WO2001029224A2 - Homologue zgpa1 de peptide de granulocyte - Google Patents

Homologue zgpa1 de peptide de granulocyte Download PDF

Info

Publication number
WO2001029224A2
WO2001029224A2 PCT/US2000/029177 US0029177W WO0129224A2 WO 2001029224 A2 WO2001029224 A2 WO 2001029224A2 US 0029177 W US0029177 W US 0029177W WO 0129224 A2 WO0129224 A2 WO 0129224A2
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
polypeptide
acid sequence
acid number
Prior art date
Application number
PCT/US2000/029177
Other languages
English (en)
Other versions
WO2001029224A3 (fr
Inventor
Darrell C. Conklin
David A. Adler
Brian A. Fox
Original Assignee
Zymogenetics, Inc.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Zymogenetics, Inc. filed Critical Zymogenetics, Inc.
Priority to AU15744/01A priority Critical patent/AU1574401A/en
Publication of WO2001029224A2 publication Critical patent/WO2001029224A2/fr
Publication of WO2001029224A3 publication Critical patent/WO2001029224A3/fr

Links

Classifications

    • 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

Definitions

  • Bacterial, and other microbial interaction with host tissues can have beneficial (symbiotic) as well as deleterious (pathogenic) consequences. Invading microbes can be pathogenic. Consequently, host biological defense strategies have evolved to protect organisms from invasion by disease-causing microorganisms.
  • Microbial infection response systems include oxidative and non-oxidative mechanisms, utilizing compounds that are enzymatically synthesized in cells, as well as peptides that are single gene products.
  • anti-microbial peptides found in polymorphonuclear leukocytes e.g., neutrophils, granulocytes, and peripheral mononuclear (PMN) cells, and the like
  • PMN peripheral mononuclear
  • One major class of anti-microbial peptides is defined by conserved cysteine residue patterns and is termed defensins.
  • mammalian defensins derived from skin, lung and intestine, exhibit antibiotic activity against a wide variety of pathogens, including Gram-positive and Gram-negative bacteria, fungi (e.g., Candida species) and viruses. See, for example, Porter et al., Infect. Immun. 65(6): 2396-401, 1997; Selsted, ME and Ouellette, AL, Trends in Cell Biol. 5:114-119, 1995; Diamond, G et al., Proc. Natl. Acad. Sci. USA 90:4596-4600, 1993. Another major class of microbial peptides is called adhesins.
  • Adhesins enable microbes to adhere to mammalian tissues, for example the oral, gastrointestinal, urogenital and respiratory tracts. For a pathogenic microorganism, this may be a primary route to colonization and/or invasion of the host. Conversely, natural microbial flora can adhere to host tissues and create beneficial symbiotic relationships such as nutritional benefits, and protection against colonization of pathogenic microbes.
  • the host defenses involved in attracting and establishing beneficial microbial colonization, as opposed to pathogenic microbial colonization, are not well understood. However, host defenses that affect this balance may have anti-microbial, immunomodulatory, inflammatory, anti- inflammatory or other properties.
  • neutrophils of cows, sheep, goats and ibex contain very large cytoplasmic granules that are distinct from classical azurophil and specific granules.
  • Polypeptides isolated therefrom are potent anti-microbial peptides and are structurally distinct from defensins.
  • antimicrobial mammalian polypeptides include arginine- rich peptides, called bactenecins; cathelicidin peptides; and a cationic tryptophan-rich tridecapeptide amide, indolicidin; as well as ⁇ -defensins that are distinct from classic defensins in primary sequence, but have similar structural and functional attributes (Gundmundsson, GH and Agerberth, B J. Immunol. Meth.
  • the currently known antimicrobial mammalian polypeptides have a range of useful activities including bactericidal, anti-fungal and antiviral activities.
  • the use of such anti-microbial peptides in therapeutic applications and drug development is important, as resistance and multi-resistance of microorganisms to classic antibiotic treatment is problematic and an ever-increasing threat to health care.
  • novel moieties having anti-microbial, or adhesin-like, immunomodulatory, inflammatory, anti-inflammatory or other properties is sought.
  • the present invention provides such polypeptides and peptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
  • the present invention provides isolated polynucleotide that encodes a polypeptide comprising a sequence of amino acid residues that is at least 96% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 8 from amino acid number 210 (Val) to 340 (Leu); (b) the amino acid sequence as shown in SEQ ID NO:8 from amino acid number 52 (Val) to 340 (Leu); (c) the amino acid sequence as shown in SEQ ID NO: 8 from amino acid number 18 (Asp) to 369 (His); (d) the amino acid sequence as shown in SEQ ID NO:5 from amino acid number 18 (Asp) to 375 (His); (e) the amino acid sequence as shown in SEQ ID NO:8 from amino acid number 1 (Met) to 369 (His); and (f) the amino acid sequence as shown in SEQ ID NO: 5 from amino acid number 1 (Met) to 375 (His).
  • the isolated polynucleotide disclosed above encodes a polypeptide that comprises a sequence of amino acid residues that is selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 8 from amino acid number 52 (Val) to 183 (Val); (b) the amino acid sequence as shown in SEQ ID NO:8 from amino acid number 210 (Val) to 340 (Leu); (c) the amino acid sequence as shown in SEQ ID NO:8 from amino acid number 52 (Val) to 340 (Leu); (d) the amino acid sequence as shown in SEQ ID NO:8 from amino acid number 18 (Asp) to 369 (His); (e) the amino acid sequence as shown in SEQ ID NO:5 from amino acid number 18 (Asp) to 375 (His); (f) the amino acid sequence as shown in SEQ ID NO:8 from amino acid number 1 (Met) to 369 (His); and (g) the amino acid sequence as shown in SEQ ID NO:5 from amino acid number 1 (
  • the isolated polynucleotide disclosed above comprises SEQ ID NO:6 or SEQ ID NO:9.
  • the isolated polynucleotide disclosed above encodes a polypeptide that has activity as measured by cell proliferation, activation of transcription of a reporter gene, anti-microbial activity, or wherein the polypeptide encoded by the polynucleotide further binds to an antibody, wherein the antibody is raised to a polypeptide comprising a sequence of amino acids from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:5 from amino acid number 18 (Asp) to 57 (Thr); (b) the amino acid sequence as shown in SEQ ID NO:8 from amino acid number 52 (Val) to 183 (Val); (c) the amino acid sequence as shown in SEQ ID NO:8 from amino acid number 184 (Gin) to 209 (Val); (d) the amino acid sequence as shown in SEQ ID NO: 8 from amino acid number 210 (Val) to 340 (Leu);
  • the isolated polynucleotide disclosed above encodes a polypeptide that further comprises a sequence of amino acids as shown in SEQ ID NO: 12.
  • the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as shown as shown in SEQ ID NO:8 from amino acid number 18 (Asp) to 369 (His); or as shown in SEQ ID NO:5 from amino acid number 18 (Asp) to 375 (His); a transcription terminator, wherein the promoter is operably linked to the DNA segment, and the DNA segment is operably linked to the transcription terminator.
  • the expression vector disclosed above further comprising a secretory signal sequence operably linked to the DNA segment.
