsigόό polypeptide; washing the immobilized antibody-polypeptide to remove unbound contaminants; exposing the immobilized antibody-polypeptide to a second antibody directed to a second epitope of a zsigόό polypeptide, wherein the second antibody is associated with a detectable label; and detecting the detectable label. Altered levels of zsigόό polypeptides in a test sample, such as serum sweat, saliva, biopsy, and the like, can be monitored as an indication of reproductive function or of cancer or disease, when compared against a normal control. Additional methods using probes or primers derived, for example, from the nucleotide sequences disclosed herein can also be used to detect zsigόό expression in a patient sample, such as a blood, saliva, sweat, tissue sample, or the like. For example, probes can be hybridized to tumor tissues and the hybridized complex detected by in situ hybridization. Zsigόό sequences can also be detected by PCR amplification using cDNA generated by reverse translation of sample mRNA as a template (PCR Primer A Laboratory Manual. Dieffenbach and Dveksler, eds., Cold Spring Harbor Press, 1995). When compared with a normal control, both increases or decreases of zsigόό expression in a patient sample, relative to that of a control, can be monitored and used as an indicator or diagnostic for disease. As a pituitary hormone, zsigόό may regulate blood pressure and water
(osmotic) balance in the human body as discussed herein. As such, zsigόό, agonists, and antagonists have great therapeutic potential in regulating blood pressure. For example, zsigόό, agonists, and antagonists have therapeutic potential in enhancing blood pressure, for example in the case of traumatic shock; or decreasing blood pressure, for example in the case of hypertension. Moreover water balance is important in maintaining electrolytes as well as affecting overall health. As such, zsigόό, agonists, and antagonists have great therapeutic potential in regulating water balance, for example after major organ surgeries, trauma nutritional or disease states that alter such balance.
Moreover, zsigόό polypeptides may modulate mammalian energy balance. The pituitary expression of zsigόό suggests that zsigόό may exhibit effects on glucose uptake, e.g. through GLUT-1, and thermogenesis (thermoregulation). For example, zsigόό polypeptides may find utility in modulating nutrient uptake, as demonstrated, for example, by 2-deoxy-glucose uptake in the brain or the like. Among other methods known in the art or described herein, mammalian energy balance may be evaluated by monitoring one or more of the following metabolic functions: adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, protein synthesis, thermogenesis, oxygen utilization or the like. These metabolic functions are monitored by techniques (assays or animal models) known to one of ordinary skill in the art, as is more fully set forth below. For example, the glucoregulatory effects of insulin are predominantly exerted in the liver, skeletal muscle and adipose tissue. Insulin binds to its cellular receptor in these three tissues and initiates tissue-specific actions that result in, for example, the inhibition of glucose production and the stimulation of glucose utilization. In the liver, insulin stimulates glucose uptake and inhibits gluconeogenesis and glycogenolysis. In skeletal muscle and adipose tissue, insulin acts to stimulate the uptake, storage and utilization of glucose.
Zsigόό polypeptide is expressed in pituitary but may exhibit extra- pituitary activity in organs that affect metabolic functions. Thus, pharmaceutical compositions of the present invention may be useful in prevention or treatment of pancreatic and other endocrine, exocrine or neurocrine disorders. For example, zsigόό may be associated with pathological regulation of the expansion of neurocrine and exocrine cells in the pancreas, as evident in IDDM, pancreatic cancer or the like, or zsigόό may be associated with pathological regulation of reproductive organs such as the ovaries. Pharmaceutical compositions of the present invention may also be involved in prevention or treatment of pancreatic conditions characterized by dysfunction associated with pathological regulation of blood glucose levels, insulin resistance or digestive function. Art-recognized methods exist for monitoring all of the metabolic functions recited above. Thus, one of ordinary skill in the art is able to evaluate zsigόό polypeptides, fragments, fusion proteins, antibodies, agonists and antagonists for metabolic modulating functions. Exemplary modulating techniques are set forth below. Adipogenesis, gluconeogenesis and glycogenolysis are interrelated components of mammalian energy balance, which may be evaluated by known techniques using, for example, ob/ob mice or db/db mice. The ob/ob mice are inbred mice that are homozygous for an inactivating mutation at the ob (obese) locus. Such ob/ob mice are hyperphagic and hypometabolic, and are believed to be deficient in production of circulating OB protein. The db/db mice are inbred mice that are homozygous for an inactivating mutation at the db (diabetes) locus. The db/db mice display a phenotype similar to that of ob/ob mice, except db/db mice display a more severe diabetic phenotype. Such db/db mice are believed to be resistant to the effects of circulating OB protein. Also, various in vitro methods of assessing these parameters are known in the art.
Insulin-stimulated lipogenesis, for example, may be monitored by measuring the incorporation of ^C-acetate into triglyceride (Mackall et al. J. Biol. Chem. 25J,:6462-6464, 1976) or triglyceride accumulation (Kletzien et al., Mol. Pharmacol. 41 :393-398. 1992). Glucose uptake may be evaluated, for example, in an assay for insulin- stimulated glucose transport. Non-transfected, differentiated L6 myotubes (maintained in the absence of G418) are placed in DMEM containing 1 g/l glucose, 0.5 or 1.0% BSA, 20 mM Hepes, and 2 mM glutamine. After two to five hours of culture, the medium is replaced with fresh, glucose-free DMEM containing 0.5 or 1.0% BSA, 20 mM Hepes, 1 mM pyruvate, and 2 mM glutamine. Appropriate concentrations of insulin or IGF-1, or a dilution series of the test substance, are added, and the cells are incubated for 20-30 minutes. -1H or 14c-labeled deoxyglucose is added to «50 1 M final concentration, and the cells are incubated for approximately 10-30 minutes. The cells are then quickly rinsed with cold buffer (e.g. PBS), then lysed with a suitable lysing agent (e.g. 1% SDS or 1 N NaOH). The cell lysate is then evaluated by counting in a scintillation counter. Cell-associated radioactivity is taken as a measure of glucose transport after subtracting non-specific binding as determined by incubating cells in the presence of cytochalasin b, an inhibitor of glucose transport. Other methods include those described by, for example, Manchester et al., Am. J. Phvsiol. 266 (Endocrinol. Metab. 29):E326-E333, 1994 (insulin-stimulated glucose transport). Protein synthesis may be evaluated, for example, by comparing precipitation of 35S-methionine-labeled proteins following incubation of the test cells with 35S-methionine and 35S-methionine and a putative modulator of protein synthesis.
Thermogenesis may be evaluated as described by B. Stanley in The Biology of Neuropeptide Y and Related Peptides. W. Colmers and C. Wahlestedt (eds.), Humana Press, Ottawa, 1993, pp. 457-509; C. Billington et al., Am. J. Phvsiol. 260:R321, 1991 ; N. Zarjevski et al., Endocrinology 133:1753, 1993; C. Billington et al.. Am. J. Phvsiol. 266:R1765. 1994; Heller et al.. Am. J. Phvsiol. 252(4 Pt 2): R661-7, 1987; and Heller et al.. Am. J. Phvsiol. 245(3): R321-8, 1983. Also, metabolic rate, which may be measured by a variety of techniques, is an indirect measurement of thermogenesis.