  • the present invention provides a cultured cell comprising 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 as shown in SEQ ID NO:5 from amino acid number 18 (Asp) to 57 (Thr); (b) the amino acid sequence as shown in SEQ ID NO:5 from amino acid number 18 (Asp) to 57 (Thr); (b) the amino acid sequence as shown in SEQ ID NO:5 from amino acid number 18 (Asp) to 57 (Thr); (b) the amino acid sequence as shown in SEQ ID NO:5 from amino acid number 18 (Asp) to 57 (Thr); (b) the amino acid sequence as shown in SEQ ID NO:5 from amino acid number 18 (Asp) to 57 (Thr); (b) the amino acid sequence as shown in SEQ ID NO:5 from amino acid number 18 (Asp) to 57 (Thr); (b) the amino acid sequence as
  • the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA construct encoding a fusion protein as disclosed above; and a transcription terminator, wherein the promoter is operably linked to the DNA construct, and the DNA construct is operably linked to the transcription terminator.
  • the present invention provides a cultured cell comprising an expression vector as disclosed above, wherein the cell expresses a polypeptide encoded by the DNA construct.
  • the present invention provides a method of producing a fusion protein comprising: culturing a cell as disclosed above; and isolating the polypeptide produced by the cell.
  • the present invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 96% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:8 from amino acid number 210 (Val) to 340 (Leu); (b) the amino acid sequence as shown in SEQ ID NO:8 from amino acid number 52 (Val) to 340 (Leu); (c) the amino acid sequence as shown in SEQ ID NO: 8 from amino acid number 18 (Asp) to 369 (His); (d) the amino acid sequence as shown in SEQ ID NO:5 from amino acid number 18 (Asp) to 375 (His); (e) the amino acid sequence as shown in SEQ ID NO:8 from amino acid number 1 (Met) to 369 (His); and (f) the amino acid sequence as shown in SEQ ID NO:5 from amino acid number 1 (Met) to 375 (His).
  • 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:8 from amino acid number 52 (Val) to 183 (Val); (b) the amino acid sequence as shown in SEQ ID NO: 8 from amino acid number 210 (Val) to 340 (Leu); (c) the amino acid sequence as shown in SEQ ID NO: 8 from amino acid number 52 (Val) to 340 (Leu); (d) the amino acid sequence as shown in SEQ ID NO:8 from amino acid number 18 (Asp) to 369 (His); (e) the amino acid sequence as shown in SEQ ID NO:5 from amino acid number 18 (Asp) to 375 (His); (f) the amino acid sequence as shown in SEQ ID NO: 8 from amino acid number 1 (Met) to 369 (His); and (g) the amino acid sequence as shown in SEQ ID NO:5 from amino acid number 1 (Met) to 375 (His).
  • the isolated polypeptide disclosed has activity as measured by cell proliferation, activation of transcription of a reporter gene, antimicrobial activity, or wherein the polypeptide further binds to an antibody, wherein the antibody is raised to a polypeptide comprising a sequence of amino acids from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:5 from amino acid number 18 (Asp) to 57 (Thr); (b) the amino acid sequence as shown in SEQ ID NO:8 from amino acid number 52 (Val) to 183 (Val); (c) the amino acid sequence as shown in SEQ ID NO:8 from amino acid number 184 (Gin) to 209 (Val); (d) the amino acid sequence as shown in SEQ ID NO: 8 from amino acid number 210 (Val) to 340 (Leu); (e) the amino acid sequence as shown in SEQ ID NO: 8 from amino acid number 52 (Val) to 340 (Leu); (f) the amino acid sequence as shown in SEQ ID NO: 8 from amino acid number 18 (A) the amino
  • the present invention provides a method of producing a polypeptide comprising: culturing a cell as disclosed above; and isolating the polypeptide produced by the cell.
  • the present invention provides a method of producing an antibody to a zgpal polypeptide comprising: inoculating an animal with a polypeptide selected from the group consisting of: (a) a polypeptide as disclosed above; (b) a polypeptide comprising an amino acid sequence as shown in SEQ ID NO:5 from amino acid number 47 (Gly) to 52 (Asp); (c) a polypeptide comprising an amino acid sequence as shown in SEQ ID NO:5 from amino acid number 18 (Asp) to 57 (Thr); (d) a polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 8 from amino acid number 205 (Ala) to 223 (Pro); (e) a polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 8 from amino acid number 184 (Gin
  • the present invention provides an antibody produced by the method as disclosed above, which binds to a polypeptide consisting of an amino acid sequence as shown in SEQ ID NO:8 from amino acid number 18 (Asp) to 369 (His); or an amino acid sequence as shown in SEQ ID NO:5 from amino acid number 18 (Asp) to 375 (His).
  • the antibody disclosed above is a monoclonal antibody.
  • the present invention provides an antibody that specifically binds to a polypeptide as disclosed above.
  • the present invention provides a method of detecting, in a test sample, the presence of an antagonist of zgpal protein activity, comprising: culturing a cell that is responsive to a zgpal -stimulated cellular pathway; and producing a zgpal polypeptide by the method as disclosed above; and exposing the zgpal polypeptide to the cell, in the presence and absence of a test sample; and comparing levels of response to the zgpal 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 antagonist of zgpal activity in the test sample.
  • the present invention provides a method of detecting, in a test sample, the presence of an agonist of zgpal protein activity, comprising: culturing a cell that is responsive to a zgpal -stimulated cellular pathway; and adding a test sample; and comparing levels of response in the presence and absence of the test sample, by a biological or biochemical assay; and determining from the comparison, the presence of the agonist of zgpal activity in the test sample.
  • 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:4 or SEQ ID NO:7 or the complement of SEQ ID NO:4 or SEQ ID NO:7, 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 method for detecting a cancer in a patient, comprising: obtaining a tissue or biological sample from a patient; incubating the tissue or biological sample with an antibody as disclosed above under conditions wherein the antibody binds to its complementary polypeptide in the tissue or biological sample; visualizing the antibody bound in the tissue or biological sample; and comparing levels of antibody bound in the tissue or biological sample from the patient to a normal control tissue or biological sample, wherein an increase in the level of antibody bound to the patient tissue or biological sample relative to the normal control tissue or biological sample is indicative of a cancer in the patient.
  • the present invention provides a method for detecting a cancer in a patient, comprising: obtaining a tissue or biological sample from a patient; labeling a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO:4 or SEQ ID NO:7 or the complement of SEQ ID NO:4 or SEQ ID NO:7; incubating the tissue or biological sample with under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence; visualizing the labeled polynucleotide in the tissue or biological sample; and comparing the level of labeled polynucleotide hybridization in the tissue or biological sample from the patient to a normal control tissue or biological sample, wherein an increase in the labeled polynucleotide hybridization to the patient tissue or biological sample relative to the normal control tissue or biological sample is indicative of a cancer in the patient.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
  • 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.
  • allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • the terms "ammo-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 ⁇ l ⁇ " M ⁇ l.
  • a "complement" of a polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence.
  • the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • corresponding to when applied to positions of amino acid residues in sequences, means corresponding positions in a plurality of sequences when the sequences are optimally aligned.
  • 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).
  • 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.
  • 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.
  • the isolated polypeptide or protein is substantially free of other polypeptides or proteins, particularly those of animal origin. It is preferred to provide the polypeptides and proteins in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • the term “isolated” does not exclude the presence of the same polypeptide or protein in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • “Operably linked” means that two or more entities are joined together such that they function in concert for their intended purposes.
  • DNA segments the phrase indicates, for example, that coding sequences are joined in the correct reading frame, and transcription initiates in the promoter and proceeds through the coding segment(s) to the terminator.
  • “operably linked” includes both covalently (e.g., by disulfide bonding) and non-covalently (e.g., by hydrogen bonding, hydrophobic interactions, or salt-bridge interactions) linked sequences, wherein the desired function(s) of the sequences are retained.
  • 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.
  • 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.
  • bp base pairs
  • nt nt
  • kb kilobases
  • 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”.
  • promoter is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
  • a “protein” is a macromolecule comprising one or more polypeptide chains.
  • a protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless. Thus, a protein "consisting of, for example, from 15 to 1500 amino acid residues may further contain one or more carbohydrate chains.
  • 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-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction.
  • Many cell-surface receptors are, in their active forms, multi-subunit structures in which the ligand-binding and signal transduction functions may reside in separate subunits. 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.
  • 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).
  • a “secretory signal sequence” is a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • a “segment” is a portion of a larger molecule (e.g., polynucleotide or polypeptide) having specified attributes.
  • a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, that, when read from the 5' to the 3' direction, encodes the sequence of amino acids of the specified polypeptide.
  • 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 DNA sequence that encodes a human polypeptide having structural similarity to proteins of the granulocyte peptide A (GP-A) precursor family, and mouse Tag7 (Kiselev, SI et al., J. Biol. Chem. 273:18633-18639, 1998).
  • the present invention provides polypeptides and peptides useful as antimicrobial agents.
  • Antimicrobial mammalian polypeptides have a range of useful activities including bactericidal, anti-fungal and antiviral activities. Such activities can be used therapeutically in vivo and ex vivo, and have numerous uses as a research reagent to prevent or curb contamination of, for example tissue samples and cell cultures, by microbial agents.
  • Bovine granulocyte peptide A precursor antimicrobial BGP-A
  • Bovine and murine granulocyte peptide A precursor are disclosed in WIPO publication WO 97/29765.
  • Bovine GP-A was isolated from a bone marrow library (WO 97/29765). GP-A exhibits broad- spectrum anti-microbial activity against Gram-positive and Gram-negative bacteria, fungi and viruses.
  • zgpal can exhibit antimicrobial (including one or more of anti-bacterial, anti-fungal, and anti-viral) activity, immune modulating activity, wound healing, chemotactic, mitogenic and stimulatory activities and is a novel member of the Tag7 and BGP-A protein family.
  • the sequence of the zgpal polypeptide was obtained from a cDNA clone that was obtained from a fetal tissue library.
  • Other libraries that might also be searched for such sequences include epithelial cells, lung, salivary gland, intestine, skin, lymphoid cells, neutrophils, PMN cells, and the like.
  • Such libraries may also be induced, as anti- bacterial polypeptides, such as defensins, are expressed in epithelial tissues, and are highly inducible upon microbial infection.
  • Nucleotide sequences of representative zgpal -encoding cDNA are described in SEQ ID NO:4 (from nucleotide 1 to 1125), with its deduced 375 amino acid sequence described in SEQ ID NO:5; SEQ ID NO:7 (from nucleotide 1 to 1107), with its deduced 369 amino acid sequence described in SEQ ID NO:8.; and in SEQ ID NO: l (from nucleotide 364 to 705), with its deduced 114 amino acid sequence described in SEQ ID NO:2.
  • the zgpal polypeptide of SEQ ID NO:5, or SEQ ID NO:7 represents a full-length polypeptide segment (residue 1 (Met) to residue 375 (His) of SEQ ID NO:5; residue 1 (Met) to residue 369 (His) of SEQ ID NO:7).
  • the sequence in SEQ ID NO:2 represents a truncated form of zgpal (residue 1 (Met) to residue 114 (Lys) of SEQ ID NO:2).
  • the domains and structural features of the zgpal polypeptides are further described below.
  • N-terminal polypeptide corresponding to amino acid residues 18 (Asp) to amino acid residue 51 (Thr) of SEQ ID NO: 8; corresponding to amino acid residues 18 (Asp) to amino acid residue 57 (Thr) of SEQ ID NO:5; and corresponding to amino acid residues 18 (Asp) to amino acid residue 51 (Thr) of SEQ ID NO:2;
  • domain 1 A first of a repeated domain, referred to hereinafter as "domain 1 ,” corresponding approximately to amino acid residues 52 (Val) to amino acid residue 183 (Val) of SEQ ID NO:8; amino acid residues 58 (Val) to amino acid residue 189 (Val) of SEQ ID NO:5; and a truncated version of domain 1 corresponding to 52 (Val) to amino acid residue 114 (Lys) of SEQ ID NO:2.
  • Domain 1 contains conserved Cys residues (when compared to domain 2, below) at positions 64, 85, 91, and 109 in SEQ ID NO:8; and at positions 70, 91, 97, and 115 in SEQ ID NO:5.
  • linker polypeptide corresponding to amino acid residues 184 (Gin) to amino acid residue 209 (Val) of SEQ ID NO:8; corresponding to amino acid residues 190 (Gin) to amino acid residue 215 (Val) of SEQ ID NO:5;
  • domain 2 A second of a repeated domain, referred to hereinafter as "domain 2," corresponding approximately to amino acid residues 210 (Val) to amino acid residue 340 (Leu) of SEQ ID NO:8; amino acid residues 216 (Val) to amino acid residue 346 (Leu) of SEQ ID NO:5.
  • Domain 2 contains conserved Cys residues (when compared to domain 1, above) at positions 222, 242, 248, and 266 in SEQ ID NO:8; and at positions 228, 248, 254, and 272 in SEQ ID N ⁇ :5.
  • C-terminal polypeptide corresponding to amino acid residues 341 (Leu) to amino acid residue 369 (His) of SEQ ID NO:8; corresponding to amino acid residues 347 (Leu) to amino acid residue 375 (His) of SEQ ID NO:5.
  • the repeated domains 1 and 2 contain a consensus sequence as shown in SEQ ID NO: 12 wherein the conserved residues are indicated by their three-letter code, and Xaa is any amino acid and the amino acid at position 15 can be absent. Moreover, domains 1 and 2 contain a conserved motif, hereinafter referred to as "motif 1" as shown in SEQ ID NO: 12 from amino acid 58 (Cys) to 79 (Asn). This motif is also conserved in BGP-A and mouse Tag7 proteins, suggesting that it is a structurally or functionally essential motif. Thus, preferred embodiments of the present invention comprise "motif 1" as shown in SEQ ID NO: 12 from amino acid 58 (Cys) to 79 (Asn). This motif is also conserved in BGP-A and mouse Tag7 proteins, suggesting that it is a structurally or functionally essential motif. Thus, preferred embodiments of the present invention comprise "motif
  • SEQ ID NO: 12 As anti-microbial polypeptides are known to act individually or associate as dimers, heterodimeric or multimeric polypeptides, the individual domains 1 and 2 of zgpal can function independently, together in a dimer-like fashion, or as heterodimers and multimers with other anti-microbial polypeptides described herein and in the art.
  • the corresponding polynucleotides encoding the zgpal polypeptide regions, domains, motifs, residues and sequences described herein are as shown in SEQ ID NO:l, SEQ ID NO:4 and SEQ ID NO:7.
  • zgpal polypeptide regions, domains, motifs, residues and sequences described herein are also as shown in SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:8.
  • Structural modeling is performed by one of skill in the art, using modelling software, such as InsightH® (Biosym MSI, San Diego, CA).
  • the presence of low variance and structural motifs generally correlate with or define important structural regions in proteins.
  • Regions of low variance e.g., hydrophobic clusters
  • regions of low variance often contain rare or infrequent amino acids, such as Tryptophan.
  • 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, cell-cell interaction, tissue localization domains and the like.
  • RT-PCR reverse transcription-polymerase chain reaction
  • degenerate DNA probes and degenerate primers can be employed to identify other human or zgpal-like polynucleotides.
  • highly degenerate primers designed from the above sequences are useful for this purpose.