Oxygen utilization may be evaluated as described by Heller et al., Pflugers Arch 369(1): 55-9, 1977. This method also involved an analysis of hypothalmic temperature and metabolic heat production. Oxygen utilization and thermoregulation have also been evaluated in humans as described by Haskell et al., J. Appl. Phvsiol. 51(4): 948-54, 1981.
The zsigόό polypeptides of the present invention may act in the neuroendocrine/exocrine cell fate decision pathway and is therefore capable of regulating the expansion of neuroendocrine and exocrine cells in the pancreas. One such regulatory use is that of islet cell regeneration. Also, it has been hypothesized that the autoimmunity that triggers IDDM starts in utero, and zsigόό polypeptide is a developmental gene involved in cell partitioning. Assays and animal models are known in the art for monitoring the exocrine/neuroendocrine cell lineage decision, for observing pancreatic cell balance and for evaluating zsigόό polypeptide, fragment, fusion protein, antibody, agonist or antagonist in the prevention or treatment of the conditions set forth above. zsigόό polypeptides can also be used to prepare antibodies that bind to zsigόό epitopes, peptides or polypeptides. The zsigόό polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response. One of skill in the art would recognize that antigenic, epitope-bearing polypeptides contain a sequence of at least 6, preferably at least 9, and more preferably at least 15 to about 30 contiguous amino acid residues of a zsigόό polypeptide (e.g., SEQ ID NO:2). Polypeptides comprising a larger portion of a zsigόό polypeptide, i.e., from 30 to 10 residues up to the entire length of the amino acid sequence are included. Antigens or immunogenic epitopes can also include attached tags, adjuvants and carriers, as described herein. Suitable antigens include the zsigόό polypeptide encoded by SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly), or a contiguous 9 to 57 amino acid fragment thereof. Other suitable antigens include motifs 1 through 4, as disclosed herein. Preferred peptides to use as antigens are hydrophilic peptides such as those predicted by one of skill in the art from a hydrophobicity plot (See Figure). Zsigόό hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: (1) amino acid number 1 (Met) to amino acid number 6 (Glu) of SEQ ID NO:2; (2) amino acid number 7 (Val) to amino acid number 12 (He) of SEQ ID NO:2; (3) amino acid number 26 (Ser) to amino acid number 32 (Ser) of SEQ ID NO:2; (4) amino acid number 29 (Asp) to amino acid number 34 (Ser) of SEQ ID NO:2; and (5) amino acid number 51 (Leu) to amino acid number 56 (Glu) of SEQ ID NO:2. Antibodies from an immune response generated from inoculation with these antigens can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al, Molecular Cloning: A Laboratory Manual. Second Edition. Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications. CRC Press, Inc., Boca Raton, FL, 1982.
As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated by inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a zsigόό polypeptide or a fragment thereof. The immunogenicity of a zsigόό polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of zsigόό or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization. As used herein, the term "antibodies" includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included. Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non- human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered" antibody). In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. Moreover, human antibodies can be produced in transgenic, non-human animals that have been engineered to contain human immunoglobulin genes as disclosed in WIPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination.
Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zsigόό protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zsigόό protein or peptide). Genes encoding polypeptides having potential zsigόό polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli. Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US Patent NO. 5,223,409; Ladner et al., US Patent NO. 4,946,778; Ladner et al., US Patent NO. 5,403,484 and Ladner et al., US Patent NO. 5,571,698) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Random peptide display libraries can be screened using the sequences disclosed herein to identify proteins which bind to zsigόό. These "binding polypeptides" which interact with zsigόό polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding polypeptides can also be used in analytical methods such as for screening expression libraries and neutralizing activity, e.g., for blocking interaction between ligand and receptor, or viral binding to a receptor. The binding polypeptides can also be used for diagnostic assays for determining circulating levels of zsigόό sequences disclosed herein to identify proteins which bind to zsigόό. These "binding polypeptides" which interact with zsigόό polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding polypeptides can also be used in analytical methods such as for screening expression libraries and neutralizing activity, e.g., for blocking interaction between ligand and receptor, or viral binding to a receptor. The binding polypeptides can also be used for diagnostic assays for determining circulating levels of zsigόό polypeptides; for detecting or quantitating soluble zsigόό polypeptides as marker of underlying pathology or disease. These binding polypeptides can also act as zsigόό "antagonists" to block zsigόό binding and signal transduction in vitro and in vivo. These anti-zsigόό binding polypeptides would be useful for inhibiting zsigόό activity or protein-binding.
Antibodies are considered to be specifically binding if: 1) they exhibit a threshold level of binding activity, and 2) they do not significantly cross-react with known related polypeptide molecules. A threshold level of binding is determined if anti-zsigόό antibodies herein bind to a zsigόό polypeptide, peptide or epitope with an affinity at least 10-fold greater than the binding affinity to control (non-zsig66) polypeptide. It is preferred that the antibodies exhibit a binding affinity (Ka) of 10 M"
1 7 -1 8 -1 or greater, preferably 10 M" or greater, more preferably 10 M" or greater, and most preferably 10 9 M -1 or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis
(Scatchard, G., Ann. NY Acad. Sci. 5J_: 660-672, 1949).
Whether anti-zsigόό antibodies do not significantly cross-react with known related polypeptide molecules is shown, for example, by the antibody detecting zsigόό polypeptide but not known related polypeptides using a standard Western blot analysis (Ausubel et al., ibid.). Examples of known related polypeptides are those disclosed in the prior art, such as known orthologs, and paralogs, and similar known members of a protein family, Screening can also be done using non-human zsigόό, and zsigόό mutant polypeptides. Moreover, antibodies can be "screened against" known related polypeptides, to isolate a population that specifically binds to the zsigόό polypeptides. For example, antibodies raised to zsigόό are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to zsigόό will flow through the matrix under the proper buffer conditions. Screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to known closely related polypeptides (Antibodies: A Laboratory Manual. Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology. Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995). Screening and isolation of specific antibodies is well known in the art. See, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principles and Practice. Goding, J.W. (eds.), Academic Press Ltd.. 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984. Specifically binding anti-zsigόό antibodies can be detected by a number of methods in the art, and disclosed below.
A variety of assays known to those skilled in the art can be utilized to detect antibodies which bind to zsigόό proteins or polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual. Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant zsigόό protein or polypeptide.
Antibodies to zsigόό and zsigόό binding polypeptides described herein may be used for tagging cells that express zsigόό; for isolating zsigόό by affinity purification; for diagnostic assays for determining circulating levels of zsigόό polypeptides; for detecting or quantitating soluble zsigόό as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; for detecting or quantitating soluble zsigόό polypeptides as marker of underlying pathology or disease. These antibodies and binding polypeptides can also act as zsigόό "antagonists" to block zsigόό binding and signal transduction in vitro and in vivo. These anti-zsigόό binding polypeptides would be useful for inhibiting zsigόό activity or protein-binding.
Antibodies to zsigόό may be used for tagging cells that express zsigόό; for isolating zsigόό by affinity purification; for diagnostic assays for determining circulating levels of zsigόό polypeptides; for detecting or quantitating soluble zsigόό as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zsigόό activity in vitro and in vivo. Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti- complement pairs as intermediates. Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. Moreover, antibodies to zsigόό or fragments thereof may be used in vitro to detect denatured zsigόό or fragments thereof in assays, for example, Western Blots or other assays known in the art. Antibodies or polypeptides herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. For instance, polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance). More specifically, zsigόό polypeptides or anti-zsigόό antibodies, or bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti- complementary molecule.
Suitable detectable molecules may be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like. Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium- 188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance). Polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion. For these purposes, biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
In another embodiment, polypeptide-toxin fusion proteins or antibody- toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues). Alternatively, if the polypeptide has multiple functional domains (i.e., an activation domain or a ligand binding domain, plus a targeting domain), a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest. In instances where the domain only fusion protein includes a complementary molecule, the anti-complementary molecule can be conjugated to a detectable or cytotoxic molecule. Such domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue- specific delivery of generic anti-complementary-detectable/ cytotoxic molecule conjugates.
In another embodiment, zsigόό-cytokine fusion proteins or antibody- cytokine fusion proteins can be used for enhancing in vivo killing of target tissues (for example, blood and bone marrow cancers), if the zsigόό polypeptide or anti-zsig66 antibody targets the hyperproliferative blood or bone marrow cell (See, generally, Hornick et al., Blood 89:4437-47, 1997). They described fusion proteins enable targeting of a cytokine to a desired site of action, thereby providing an elevated local concentration of cytokine. Suitable zsigόό polypeptides or anti-zsigόό antibodies target an undesirable cell or tissue (i.e., a tumor or a leukemia), and the fused cytokine mediated improved target cell lysis by effector cells. Suitable cytokines for this purpose include interleukin 2 and granulocyte-macrophage colony-stimulating factor (GM- CSF), for instance. Differentiation is a progressive and dynamic process, beginning with pluripotent stem cells and ending with terminally differentiated cells. Pluripotent stem cells that can regenerate without commitment to a lineage express a set of differentiation markers that are lost when commitment to a cell lineage is made. Progenitor cells express a set of differentiation markers that may or may not continue to be expressed as the cells progress down the cell lineage pathway toward maturation. Differentiation markers that are expressed exclusively by mature cells are usually functional properties such as cell products, enzymes to produce cell products, and receptors. The stage of a cell population's differentiation is monitored by identification of markers present in the cell population. Myocytes, osteoblasts, adipocytes, chrondrocytes, fibroblasts and reticular cells are believed to originate from a common mesenchymal stem cell (Owen et al., Ciba Fdn. Svmp. 136:42-46, 1988). Markers for mesenchymal stem cells have not been well identified (Owen et al., J. of Cell Sci. 87:731-738, 1987), so identification is usually made at the progenitor and mature cell stages. The novel polypeptides of the present invention may be useful for studies to isolate mesenchymal stem cells and myocyte or other progenitor cells, both in vivo and ex vivo.
There is evidence to suggest that factors that stimulate specific cell types down a pathway towards terminal differentiation or dedifferentiation affect the entire cell population originating from a common precursor or stem cell. Thus, the present invention includes stimulating or inhibiting the proliferation of myocytes, smooth muscle cells, osteoblasts, adipocytes, chrondrocytes, neuronal and endothelial cells. Molecules of the present invention for example, may while stimulating proliferation or differentiation of cardiac myocytes, inhibit proliferation or differentiation of adipocytes, by virtue of the affect on their common precursor/stem cells. Thus molecules of the present invention may have use in inhibiting chondrosarcomas, atherosclerosis, restenosis and obesity.
Assays measuring differentiation include, for example, measuring cell markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses. 161-171, 1989; all incorporated herein by reference). Alternatively, ZSIGόό polypeptide itself can serve as an additional cell-surface or secreted marker associated with stage-specific expression of a tissue. As such, direct measurement of ZSIG66 polypeptide, or its loss of expression in a tissue as it differentiates, can serve as a marker for differentiation of tissues. Similarly, direct measurement of ZSIG66 polypeptide, or its loss of expression in a tissue can be determined in a tissue or cells as they undergo tumor progression. Increases in invasiveness and motility of cells, or the gain or loss of expression of ZSIGόό in a pre-cancerous or cancerous condition, in comparison to normal tissue, can serve as a diagnostic for transformation, invasion and metastasis in tumor progression. As such, knowledge of a tumor's stage of progression or metastasis will aid the physician in choosing the most proper therapy, or aggressiveness of treatment, for a given individual cancer patient. Methods of measuring gain and loss of expression (of either mRNA or protein) are well known in the art and described herein and can be applied to ZSIG66 expression. For example, appearance or disappearance of polypeptides that regulate cell motility can be used to aid diagnosis and prognosis of prostate cancer (Banyard, J. and Zetter, B.R., Cancer and Metast. Rev. 17:449-458, 1999). As an effector of cell motility, ZSIG66 gain or loss of expression may serve as a diagnostic for prostate and other cancers.
Moreover, the activity and effect of zsigόό on tumor progression and metastasis can be measured in vivo. Several syngeneic mouse models have been developed to study the influence of polypeptides, compounds or other treatments on tumor progression. In these models, tumor cells passaged in culture are implanted into mice of the same strain as the tumor donor. The cells will develop into tumors having similar characteristics in the recipient mice, and metastasis will also occur in some of the models. Appropriate tumor models for our studies include the Lewis lung carcinoma (ATCC No. CRL- 1642) and B 16 melanoma (ATCC No. CRL-6323), amongst others. These are both commonly used tumor lines, syngeneic to the C57BL6 mouse, that are readily cultured and manipulated in vitro. Tumors resulting from implantation of either of these cell lines are capable of metastasis to the lung in C57BL6 mice. The Lewis lung carcinoma model has recently been used in mice to identify an inhibitor of angiogenesis (O'Reilly MS, et al. C l 79: 315-328,1994).