  • Polypeptides of the present invention comprise at least 6, preferably at least 9, more preferably at least 15 contiguous amino acid residues of SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:8.
  • the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:8, up to the entire predicted mature polypeptide (residues 18-114 of SEQ ID NO:2, 18-375 of SEQ ID NO:5 or 18-369 of SEQ ID NO:8) or the primary translation product (residues 1-114 of SEQ ID NO:2, 1-375 of SEQ ID NO:5 or 1-369 of SEQ ID NO:8).
  • these polypeptides can further comprise additional, non-zgpal, polypeptide sequence(s).
  • polypeptides of the present invention are polypeptides that comprise an epitope-bearing portion of a protein as shown in SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:8.
  • An "epitope" is a region of a protein to which an antibody can bind. See, for example, Geysen et al., Proc. Natl Acad. Sci. USA 81:3998-4002, 1984.
  • Epitopes can be linear or conformational, the latter being composed of discontinuous regions of the protein that form an epitope upon folding of the protein. Linear epitopes are generally at least 6 amino acid residues in length.
  • Antibodies to linear epitopes are also useful for detecting fragments of zgpal, such as might occur in body fluids or cell culture media.
  • the amino acid sequence of the epitope-bearing polypeptide is selected to provide substantial solubility in aqueous solvents, that is the sequence includes relatively hydrophilic residues, and hydrophobic residues are substantially avoided.
  • the amino termini of such zgpal hexapeptides include residues 92, 117, 150, 151, and 152 of SEQ ID NO:8; and 98, 123, 156, 157, and 158 of SEQ ID NO:5.
  • longer polypeptides comprising these hexapeptides can also be used and will often be preferred.
  • those antigenic epitopes that comprise residues predicted from a Jameson- Wolf plot as described herein are also preferred epitopes.
  • Antigenic, epitope-bearing polypeptides of the present invention are useful for raising antibodies, including monoclonal antibodies that specifically bind to a zgpal protein.
  • Antigenic, epitope-bearing polypeptides contain a sequence of at least six, preferably at least nine, more preferably from 15 to about 30 contiguous amino acid residues of a zgpal protein (e.g., SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:8).
  • Polypeptides comprising a larger portion of a zgpal protein, i.e. from 30 to 50 residues up to the entire sequence, are included.
  • amino acid sequence of the epitope-bearing polypeptide is selected to provide substantial solubility in aqueous solvents, that is the sequence includes relatively hydrophilic residues, and hydrophobic residues are substantially avoided.
  • Preferred such regions include the interdomain loops of zgpal and fragments thereof.
  • Polypeptides of the present invention can be prepared with one or more amino acid substitutions, deletions or additions as compared to SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:8. These changes are preferably of a minor nature, that is conservative amino acid substitutions and other changes that do not significantly affect the folding or activity of the protein or polypeptide, and include amino- or carboxyl- terminal extensions, such as an amino-terminal methionine residue, an amino or carboxyl-terminal cysteine residue to facilitate subsequent linking to maleimide- activated keyhole limpet hemocyanin, a small linker peptide of up to about 20-25 residues, or an extension that facilitates purification (an affinity tag) as disclosed above. Two or more affinity tags may be used in combination.
  • Polypeptides comprising affinity tags can further comprise a polypeptide linker and/or a proteolytic cleavage site between the zgpal polypeptide and the affinity tag.
  • Preferred cleavage sites include thrombin cleavage sites and factor Xa cleavage sites.
  • the present invention further provides a variety of other polypeptide fusions.
  • a zgpal 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-zgpal polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric zgpal analogs.
  • a zgpal polypeptide can be joined to another bioactive molecule, such as a cytokine, to provide a multifunctional molecule.
  • One or more domains of a zgpal polypeptide can be joined to another anti-microbial polypeptide or a known cytokine, or the like, to enhance or otherwise modify its biological properties.
  • Auxiliary domains can be fused to zgpal polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen).
  • a zgpal polypeptide or protein can be targeted to a predetermined cell type by fusing a zgpal 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 zgpal 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.
  • polypeptides of the present invention comprise at least 6 contiguous residues of SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:8. These polypeptides may further comprise additional residues as shown in SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:8, a variant of SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:8, or another protein as disclosed herein.
  • the resulting polypeptide will preferably be at least 90%, more preferably at least 95% identical to the corresponding region of SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:8. Percent sequence identity is determined by conventional methods.
  • 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.
  • 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 FASTA program parameters set as default.
  • the present invention includes polypeptides having one or more conservative amino acid changes as compared with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:8.
  • the BLOSUM62 matrix (Table 1) 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, ibid.). Thus, 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 term "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 one 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • the proteins of the present invention can also comprise non-naturally occuring amino acid residues.
  • Non-naturally occuring amino acids include, without limitation, tr ⁇ n.s-3-methylproline, 2,4-methanoproline, -4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, ⁇ //o-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, , 4- azaphenylalanine, and 4-fluorophenylalanine.
  • Several methods are known in the art for incorporating non-naturally occuring amino acid residues into proteins.
  • 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; Ellman et al., Methods Enzymol.
  • non-naturally occuring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994. Naturally occuring amino acid residues can be converted to non-naturally occuring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).
  • Amino acid sequence changes are made in zgpal polypeptides so as to minimize disruption of higher order structure essential to biological activity.
  • Amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can identify specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule.
  • Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity, secondary structure propensities, binary patterns, complementary packing, and buried polar interactions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al., Current Opin. Struct. Biol. 6:3-10, 1996).
  • Protein folding can be measured by circular dichroism (CD). Measuring and comparing the CD spectra generated by a modified molecule and standard molecule are routine in the art (Johnson, Proteins 7:205-214, 1990). Crystallography is another well known and accepted method for analyzing folding and structure. Nuclear magnetic resonance (NMR), digestive peptide mapping and epitope mapping are other known methods for analyzing folding and structural similarities between proteins and polypeptides (Schaanan et al., Science 257:961-964. 1992).
  • NMR nuclear magnetic resonance
  • digestive peptide mapping and epitope mapping are other known methods for analyzing folding and structural similarities between proteins and polypeptides (Schaanan et al., Science 257:961-964. 1992).
  • Mass spectrometry and chemical modification using reduction and alkylation can be used to identify cysteine residues that are associated with disulfide bonds or are free of such associations (Bean et al., Anal. Biochem. 201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Patterson et al., Anal. Chem. 66:3727-3732, 1994). Alterations in disulfide bonding will be expected to affect protein folding. These techniques can be employed individually or in combination to analyze and compare the structural features that affect folding of a variant protein or polypeptide to a standard molecule to determine whether such modifications would be significant.
  • a Hopp/Woods hydrophilicity profile of the zgpal protein sequence as shown in SEQ ID NO:8 or SEQ ID NO:5 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 j _: 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.
  • hydrophilic regions include: (1) amino acid number 92 (Ser) to amino acid number 97 (Glu) of SEQ ID NO: 8 (98 (Ser) to amino acid number 103 (Glu) of SEQ ID NO:5); (2) amino acid number 117 (Val) to amino acid number 122 (Arg) of SEQ ID NO: 8 (123 (Val) to amino acid number 128 (Arg) of SEQ ID NO:5); (3) amino acid number 150 (Gly) to amino acid number 155 (His) of SEQ ID NO:8 (156 (Gly) to amino acid number 161 (His) of SEQ ID NO:5); (4) amino acid number 151 (Thr) to amino acid number 156 (Ser) of SEQ ID NO:8 (157 (Thr) to amino acid number 162 (Ser) of SEQ ID NO: 5); and (5) amino acid 152 (Lys) to amino acid number 157 (Pro) of SEQ ED NO: 8 (158 (Lys) to amino acid number 163 (Pro) of SEQ ED NO:
  • hydrophilicity or hydrophobicity will be taken into account when designing modifications in the amino acid sequence of a zgpal 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,
  • residues tolerant of substitution could include these hydrophobic residues as shown in SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO: 8. Cysteine residues that are involved in disulfide bonds will be relatively intolerant of substitution.