C57BL6/J mice are treated with an experimental agent either through daily injection of recombinant protein, agonist or antagonist or a one time injection of recombinant adenovirus. Three days following this treatment, 105 to 10 cells are implanted under the dorsal skin. Alternatively, the cells themselves may be infected with recombinant adenovirus, such as one expressing zsigόό, before implantation so that the protein is synthesized at the tumor site or intracellularly, rather than systemically. The mice normally develop visible tumors within 5 days. The tumors are allowed to grow for a period of up to 3 weeks, during which time they may reach a size of 1500 - 1800 mm in the control treated group. Tumor size and body weight are carefully monitored throughout the experiment. At the time of sacrifice, the tumor is removed and weighed along with the lungs and the liver. The lung weight has been shown to correlate well with metastatic tumor burden. As an additional measure, lung surface metastases are counted. The resected tumor, lungs and liver are prepared for histopathological examination, immunohistochemistry, and in situ hybridization, using methods known in the art and described herein. The influence of the expressed polypeptide in question, e.g., zsigόό, on the ability of the tumor to recruit vasculature and undergo metastasis can thus be assessed. In addition, aside from using adenovirus, the implanted cells can be transiently transfected with zsigόό. Use of stable zsigόό transfectants as well as use of induceable promoters to activate zsigόό expression in vivo are known in the art and can be used in this system to assess zsigόό induction of metastasis. Moreover, purified zsigόό or zsigόό conditioned media can be directly injected in to this mouse model, and hence be used in this system. For general reference see, O'Reilly MS, et al. Cell 79:315-328, 1994; and Rusciano D, et al. Murine Models of Liver Metastasis. Invasion Metastasis 14:349-361, 1995.
In yet another embodiment, if the zsigόό polypeptide or anti- zsigόό antibody targets vascular cells or tissues, such polypeptide or antibody may be conjugated with a radionuclide, and particularly with a beta-emitting radionuclide, to reduce restenosis. Such therapeutic approach poses less danger to clinicians who administer the radioactive therapy. For instance, iridium-192 impregnated ribbons placed into stented vessels of patients until the required radiation dose was delivered showed decreased tissue growth in the vessel and greater luminal diameter than the control group, which received placebo ribbons. Further, revascularisation and stent thrombosis were significantly lower in the treatment group. Similar results are predicted with targeting of a bioactive conjugate containing a radionuclide, as described herein. The bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action.
Molecules of the present invention can be used to identify and isolate receptors that bind zsigόό. For example, proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column (Immobilized Affinity Ligand Techniques. Hermanson et al., eds., Academic Press, San Diego, CA, 1992, pp.195-202). Proteins and peptides can also be radiolabeled (Methods in Enzymol.. vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990, 721-37) or photoaffinity labeled (Brunner et al., Ann- Rev. Biochem. 62:483-514, 1993 and Fedan et al., Biochem. Pharmacol. 33:1167-80, 1984) and specific cell-surface proteins can be identified.
The molecules of the present invention, although expressed in pituitary, may exert their effects elsewhere and hence may be useful for treating diabetes, and pancreatic cancer. The polypeptides, nucleic acid and/or antibodies of the present invention can be used in treatment of disorders and complications associated with these diseases. The molecules of the present invention can be used to modulate insulin, glucagon, and the like, or to treat or prevent development of pathological conditions in such diverse tissue as pancreas and other endocrine tissues. In particular, certain syndromes and diseases may be amenable to such diagnosis, treatment or prevention.
Since zsigόό appears to have structural similarity to vasopressin and oxytocin, zsigόό could be useful as modulator blood pressure, muscle tension or and osmotic balance. For example, blood pressure modification is important in situations such as heart attack, stroke, traumatic shock, surgery, and any number of bleeding complications. As a modulator of blood pressure, muscle tension or and osmotic balance, zsigόό may modulate contractility in the organ systems and tissues that it effects. Thus, The activity of molecules of the present invention can be measured using a variety of assays that measure cell contractility. Such assays are well known in the art. As zsigόό is structurally related to vasopressin, it can be used to modualte tissues that contract. For example contractile tissues upon which zsigόό may act include skeletal muscle, uterus; tissues in testis, e.g., vas deferens; prostate tissues; gastrointestinal tissues, e.g., colon and small intestine; and heart. The effects of zsigόό polypeptide, its antagonists and agonists, on tissue contractility can be measured in vitro using a tensiometer with or without electrical field stimulation. Such assays are known in the art and can be applied to tissue samples, such as aortic rings, vas deferens, ilium, uterine and other contractile tissue samples, as well as to organ systems, such as atria, and can be used to determine whether zsigόό polypeptide, its agomsts or antagonists, enhance or depress contractility. Molecules of the present invention are hence useful for treating dysfunction associated with contractile tissues or can be used to suppress or enhance contractility in vivo. As such, molecules of the present invention have utility in treating cardiovascular disease, infertility, in vitro fertilization, birth control, treating impotence or other male reproductive dysfunction, as well as inducing birth.
The effect of the zsigόό polypeptides, antagonists and agonists of the present invention on contractility of tissues including uterus, prostate, testis, gastrointestinal tissues, and heart can be measured in a tensiometer that measures contractility and relaxation in tissues. See, Dainty et al., J. Pharmacol. 100:767. 1990; Rhee et al, Neurotox. 16: 179, 1995; Anderson, M.B., Endocrinol. 114:364-368, 1984; and Downing, S.J. and Sherwood, O.D, Endocrinol. 116:1206-1214, 1985. For example, measuring vasodilatation of aortic rings is well known in the art. Briefly, aortic rings are taken from 4 month old Sprague Dawley rats and placed in a buffer solution, such as modified Krebs solution (118.5 mM NaCI, 4.6 mM KCI, 1.2 mM MgSO4.7H2O, 1.2 mM KH2PO4, 2.5 mM CaCl2.2H2O, 24.8 mM NaHCO3 and 10 mM glucose). One of skill in the art would recognize that this method can be used with other animals, such as rabbits, other rat strains, Guinea pigs, and the like. The rings are then attached to an isometric force transducer (Radnoti Inc., Monrovia, CA) and the data recorded with a Ponemah physiology platform (Gould Instrument systems, Inc., Valley View, OH) and placed in an oxygenated (95% O2, 5% CO2) tissue bath containing the buffer solution. The tissues are adjusted to 1 gram resting tension and allowed to stabilize for about one hour before testing. The integrity of the rings can be tested with norepinepherin (Sigma Co., St. Louis, MO) and Carbachol, a muscarinic acetylcholine agonist (Sigma Co.). After integrity is checked, the rings are washed three times with fresh buffer and allowed to rest for about one hour. To test a sample for vasodilatation, or relaxation of the aortic ring tissue, the rings are contracted to two grams tension and allowed to stabilize for fifteen minutes. A zsigόό polypeptide sample is then added to 1 , 2 or 3 of the 4 baths, without flushing, and tension on the rings recorded and compared to the control rings containing buffer only. Enhancement or relaxation of contractility by zsigόό polypeptides, their agonists and antagonists is directly measured by this method, and it can be applied to other contractile tissues such as uterus, prostate, and testis.