  • identities of essential amino acids can also be inferred from analysis of sequence similarity between family members with zgpal. 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 zgpal polynucleotide on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant zgpal polynucleotide can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, as discussed above.
  • Essential amino acids in the polypeptides of the present invention can be identified experimentally according to procedures known in the art, such as site- directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991).
  • site- directed mutagenesis or alanine-scanning mutagenesis
  • alanine-scanning mutagenesis Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991.
  • single alanine mutations are introduced throughout the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule.
  • Variants of the disclosed zgpal DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391. 1994 and Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751, 1994. Briefly, variant genes are generated by in vitro homologous recombination by random fragmentation of a parent gene followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent genes, such as allelic variants or genes from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
  • the structure of the final polypeptide product will result from processing of the nascent polypeptide chain by the host cell, thus the final sequence of a zgpal polypeptide produced by a host cell will not always correspond to the full sequence encoded by the expressed polynucleotide.
  • expressing the complete zgpal sequence in a cultured mammalian cell is expected to result in removal of at least the secretory peptide, while the same polypeptide produced in a prokaryotic host would not be expected to be cleaved.
  • Differential processing of individual chains may result in heterogeneity of expressed polypeptides.
  • Zgpal proteins of the present invention are characterized by their activity, that is, antimicrobial activity, or ability to lyse cells, induce immune cell migration or induce apoptosis.
  • Biological activity of zgpal proteins is assayed using in vitro or in vivo assays designed to detect protection of cells against microbial invasion or contamination, ion flux, cell lysis, cell migration , apoptosis, and the like.
  • Many suitable assays are known in the art, and representative assays are disclosed herein. Assays using cultured cells, or conditioned medium from cultured cells, are most convenient for screening, such as for determining the effects of amino acid substitutions, deletions, or insertions.
  • assays will generally be employed to confirm and further characterize biological activity.
  • Assays can be performed using exogenously produced proteins, or may be carried out in vivo or in vitro using cells expressing the polypeptide(s) of interest.
  • Assays can be conducted using zgpal proteins alone or in combination with other proteins such as other anti- microbial polypeptides, e.g., defensins, and the like. Representative assays are disclosed below.
  • Mutagenesis methods as disclosed above can be combined with high volume or high-throughput screening methods to detect biological activity of zgpal variant polypeptides.
  • Assays that can be scaled up for high throughput include _ion flux, cell lysis, migration and signaling assays amongst others, which can be run in a
  • Mutagenized DNA molecules that encode active zgpal polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure. Using the methods discussed above, one of ordinary skill in the art can prepare a variety of polypeptide fragments or variants of SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:8 that retain the activity of wild-type zgpal.
  • the present invention also provides zgpal polynucleotide molecules.
  • These polynucleotides include DNA and RNA, both single- and double-stranded, the former encompassing both the sense strand and the antisense strand.
  • a representative DNA sequence encoding the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:8 is shown in SEQ ID NO:l, SEQ ID NO:4 or SEQ ID NO:7.
  • SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9 are degenerate DNA sequences that encompass all DNAs that encode the zgpal polypeptide of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8 respectively.
  • the degenerate sequence of SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9 also provides] all RNA sequences encoding SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8 by substituting U for T.
  • zgpal polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 342 of SEQ ID NO:3, nucleotide 1 to nucleotide 1125 of SEQ ID NO:6, and nucleotide 1 to nucleotide 1107 of SEQ ID NO:9 and their RNA equivalents are contemplated by the present invention, as are segments thereof encoding other zgpal polypeptides disclosed herein.
  • Table 2 sets forth the one-letter codes used within SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9 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 2 sets forth the
  • degenerate codons used in SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9, encompassing all possible codons for a given amino acid, are set forth in Table 3, below.
  • degenerate codon representative of all possible codons encoding each amino acid.
  • WSN can, in some circumstances, encode arginine
  • MGN can, in some circumstances, encode serine
  • polynucleotides encompassed by a degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:6 or SEQ ID NO:9. Variant sequences can be readily tested for functionality as described herein.
  • the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO: 1 , SEQ ID NO:4,
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake, MN) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto, CA), as well as sites on the Internet, are available tools for analyzing a given sequence and calculating T m based on user defined criteria. Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences. Typically, hybridization of longer polynucleotide sequences, >50 base pairs, is performed at temperatures of about 20-25°C below the calculated T m .
  • hybridization is typically carried out at the T m or 5-10°C below. This allows for the maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids. Higher degrees of stringency at lower temperatures can be achieved with the addition of formamide which reduces the T m of the hybrid about 1°C for each 1% formamide in the buffer solution. 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 6X SSC, about 5X Denhardt's solution, zero up to about 10% dextran sulfate, and about 10-20 ⁇ g/ml denatured commercially-available carrier DNA.
  • such stringent conditions include temperatures of 20-70°C and a hybridization buffer containing up to 6x SSC and 0-50% formamide; 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. Different degrees of stringency can be used during hybridization and washing to achieve maximum specific binding to the target sequence.
  • the washes following hybridization are performed at increasing degrees of stringency to remove non-hybridized polynucleotide probes from hybridized complexes.
  • 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 zgpal RNA.
  • Such tissues and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), and may include epithelial tissues, fetal tissues, gastrointestinal tissues, skin, lung, and include whole tissue extracts or sub-fractions of tissues including specialized cells, such as columnar epithelial cells, airway epithelial cells, cervical cells, and the like, although DNA can also be prepared using RNA from other tissues or isolated as genomic DNA.
  • 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 zgpal polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • Full-length clones encoding zgpal 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 zgpal, receptor fragments, or other specific binding partners.
  • Zgpal polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a zgpal gene.
  • Promoter elements from a zgpal gene can thus be used to direct the expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy.
  • Cloning of 5' flanking sequences also facilitates production of zgpal proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous zgpal gene in a cell is altered by introducing into the zgpal 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 zgpal 5' non-coding sequence that permits homologous recombination of the construct with the endogenous zgpal locus, whereby the sequences within the construct become operably linked with the endogenous zgpal coding sequence.
  • an endogenous zgpal promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue- specific, or otherwise regulated expression.
  • zgpal is specifically-expressed in cervix, fetal skin, salivary gland, keratinocytes, trachea, lung tumor, ovarian tumor, esophageal tumor and rectal tumor tissues, but generally not in not others, the regulatory regions described above are expected to provide for tissue-specific expression.
  • comparison of the zgpal cDNA sequences can be used to identify the intron-exon junctions in zgpal genomic DNA (Genbank Accession No. ACO 11666), as well as promoter and regulatory regions therein.
  • sequences disclosed in SEQ ID NO: l, SEQ ID NO:4 and SEQ ID NO:7 represent a single allele of human zgpal. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.
  • the present invention further provides counterpart polypeptides and polynucleotides from other species ("orthologs").
  • zgpal polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides.
  • non-human zgpal polypeptides and polynucleotides, as well as antagonists thereof and other related molecules can be used, inter alia, in veterinary medicine.