The activity of molecules of the present invention can be measured using a variety of assays that measure stimulation of gastrointestinal cell contractility, modulation of nutrient uptake and/or secretion of digestive enzymes. Of particular interest are changes in contractility of smooth muscle cells. For example, the contractile response of segments of mammalian duodenum or other gastrointestinal smooth muscles tissue (Depoortere et al., J. Gastrointestinal Motility 1:150-159, 1989, incorporated herein by reference). An exemplary in vivo assay uses an ultrasonic micrometer to measure the dimensional changes radially between commissures and longiturdinally to the plane of the valve base (Hansen et al., Society of Thoracic Surgeons 60:S384-390. 1995).
Gastric motility is generally measured in the clinical setting as the time required for gastric emptying and subsequent transit time through the gastrointestinal tract. Gastric emptying scans are well known to those skilled in the art, and briefly, comprise use of an oral contrast agent, such as barium, or a radiolabeled meal. Solids and liquids can be measured independently. A test food or liquid is radiolabeled with an isotope (e.g. 99mTc), and after ingestion or administration, transit time through the gastrointestinal tract and gastric emptying are measured by visualization using gamma cameras (Mever et al.. Am. J. Dig. Pis. 21 :296. 1976; Collins et al, Gut 24:1117, 1983; Maughan et al, Diabet. Med. 13 9 Supp. 5:S6-10. 1996 and Horowitz et al., Arch. Intern. Med. 145:1467-1472, 1985). These studies may be performed before and after the administration of a pro-motility agent to quantify the efficacy of the drug.
The polypeptides, antagonists, agonists, nucleic acid and/or antibodies of the present invention can also be used in treatment of disorders associated with gastrointestinal cell contractility, secretion of digestive enzymes and acids, gastrointestinal motility, recruitment of digestive enzymes; inflammation, particularly as it affects the gastrointestinal system; reflux disease and regulation of nutrient absorption. Specific conditions that will benefit from treatment with molecules of the present invention include, but are not limited to, diabetic gastroparesis, post-surgical gastroparesis, vagotomy, chronic idiopathic intestinal pseudo-obstruction and gastroesophageal reflux disease. Additional uses include, gastric emptying for radiological studies, stimulating gallbladder contraction and antrectomy.
The motor and neurological affects of molecules of the present invention make it useful for treatment of obesity and other metabolic disorders where neurological feedback modulates nutritional absorption. The molecules of the present invention are useful for regulating satiety, glucose absorption and metabolism, and neuropathy-associated gastrointestinal disorders. Molecules of the present invention are also useful as additives to anti-hypoglycemic preparations containing glucose and as adsorption enhancers for oral drugs which require fast nutrient action. Additionally, molecules of the present invention can be used to stimulate glucose-induced insulin release.
Polynucleotides encoding zsigόό polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zsigόό activity. If a mammal has a mutated or absent zsigόό gene, the zsigόό gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zsigόό polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. A defective virus is not infectious after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30, 1992; and a defective adeno-associated virus vector (Samulski et al., J. Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).
In another embodiment, a zsigόό gene can be introduced in a retroviral vector, e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No.
4,980,289; Markowitz et al., J. Virol. 62:1 120, 1988; Temin et al, U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al.; and Kuo et al., Blood 82:845, 1993. Alternatively, the vector can be introduced by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. More particularly, directing transfection to particular cells represents one area of benefit. For instance, directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the purpose of targeting. Targeted peptides (e.g., hormones or neurotransmitters), proteins such as antibodies, or non- peptide molecules can be coupled to liposomes chemically. It is possible to remove the target cells from the body; to introduce the vector as a naked DNA plasmid; and then to re-implant the transformed cells into the body. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem. 267:963-7,
1992: Wu et al.. J. Biol. Chem. 263:14621-4. 1988.
Antisense methodology can be used to inhibit zsigόό gene transcription, such as to inhibit cell proliferation in vivo. Polynucleotides that are complementary to a segment of a zsigόό-encoding polynucleotide (e.g., a polynucleotide as set froth in SEQ ID NO:l) are designed to bind to zsigόό-encoding mRNA and to inhibit translation of such mRNA. Such antisense polynucleotides are used to inhibit expression of zsigόό polypeptide-encoding genes in cell culture or in a subject.
The present invention also provides reagents which will find use in diagnostic applications. For example, the zsigόό gene, a probe comprising zsigόό DNA or RNA or a subsequence thereof can be used to determine if the zsigόό gene is present on a chromosome or if a mutation has occurred. Detectable chromosomal aberrations at the zsigόό gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid. ; Ausubel et. al., ibid. ; Marian, Chest 108:255-65, 1995).
Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes (Cox et al., Science 250:245-50, 1990). Partial or full knowledge of a gene's sequence allows one to design PCR primers suitable for use with chromosomal radiation hybrid mapping panels. Radiation hybrid mapping panels are commercially available which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL). These panels enable rapid, PCR-based chromosomal localizations and ordering of genes, sequence-tagged sites (STSs), and other nonpolymorphic and polymorphic markers within a region of interest. This includes establishing directly proportional physical distances between newly discovered genes of interest and previously mapped markers.
The precise knowledge of a gene's position can be useful for a number of purposes, including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have.
Sequence tagged sites (STSs) can also be used independently for chromosomal localization. An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome. An STS is defined by a pair of oligonucleotide primers that are used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Since STSs are based solely on DNA sequence they can be completely described within an electronic database, for example, Database of Sequence Tagged Sites (dbSTS), GenBank, (National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), and can be searched with a gene sequence of interest for the mapping data contained within these short genomic landmark STS sequences.
The zsigόό gene can be used to detect genes of known function map to the same region of the human chromosome. In addition, zsigόό polynucleotide probes can be used to detect abnormalities or genotypes associated with human disease states as well as map to the same region of the human genome. See the Online Mendellian Inheritance of Man (OMIM) gene map, on a publicly available WWW server (http://www3.ncbi.nlm.nih.gov/htbin-post/Omim). Candidate genes for an inheritable disease which show linkage to the same chromosomal region as the zsigόό gene can hence be identified.