  • Orthologs of human zgpal 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 zgpal as disclosed above.
  • a library is then prepared from mRNA of a positive tissue or cell line.
  • a zgpal -encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequence.
  • a cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human zgpal sequence disclosed herein.
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to zgpal polypeptide. Similar techniques can also be applied to the isolation of genomic clones. For any zgpal 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 2 and 3, above. Moreover, those of skill in the art can use standard software to devise zgpal variants based upon the nucleotide and amino acid sequences described herein.
  • the present invention thus provides a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ ID NO: 1 through SEQ ID NO:9 and portions thereof.
  • Suitable forms of computer-readable media include magnetic media and optically-readable media. Examples of magnetic media include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT), a disk cache, and a ZIPTM disk.
  • Optically readable media are exemplified by compact discs (e.g., CD-read only memory (ROM), CD-rewritable (RW), and CD- recordable), and digital versatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).
  • the zgpal polypeptides of the present invention can be produced according to conventional techniques using cells into which have been introduced an expression vector encoding the polypeptide.
  • cells into which have been introduced an expression vector include both cells that have been directly manipulated by the introduction of exogenous DNA molecules and progeny thereof that contain the introduced DNA.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells.
  • a DNA sequence encoding a zgpal polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers can be provided on separate vectors, and replication of the exogenous DNA is provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
  • 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 zgpal (residues 1-17 of SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:8) or may be derived from another secreted protein (e.g., t-PA; see, U.S. Patent No. 5,641,655) or synthesized de novo.
  • the secretory signal sequence is operably linked to the zgpal 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 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).
  • polypeptide fusions, or hybrid zgpal proteins are constructed using regions or domains of the inventive zgpal in combination with those of other human cytokine family proteins (e.g. interleukins or GM-CSF), or heterologous proteins (Sambrook et al., ibid., Altschul et al., ibid., Picard, Cur. Opin. Biology, 5:511-5, 1994, and references therein). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest.
  • hybrids may alter reaction kinetics, binding, alter cell migration or proliferative activity, constrict or expand the substrate or anti-microbial specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure.
  • Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them.
  • a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein.
  • part or all of a domain, motif, or region conferring a biological function may be swapped between zgpal of the present invention with the functionally equivalent domain, motif, or region from another family member, such as bGP-A, or functionally related family, such as ⁇ -defensins, indolicidin, and ⁇ -defensins.
  • 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 known anti-microbial 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 zgpal polypeptide and those polypeptides to which they are fused.
  • a DNA segment that encodes a domain of interest e.g., zgpal domain 1, domain 2, or other regions, motifs or domains described herein, is operably linked in frame to at least one other DNA segment encoding an additional polypeptide (for instance a domain or region from another anti-microbial polypeptide, such as bGP-A, defensin, or the like), and inserted into an appropriate expression vector, as 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 containing functionally active domain 1, followed by domain 2; or a signal polypeptide followed by a polypeptide containing functionally active zgpal domain 1, followed by a functionally active bGP-A, defensin, indolicidin, or other antimicrobial polypeptide; or a signal polypeptide followed by a functionally active bGP-A, defensin, indolicidin, or other anti-microbial polypeptide followed by a polypeptide containing functionally active zgpal domain 2; or for example, any of the above as interchanged with equivalent regions from another antimicrobial polypeptide.
  • Such fusion proteins can be expressed,
  • fusion proteins can be used to express and secrete fragments of the zgpal polypeptide, to be used, for example to inoculate an animal to generate anti- zgpal antibodies as described herein.
  • a secretory signal sequence can be operably linked to zgpal domain 1, domain 2, or other regions, motifs or domains described herein or a combination thereof (e.g., operably linked polypeptides comprising zgpal domain 1 through domain 2, or other regions, zgpal polypeptide fragments, motifs or domains described herein) to secrete a fragment of zgpal polypeptide that can be purified as described herein and serve as an antigen to be inoculated into an animal to produce anti-zgpal antibodies, as described herein.
  • Zgpal polypeptides or fragments thereof may also be prepared through chemical synthesis, zgpal 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.
  • the polypeptides can be prepared by solid phase peptide synthesis, for example as described by Merrifield, J. Am. Chem. Soc. 85:2149, 1963. Cultured mammalian cells can be used as hosts within the present invention.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. L841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al., ibid.), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993).
  • 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
  • Chinese hamster ovary e.g. CHO-Kl, ATCC No. CCL 61; or CHO DG44, Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986
  • Additional 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.
  • promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
  • Expression vectors for use in mammalian cells include pZP-1 and pZP-9, which have been deposited with the American Type Culture Collection, Manassas, VA USA under accession numbers 98669 and 98668, respectively, and derivatives thereof.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants”. Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.”
  • 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
  • hygromycin resistance multi-drug resistance
  • puromycin acetyltransferase can also be used.
  • Alternative markers that produce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, and placental alkaline phosphatase, can be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
  • the adenovirus system can also be used for protein production in vitro.
  • 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 Garnier 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.
  • non- secreted proteins can also be effectively obtained.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa calif omica nuclear polyhedrosis virus (AcNPV) according to methods known in the art, such as the transposon-based system described by Luckow et al. (J. Virol. 67:4566-4579, 1993).
  • This system which utilizes transfer vectors, is commercially available in kit form (Bac-to-BacTM kit; Life Technologies, Rockville, MD).
  • the transfer vector e.g., pFastBaclTM; Life Technologies
  • transfer vectors can include an in-frame fusion with DNA encoding a polypeptide extension or affinity tag as disclosed above. Using techniques known in the art, a transfer vector containing a zgpal -encoding sequence is transformed into E.
  • coli host cells and the cells are screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, such as Sf9 cells.
  • Recombinant virus that expresses zgpal protein is subsequently produced.
  • Recombinant viral stocks are made by methods commonly used the art.
  • the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., High FiveTM cells; Invitrogen, Carlsbad, CA).
  • host cells typically a cell line derived from the fall armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., High FiveTM cells; Invitrogen, Carlsbad, CA).
  • Serum-free media are used to grow and maintain the cells. Suitable media formulations are known in the art and can be obtained from commercial suppliers. The cells are grown up from an inoculation density of approximately 2-5 x 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. Procedures used are generally known in the art.
  • eukaryotic cells can also be used as hosts, including plant 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) 11:47-58, 1987.
  • Fungal cells, including yeast cells can also be used within the present invention.
  • yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No.
  • 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 POTl vector system disclosed by Kawasaki et al. (U.S. Patent No.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.
  • Transformation systems for other yeasts including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol 132:3459-3465, 1986; Cregg, U.S. Patent No. 4,882,279; and Raymond et al., Yeast 14,
  • Aspergillus cells can be utilized according to the methods of Mc Knight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533. Production of recombinant proteins in Pichia methanolica is disclosed in U.S. Patents Nos. 5,716,808, 5,736,383, 5,854,039, and 5,888,768.
  • Prokaryotic cells including strains of the bacteria Escherichia coli, Bacillus and other genera can also be used as hosts 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 zgpal 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.
  • Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors.
  • Zgpal polypeptides can also be prepared through chemical synthesis according to methods known in the art, including exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, DL, 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989. In vitro synthesis is particularly advantageous for the preparation of smaller polypeptides.
  • polypeptides and proteins of the present invention it is preferred to purify the polypeptides and proteins 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 or protein is substantially free of other polypeptides or proteins, particularly those of animal origin.
  • Zgpal proteins are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See, in general, Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice, Springer- Verlag, New York, 1994. Proteins comprising a polyhistidine affinity tag (typically about 6 histidine residues) are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bio Technol 6: 1321-1325, 1988.
  • Proteins comprising a glu- glu tag can be purified by immunoaff ⁇ nity chromatography according to conventional procedures. See, for example, Grussenmeyer et al., ibid. Maltose binding protein fusions are purified on an amylose column according to methods known in the art.
  • zgpal proteins can be prepared as monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.
  • Molecules of the present invention can be used to identify and isolate receptors that bind zgpal polypeptide.
  • 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.
  • radiolabeled zgpal proteins and polypeptides can be used to clone the cognate receptor in binding assays using cells transfected with an expression cDNA library.
  • 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 (BIAcoreTM, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed.
  • a ligand-binding receptor or an antibody, one member of a complement/ anti-complement pair
  • a commercially available biosensor instrument (BIAcoreTM, Pharmacia Biosensor, Piscataway, NJ)
  • 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. 51: 660-72, 1949) and calorimetric assays (Cunningham et al., Science 253:545-48, 1991; Cunningham et al.. Science 245:821-25, 1991).
  • 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.
  • zgpal -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 zgpal polypeptide or anti-zgpal antibody targets the hyperproliferative blood or bone marrow cell (See, generally, Hornick et al., Blood 89:4437-47, 1997). Hornick et al.
  • Suitable zgpal polypeptides or anti-zgpal antibodies can target an undesirable cell or tissue (i.e., a tumor or a leukemia), and the fused cytokine can mediate 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.
  • zgpal is specifically expressed in various epithelial tumor tissues, such as lung tumor, ovarian tumor, esophageal tumor and rectal tumors, such tissues can serve as target tissues for zgpal -cytokine fusion proteins or antibody- cytokine fusion proteins as described above.
  • the zgpal polypeptide or anti-zgpal 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 or provide anti-cancer radioactive therapy.
  • 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.
  • 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.
  • Anti-microbial activity of zgpal polypeptides is evaluated by techniques that are known in the art. For example, anti-microbial activity can be assayed by evaluating the sensitivity of microbial cell cultures to test agents or by evaluating the protective effect of test agents on infected mice. See, for example, Musiek et al., Antimicrob. Agents Chemothr. 3:40, 1973. Antiviral activity can also be assessed by protection of mammalian cell cultures. Known techniques for evaluating anti-microbial activity are disclosed by, for example, Barsum et al., Eur. Respir. J. 8:709-714, 1995; Sandovsky-Losica et al., J. Med. Vet.
  • anti-microbial activity of zgpal polypeptides, fragments, fusions, antibodies, agonists and antagonists can be evaluated by techniques that are known in the art. More specifically, anti-microbial activity can be assayed by evaluating the sensitivity of microbial cell cultures to test agents and by evaluating the protective effect of test agents on infected mice. See, for example, Musiek et al., Antimicrob. Agents Chemothr. 3: 40, 1973. Antiviral activity can also be assessed by protection of mammalian cell cultures. Known techniques for evaluating anti-microbial activity include, for example, Barsum et al., Eur. Respir. J.
  • Panlabs, Inc. of Bothell, Washington offer in vitro or in vivo testing for bacteria, gram negative (Enterobacter cloacae, Escherichia coli, Klebsiella pneumonia, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhimurium and Serratia marcescens), gram positive (Bacillus subtilis, Brevebacterium ammoniagenes, Corynebacterium minutissimum, Micrococcus luteus, Mycobacterium ranae, Staphylococcus strains and Streptococcus strains) and anaerobic organisms (Actinomyces viscosus, Bacteroides fragilis, Clostridium sporogenes, Corynebacterium acnes, Helicobacter pylori and Porphyromonas gingivalis), as well as for protozoa (Trichomona
  • zgpal polypeptide, fragment, fusion protein, agonist, antagonist or antibody performance in this regard can be compared to proteins known to be functional in this regard, such as defensins, indolicidins, bactenicins, proline-rich proteins, lysozyme, histatins, lactoperoxidase or the like.
  • proteins known to be functional in this regard such as defensins, indolicidins, bactenicins, proline-rich proteins, lysozyme, histatins, lactoperoxidase or the like.
  • zgpal polypeptide, fragment, fusion protein, antibody, agonist or antagonist may be evaluated in combination with one or more anti-microbial agents to identify synergistic effects.
  • the anti-microbial polypeptides of the present invention can be used for treatment of dental carries (tooth decay), periodontal disease, thrush, and gastrointestinal disease. Other applications can be used in urinary tract infections, respiratory infections, vaginal infections, prevention of infection in skin and other epithelial wounds. As such, the polypeptides of the present invention can help establish normal microflora and protect against pathogenic colonization and invasion.
  • microorganisms have specific infective stages where they decorate their surface with proline coil containing proteins.
  • One theory is that they are decoying or evading the host immune system by expressing host-like, but inactive, proteins on their surfaces.
  • the zgpal polypeptide of the present invention can be one of these host polypeptides that the microorganism is mimicking.
  • Such immune activation by zgpal polypeptides can be assessed by assays that are well known in the art.
  • compositions of the present invention may also be used when pro-inflammatory activity is desired.
  • applications for such pro-inflammatory activity include the treatment of chronic tissue damage, particularly in areas having a limited or damaged vascular system, e.g., damage in extremities associated with diabetes.
  • antagonists to zgpal polypeptides may be useful as anti-inflammatory agents.
  • Zgpal polypeptide pharmaceutical compositions of the present invention may also be used in the treatment of conditions where stimulation of immune responsiveness is desired.
  • Such conditions include the treatment of patients having incompetent immune systems, such as AIDS patients or individuals that have undergone chemotherapy, radiation treatment or the like.
  • zgpal polypeptide has a structure indicative anti-microbial function, and cystic fibrosis is characterized by frequent microbial infection
  • pharmaceutical compositions containing zgpal polypeptide are also contemplated for use in the treatment of lung infections associated with cystic fibrosis.
  • zgpal polypeptides are sensitive to salt concentration
  • engineered zgpal polypeptides that are characterized by decreased sensitivity to salt concentration are contemplated by the present invention. Decreased sensitivity to high salt concentration will preserve anti-microbial activity of engineered zgpal polypeptides in high salt environments, such as in the lung airways of patients suffering from cystic fibrosis.
  • compositions containing engineered zgpal polypeptides that are formulated for delivery to the lungs can be used to treat lung infections associated with cystic fibrosis.
  • Another aspect of the present invention involves the detection of zgpal polypeptides in cell culture or in a serum sample or tissue biopsy of a patient undergoing evaluation for microbial infection, SPG, Chediak-Higashi syndrome, or other conditions characterized by an alteration in defensin concentration.
  • Zgpal polypeptides can be detected using immunoassay techniques and antibodies capable of recognizing a zgpal polypeptide epitope, as described herein.
  • the present invention contemplates methods for detecting zgpal polypeptide comprising: exposing a solution or sample or cell culture lysate or supernatant, possibly containing zgpal polypeptide, to an antibody attached to a solid support, wherein said antibody binds to a first epitope of a zgpal polypeptide; washing said immobilized antibody-polypeptide to remove unbound contaminants; exposing the immobilized antibody-polypeptide to a second antibody directed to a second epitope of a zgpal polypeptide, wherein the second antibody is associated with a detectable label; and detecting the detectable label.
  • Zgpal polypeptide concentration differing from that of controls may be indicative of SPG, Chediak-Higashi syndrome, microbial infection or other conditions characterized by an alteration in zgpal concentration.
  • expression of zgpal may be monitored in cystic fibrosis patients as a predictor of the onset of infectious crises.