The zsigόό gene is located at the 4q28-q31 region of chromosome 4 (Example 3). Several known genes map to this locus and are associated with human disease states: The Fibrinogin alpha, beta, and gamma gene cluster is located at 4q28, and mutations therein are associated with diseases such as dysfibrinogenemia, hypofibrinogenemia, and fibrinogenemia which result in blood clotting malfunction and other complications (See, for example, Berg, K. and Keirulf, P. Clin. Genet. 36: 229- 235, 1989). Zsigόό polynucleotide probes can be used to detect abnormalities or genotypes associated with these fibrinogen associated markers. Further, zsigόό polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 4q28-q31 deletions and translocations associated with human diseases, such as Reiger syndrome (deletion in 4q); loss of heterozygosity, or translocation between 4q28-q31 and another chromosome; translocations involved with malignant progression of tumors; or mutations, which are expected to be involved in chromosome rearrangements in malignancy; or deletions and translocations in other cancers. Similarly, zsigόό polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 4q28-31 trisomy and chromosome loss associated with human diseases such as Reiger syndrome (above). Moreover, amongst other genetic loci, those for autosomal dominant pseudohypoaldosteroidism (PHA) type I (4q31.1), anterior segment mesenchymal dysgenesis and Reiger syndrome (4q28- q31), Hepatitis B virus associated hepatocellular carcinoma susceptability (4q32.1), and others, all manifest themselves in human disease states as well as map to this region of the human genome. See the Online Mendellian Inheritance of Man (OMIM) gene map, and references therein, for this region of chromosome 4 on a publicly available WWW server (http://www3.ncbi.nlm.nih.gov/htbin-post/Omim/getmap?chromosome=4q28, and surrounding regions through 4q31). All of these serve as possible candidate genes for an inheritable disease which show linkage to the same chromosomal region as the zsigόό gene. Thus, zsigόό polynucleotide probes can be used to detect abnormalities or genotypes associated with these defects.
Similarly, defects in the zsigόό locus itself may result in a heritable human disease state. As a pituitary hormone, defects the zsigόό molecules of the present invention can have broad reaching effects in the human body. Molecules of the present invention, such as the polypeptides, antagonists, agonists, polynucleotides and antibodies of the present invention would aid in the detection, diagnosis prevention, and treatment associated with a zsigόό genetic defect. A diagnostic could assist physicians in determining the type of genetic disease and appropriate associated therapy, or assistance in genetic counseling. As such, the inventive anti-zsigόό antibodies, polynucleotides, and polypeptides can be used for the detection of zsigόό polypeptide, mRNA or anti-zsigόό antibodies, thus serving as markers and be directly used for detecting or diagnosing known or yet to be discovered diseases, or cancers, as described herein, using methods known in the art and described herein.
In addition, zsigόό polynucleotide probes can be used to detect allelic differences between diseased or non-diseased individuals at the zsigόό chromosomal locus. As such, the zsigόό sequences can be used as diagnostics in forensic DNA profiling. Such profiling can be applied to commercial animals as well for use in breeding programs.
In general, the diagnostic methods used in genetic linkage analysis, to detect a genetic abnormality or aberration in a patient, are known in the art. Most diagnostic methods comprise the steps of (a) obtaining a genetic sample from a potentially diseased patient, diseased patient or potential non-diseased carrier of a recessive disease allele; (b) producing a first reaction product by incubating the genetic sample with a ZSMF16 polynucleotide probe wherein the polynucleotide will hybridize to complementary polynucleotide sequence, such as in RFLP analysis or by incubating the genetic sample with sense and antisense primers in a PCR reaction under appropriate PCR reaction conditions; (iii) Visualizing the first reaction product by gel electrophoresis and/or other known method such as visualizing the first reaction product with a ZSMF16 polynucleotide probe wherein the polynucleotide will hybridize to the complementary polynucleotide sequence of the first reaction; and (iv) comparing the visualized first reaction product to a second control reaction product of a genetic sample from wild type patient. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the diseased or potentially diseased patient, or the presence of a heterozygous recessive carrier phenotype for a non-diseased patient, or the presence of a genetic defect in a tumor from a diseased patient, or the presence of a genetic abnormality in a fetus or pre- implantation embryo. For example, a difference in restriction fragment pattern, length of PCR products, length of repetitive sequences at the ZSIG66 genetic locus, and the like, are indicative of a genetic abnormality, genetic aberration, or allelic difference in comparison to the normal wild type control. Controls can be from unaffected family members, or unrelated individuals, depending on the test and availability of samples. Genetic samples for use within the present invention include genomic DNA, mRNA, and cDNA isolated form any tissue or other biological sample from a patient, such as but not limited to, blood, saliva, semen, embryonic cells, amniotic fluid, and the like. The polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO:l, the complement of SEQ ID NO: 1, or an RNA equivalent thereof. Such methods of showing genetic linkage analysis to human disease phenotypes are well known in the art. For reference to PCR based methods in diagnostics see see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methods and Applications (Humana Press, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).
Abeπations associated with the ZSIG66 locus can be detected using nucleic acid molecules of the present invention by employing standard methods for direct mutation analysis, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc. 1996), Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren (ed.), Laboratory Protocols for Mutation Detection (Oxford University Press 1996), Birren et al. (eds.), Genome Analysis, Vol. 2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998), Dracopoli et al. (eds.), Current Protocols in Human Genetics (John Wiley & Sons 1998), and Richards and Ward, "Molecular Diagnostic Testing," in Principles of Molecular Medicine, pages 83- 88 (Humana Press, Inc. 1998)). Direct analysis of an ZSIG66 gene for a mutation can be performed using a subject's genomic DNA. Methods for amplifying genomic DNA, obtained for example from peripheral blood lymphocytes, are well-known to those of skill in the art (see, for example, Dracopoli et al. (eds.), Current Protocols in Human Genetics, at pages 7.1.6 to 7.1.7 (John Wiley & Sons 1998)).
Mice engineered to express the zsigόό gene, referred to as "transgenic mice," and mice that exhibit a complete absence of zsigόό gene function, referred to as "knockout mice," may also be generated (Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature 366:740-42, 1993; Capecchi, M.R., Science 244: 1288-1292, 1989; Palmiter, R.D. et al. Annu Rev Genet. 20: 465-499, 1986). For example, transgenic mice that over-express zsigόό, either ubiquitously or under a tissue-specific or tissue-restricted promoter can be used to ask whether over-expression causes a phenotype. For example, over-expression of a wild-type zsigόό polypeptide, polypeptide fragment or a mutant thereof may alter normal cellular processes, resulting in a phenotype that identifies a tissue in which zsigόό expression is functionally relevant and may indicate a therapeutic target for the zsigόό, its agonists or antagonists. For example, a preferred transgenic mouse to engineer is one that over-expresses the zsigόό mature polypeptide (approximately amino acids 28 (Ala) to 84 (Gly) of SEQ ID NO:2). Moreover, such over-expression may result in a phenotype that shows similarity with human diseases. Similarly, knockout zsigόό mice can be used to determine where zsigόό is absolutely required in vivo. The phenotype of knockout mice is predictive of the in vivo effects of that a zsigόό antagonist, such as those described herein, may have. The human zsigόό cDNA can be used to isolate murine zsigόό mRNA, cDNA and genomic DNA, which are subsequently used to generate knockout mice. These mice may be employed to study the zsigόό gene and the protein encoded thereby in an in vivo system, and can be used as in vivo models for corresponding human diseases. Moreover, transgenic mice expression of zsigόό antisense polynucleotides or ribozymes directed against zsigόό, described herein, can be used analogously to transgenic mice described above.
For pharmaceutical use, the proteins of the present invention are formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. In general, pharmaceutical formulations will include a zsigόό protein in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy. Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995. Therapeutic doses will generally be in the range of 0.1 to 100 μg/kg of patient weight per day, preferably 0.5-20 _g/kg per day, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art. The proteins may be administered for acute treatment, over one week or less, often over a period of one to three days or may be used in chronic treatment, over several months or years.
The invention is further illustrated by the following non-limiting examples.
EXAMPLES
Example 1 Identification of zsigόό
A. Using an EST Sequence to Obtain Full-length zsigόό Scanning of translated cDNA library databases using a signal trap as a query resulted in identification of an expressed sequence tag (EST) sequence from a pituitary library found to be homologous to a human secretory signal sequence. Confirmation of the EST sequence was made by sequence analyses of the cDNA from which the EST originated. This cDNA was contained in a plasmid from a human pituitary library, and was sequenced using the following primers to generate complete double stranded sequence of this clone: ZC6,768 (SEQ ID NO:12), ZC20,137 (SEQ ID NO:13), ZC20,136 (SEQ ID NO: 14), ZC20,132 (SEQ ID NO:15), ZC20,134 (SEQ ID NO: 16), ZC19,964 (SEQ ID NO: 17), ZC19,965 (SEQ ID NO:18), ZC694 (SEQ ID NO: 19).
Example 2
Tissue Distribution Northern blot analysis was performed using Human Multiple Tissue
Northern™ Blots (MTN I, MTN II, and MTN III) (Clontech). The cDNA described in
Example 1 was used in a PCR reaction using oligos ZC21,336 (SEQ ID NO:20) and
ZC21.338 (SEQ ID NO:21) as primers. PCR conditions were as follows: 94°C for 1.5 minutes; 35 cycles at 94°C for 15 seconds , 66°C for 30 seconds; 72°C for 30 seconds, one cycle at 72°C for 5 minutes, followed by a 4°C hold. A sample of the PCR reaction product was run on a 4% agarose gel. A band of the expected size of about 220 bp was seen. The 220 bp PCR fragment, was gel purified using a commercially available kit (QiaexII™; Qiagen) and then radioactively labeled with 32P-dCTP using Rediprime II™ (Amersham), a random prime labeling system, according to the manufacturer's specifications. The probe was then purified using a Nuc-Trap™ column (Stratagene) according to the manufacturer's instructions. ExpressHyb™ (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 55°C using 1-2 x 106 cpm/ml of labeled probe. The blots were then washed for 30 minutes in 2X SSC/0.1% SDS at room temperature, followed by a wash in 2X SSC/0.1% SDS at 65°C , followed by a wash in 0.1X SSC/0.1% SDS at 65°C. The blots were exposed 7 days. No transcript signals were observed in the tissues represented on the blots. Moreover, an in-house produced Northern blot containing RNA from pituitary gland was also probed, following the methods and conditions described above. Again, no signals were observed, suggesting that the mRNA for zsigόό is relatively rare and/or expressed in only specific physiologic conditions.
Dot Blots were also performed using Human RNA Master Blots™ (Clontech). The methods and conditions for the Dot Blots are the same as for the Multiple Tissue Blots described above. A signal was observed in genomic DNA.
Example 3
Chromosomal Assignment and Placement of Zsigόό Zsigόό was mapped to chromosome 4 using the commercially available version of the "Stanford G3 Radiation Hybrid Mapping Panel" (Research Genetics, Inc., Huntsville, AL). The "Stanford G3 RH Panel" contains DNAs from each of 83 radiation hybrid clones of the whole human genome, plus two control DNAs (the RM donor and the A3 recipient). A publicly available WWW server (http://shgc- www.stanford.edu) allows chromosomal localization of markers.
For the mapping of Zsigόό with the "Stanford G3 RH Panel", 20 μl reactions were set up in a 96-well microtiter plate (Stratagene, La Jolla, CA) and used in a "RoboCycler Gradient 96" thermal cycler (Stratagene). Each of the 85 PCR reactions consisted of 2 μl 10X KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, CA), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, CA), 1 μl sense primer, ZC21,450 (SEQ ID NO:22), 1 μl antisense primer, ZC21,451 (SEQ ID NO:23), 2 μl "RediLoad" (Research Genetics, Inc., Huntsville, AL), 0.4 μl 50X Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and ddH2O for a total volume of 20 μl. The reactions were overlaid with an equal amount of mineral oil and sealed. The PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 94°C, 35 cycles of a 45 seconds denaturation at 94°C, 45 seconds annealing at 60°C and 1 minute AND
15 seconds extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72°C. The reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD).
The results showed linkage of Zsigόό to the chromosome 4 marker SHGC-57096 with a LOD score of >11 and at a distance of 0 cR_10000 from the marker. The use of surrounding genes, which have been cytogenetically mapped, positions Zsigόό in the 4q28-q31 chromosomal region.
From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

What is claimed is:
1. An isolated polynucleotide encoding a zsigόό polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser);
(b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser);
(c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 84 (Gly);
(d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and
(e) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID NO:2, wherein the amino acid percent identity is determined using a FASTA program with ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62, with other parameters set as default.
2. An isolated polynucleotide according to claim 1, wherein the polynucleotide is selected from the group consisting of:
(a) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 431 to nucleotide 472;
(b) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 425 to nucleotide 472;
(c) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 425 to nucleotide 475;
(d) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 305 to nucleotide 475; (e) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 224 to nucleotide 475; and
(f) a polynucleotide sequence complementary to (a) or (b).
3. An isolated polynucleotide sequence according to claim 1, wherein the polynucleotide comprises nucleotide 1 to nucleotide 252 of SEQ ID NO:l 1.
4. An isolated polynucleotide according to claim 1, wherein the zsigόό polypeptide comprises a sequence of amino acid residues selected from the group consisting of:
(a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser);
(b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser);
(c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 84 (Gly);
(d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and
(e) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID NO:2.
5. The isolated polynucleotide molecule of claim 1, wherein the polynucleotide encodes a polypeptide that contains motifs 1 through 4 spaced apart from N- terminus to C-terminus in a configuration Ala28-{7}-Ml-{3}-M2-{5}-M3-{6}-M4-{10}- Gly84, wherein Ml is "motif 1," a sequence of amino acids as shown in amino acids 36 to 41 of SEQ ID NO:2,
M2 is "motif 2," a sequence of amino acids as shown in amino acids 45 to 50 of SEQ ID NO:2, M3 is "motif 3," a sequence of amino acids as shown in amino acids 56 to 61 of SEQ ID NO:2,
M4 is "motif 4," a sequence of amino acids as shown in amino acids 68 to 73 of SEQ ID NO:2, and
Ala28 is the Alanine residue at amino acid number 28 in SEQ ID NO:2, Gly 84 is the Glycine residue at amino acid number 84 in SEQ ID NO:2, and {#} denotes the number of amino acids between the motifs.
An expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a zsigόό polypeptide comprising an amino acid sequence as shown in SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and a transcription terminator.
7. An expression vector according to claim 6, further comprising a secretory signal sequence operably linked to the DNA segment.
8. A cultured cell into which has been introduced an expression vector according to claim 6, wherein the cell expresses a polypeptide encoded by the DNA segment.
9. A DNA construct encoding a fusion protein, the DNA construct comprising: a first DNA segment encoding a polypeptide comprising a sequence of amino acid residues selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 2 from residue number 28 (Ala) to amino acid number 84 (Gly); and at least one other DNA segment encoding an additional polypeptide, wherein the first and other DNA segments are connected in-frame; and encode the fusion protein.
10. A fusion protein produced by a method comprising: culturing a host cell into which has been introduced a vector comprising the following operably linked elements:
(a) a transcriptional promoter;
(b) a DNA construct encoding a fusion protein according to claim 9; and
(c) a transcriptional terminator; and recovering the protein encoded by the DNA segment.
11. An isolated polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser);
(b) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 69 (Phe) to amino acid number 83 (Ser);
(c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 84 (Gly);
(d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and
(e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID NO:2, wherein the amino acid percent identity is determined using a FASTA program with ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62, with other parameters set as default.
12. An isolated polypeptide according to claim 11, wherein the polypeptide further comprises motifs 1 through 4 spaced apart from N-terminus to C-terminus in a configuration Ala28-{7}-Ml-{3}-M2-{5}-M3-{6}-M4-{10}- Gly84, wherein Ml is "motif 1," a sequence of amino acids as shown in amino acids 36 to 41 of SEQ ID NO:2,
M2 is "motif 2," a sequence of amino acids as shown in amino acids 45 to 50 of SEQ ID NO:2,
M3 is "motif 3," a sequence of amino acids as shown in amino acids 56 to 61 of SEQ ID NO:2,
M4 is "motif 4," a sequence of amino acids as shown in amino acids 68 to 73 of SEQ ID NO:2, and
Ala28 is the Alanine residue at amino acid number 28 in SEQ ID NO:2,
Gly84 is the Glycine residue at amino acid number 84 in SEQ ID NO:2, and
{#} denotes the number of amino acids between the motifs.
13. An isolated polypeptide according to claim 11, wherein the polypeptide comprises a sequence of amino acid residues selected from the group consisting of:
(a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 70 (Cys) to amino acid number 83 (Ser);
(b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 83 (Ser);
(c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 69 (Phe) to amino acid number 84 (Gly);
(d) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 28 (Ala) to amino acid number 84 (Gly); and
(e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 84 (Gly), to of SEQ ID NO:2.
14. An isolated polypeptide according to claim 11, wherein the polypeptide further comprises an amidated C-terminal Serine residue.
15. A method of producing a zsigόό polypeptide comprising: culturing a cell according to claim 8; and isolating the zsigόό polypeptide produced by the cell.
16. A method of detecting, in a test sample, the presence of a modulator of zsigόό protein activity, comprising: transfecting a zsigόό-responsive cell, with a reporter gene construct that is responsive to a zsigόό-stimulated cellular pathway; and producing a zsigόό polypeptide by the method of claim 15; and adding the zsigόό polypeptide to the cell, in the presence and absence of a test sample; and comparing levels of response to the zsigόό polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and determining from the comparison, the presence of the modulator of zsigόό activity in the test sample.
17. A method of producing an antibody to zsigόό polypeptide comprising the following steps in order: inoculating an animal with a polypeptide selected from the group consisting of:
(a) a polypeptide consisting of 9 to 57 amino acids, wherein the polypeptide has a contiguous sequence of amino acids as shown within SEQ ID NO:2 from amino acid number 28 (Ala) to amino acid number 84 (Gly);
(b) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 from residue number 28 (Ala) to amino acid number 84 (Gly);
(c) a polypeptide according to claim 11 ;
(d) a polypeptide consisting of amino acid number 1 (Met) to amino acid number 6 (Glu) of SEQ ID NO:2;
(e) a polypeptide consisting of amino acid number 7 (Val) to amino acid number 12 (He) of SEQ ID NO:2; (f) a polypeptide consisting of amino acid number 26 (Ser) to amino acid number 32 (Ser) of SEQ ID NO:2;
(g) a polypeptide consisting of amino acid number 29 (Asp) to amino acid number 34 (Ser) of SEQ ID NO:2; and
(h) a polypeptide consisting of amino acid number 51 (Leu) to amino acid number 56 (Glu) of SEQ ID NO:2; and wherein the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal.
18. An antibody produced by the method of claim 17, which binds to a zsigόό polypeptide.
19. The antibody of claim 18, wherein the antibody is a monoclonal antibody.
20. An antibody which specifically binds to a polypeptide of claim 11.
21. A method for detecting a genetic abnormality in a patient, comprising: obtaining a genetic sample from a patient; producing a first reaction product by incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO:l or the complement of SEQ ID NO:l, under conditions wherein said polynucleotide will hybridize to complementary polynucleotide sequence; visualizing the first reaction product; and comparing said first reaction product to a control reaction product from a wild type patient, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient.
22. A pharmaceutical composition comprising an isolated polypeptide of claim 11, wherein the polypeptide is in combination with a pharmaceutically acceptable vehicle.
EP99966234A 1998-12-16 1999-12-14 Pancreatic polypeptide zsig66 Withdrawn EP1141287A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US21294798A 1998-12-16 1998-12-16
US212947 1998-12-16
PCT/US1999/029669 WO2000036104A1 (en) 1998-12-16 1999-12-14 Pancreatic polypeptide zsig66

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EP (1) EP1141287A1 (en)
JP (1) JP2002532091A (en)
AU (1) AU2182900A (en)
CA (1) CA2355039A1 (en)
IL (1) IL143483A0 (en)
WO (1) WO2000036104A1 (en)
ZA (1) ZA200104393B (en)

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JP2003252801A (en) * 2002-02-27 2003-09-10 Japan Science & Technology Corp Method for using pituitary-specific gene

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
[online] 9 September 1998 (1998-09-09), MAHAIRAS ET AL.: "Sequence-tagged connectors: a sequence approach to mapping and scanning the human genome.", retrieved from EBI Database accession no. AQ153386 *
MAHAIRAS ET AL.: "Sequence-tagged connectors: a sequence approach to mapping and scanning the human genome.", PROC. NATL. ACAD. SCI. U.S.A., vol. 96, no. 17, August 1999 (1999-08-01), pages 9739 - 9744 *
See also references of WO0036104A1 *

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IL143483A0 (en) 2002-04-21
CA2355039A1 (en) 2000-06-22
WO2000036104A1 (en) 2000-06-22
AU2182900A (en) 2000-07-03
JP2002532091A (en) 2002-10-02
ZA200104393B (en) 2001-12-13

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