  • high defensin levels have been associated with cytotoxic effects in lung, indicating that other host anti-microbial polypeptides, such as zgpal polypeptide levels can be used as indicators for disease onset and cytotoxicity, and used to direct treatment for averting or addressing such cytotoxicity.
  • antibodies directed to zgpal polypeptide can be administered to inactivate the same in a treatment modality.
  • antibodies or synthesized binding proteins e.g., those generated by phage display, E. coli Fab, and the like
  • Such antibodies are useful for, among other uses as described herein, preparation of anti- idiotypic antibodies.
  • Synthesized binding proteins may be produced by phage display using commercially available kits, such as the Ph.D.TM Phage Display Peptide Library Kits available from New England Biolabs, Inc. (Beverly, Massachusetts). Phage display techniques are described, for example, in US Patent Nos. 5,223,409, 5,403,484 and 5,571,698.
  • An additional aspect of the present invention provides methods for identifying agonists or antagonists of the zgpal polypeptides disclosed above, which agonists or antagonists may have valuable properties as discussed further herein.
  • a method of identifying zgpal polypeptide agonists comprising providing cells responsive thereto, culturing the cells in the presence of a test compound and comparing the cellular response with the cell cultured in the presence of the zgpal polypeptide, and selecting the test compounds for which the cellular response is of the same type.
  • a method of identifying antagonists of zgpal polypeptide comprising providing cells responsive to a zgpal polypeptide, culturing a first portion of the cells in the presence of zgpal polypeptide, culturing a second portion of the cells in the presence of the zgpal polypeptide and a test compound, and detecting a decrease in a cellular response of the second portion of the cells as compared to the first portion of the cells.
  • a further aspect of the invention provides a method of studying chemoattraction of monocytes in cell culture, comprising incubating monocytes in a culture medium comprising a zgpal polypeptide, fragment, fusion protein, antibody, agonist or antagonist to study or evaluate monocyte chemoattraction. Such evaluation may be conducted using methods known in the art.
  • Melanocortin receptors are G-protein coupled receptors that activate adenylate cyclase and cause calcium flux.
  • the agouti protein (which contains a 36 amino acid domain that is toxin-like) is thought to inhibit the binding of MSH-alpha to MCI and MC4.
  • the agouti protein is thought to be an antagonist of calcium channels, and certain toxins are believed to modulate ion flux.
  • Experimental evidence has been generated, suggesting that defensins are capable of blocking calcium channels.
  • zgpal polypeptides could have such properties.
  • a further aspect of the invention provides a method of studying activity of the melanocortin family of receptors in cell culture, comprising incubating cells that endogenously bear such receptors (e.g., ACTH receptors or the like) or cells that have been engineered to bear such receptors in a culture medium comprising a ligand or putative ligand and zgpal polypeptide, fragment, fusion protein, antibody, agonist or antagonist to study or evaluate ligand or putative ligand binding and/or ion flux regulation or modulation.
  • Such evaluation may be conducted using methods known in the art.
  • a further aspect of the invention provides a method of studying ion flux, such as calcium flux, sodium flux, potassium flux or the like, in cell culture, comprising incubating cells that are capable of ion flux in a culture medium comprising zgpal polypeptide, fragment, fusion protein, antibody, agonist or antagonist to study or evaluate ion flux regulation or modulation.
  • ion flux such as calcium flux, sodium flux, potassium flux or the like
  • anti-microbial polypeptides such as defensins
  • defensins have been shown to directly bind and disrupt membranes by forming ion-permeable channels in lipid bilayers in a voltage dependant manner, consistent with the formation of multimeric ion pores; fusion and lysis of small unilamellar phospholipid vesicles; and permeablization of E. coli membranes.
  • methods that measure ion flux across cell membranes, lipid fusion and small and large unilamellar vesicles can be employed to directly assay the activity of zgpal polynucleotides or polypeptides. Such methods are known in the art, and are disclosed in Lehrer, RI et al., J. Clin.
  • the activity of zgpal polypeptide can be measured by a silicon-based biosensor microphysiometer that measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses.
  • An exemplary device is the CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
  • a variety of cellular responses 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.
  • the microphysiometer By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including zgpal polypeptide, its agonists, or antagonists. Preferably, the microphysiometer is used to measure responses of a zgpal- responsive cell, compared to a control cell that does not respond to zgpal polypeptide.
  • ZGPA1 -responsive cells comprise cells into which a receptor for zgpal has been transfected creating a cell that is responsive to zgpal; or cells naturally responsive to zgpal such as cells derived from gastrointestinal, skin, mucosal, epithelial, and lung tissue; or as applied to non-mammalian cells, responsive cells that are permeabilized or lyse in the presence of zgpal such as fungal or bacterial cells. Differences, measured by a change, for example, an increase or diminution in extracellular acidification, in the response of cells exposed to zgpal polypeptide, relative to a control not exposed to zgpal, are a direct measurement of zgpal -modulated cellular responses.
  • zgpal -modulated responses can be assayed under a variety of stimuli.
  • a method of identifying agonists of zgpal polypeptide comprising providing cells responsive to a zgpal 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 zgpal polypeptide and the absence of a test compound can be used as a positive control for the zgpal -responsive cells, and as a control to compare the agonist activity of a test compound with that of the zgpal polypeptide.
  • a method of identifying antagonists of zgpal polypeptide comprising providing cells responsive to a zgpal polypeptide, culturing a first portion of the cells in the presence of zgpal and the absence of a test compound, culturing a second portion of the cells in the presence of zgpal 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 zgpal polypeptide can be rapidly identified using this method.
  • zgpal can be used to identify cells, tissues, or cell lines which respond to a zgpal -stimulated pathway.
  • the microphysiometer, described above can be used to rapidly identify ligand-responsive cells, such as cells responsive to zgpal of the present invention.
  • Cells can be cultured in the presence or absence of zgpal polypeptide. Those cells that elicit a measurable change in extracellular acidification in the presence of zgpal are responsive to zgpal.
  • Such cells can be used to identify antagonists and agonists of zgpal polypeptide as described above.
  • the activity of molecules of the present invention can be measured using a variety of assays that measure ion flux or voltage conductance. Of particular interest is measuring ion transfer cross cell membranes. Such assays are well known in the art. Specific assays to assess ion flux or voltage conductance activity include, but are not limited to, bioassays measuring voltage-dependent conductance in Xenopus laevis oocytes (see, Rudy, B., and Iverson, L.E., eds., Meth. Enzymol., vol. 207, Academic Press, San Diego, CA, 1992; Hamill, O.P et al., Pfluegers Arch. 391:85-
  • This method involves injecting in vitro expressed mRNAs into isolated oocytes and assessing voltage-dependent conductance using a patch-clamp technique.
  • This system may be applied to other cell types, such as insect and mammalian cells (see, Rudy, B., Iverson, L.E., eds., ibid.).
  • Ion flux or voltage conductance activity can also be monitored by using a radiolabeled ion, such as a l25 I efflux assay (Xia, Y. et al., J.
  • antimicrobial polypeptides such as defensins
  • zgpal may also be a signaling polypeptide that affects chemotaxis, proliferation and immune cell migration.
  • cells are cultured in the presence or absence of a test compound, and cell proliferation is detected by, for example, measuring incorporation of tritiated thymidine or by colorimetric assay based on the reduction or metabolic breakdown of Alymar BlueTM (AccuMed, Chicago, IL) or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Mosman, J. Immunol. Meth. 65: 55-63, 1983).
  • An alternative assay format uses cells that are further engineered to express a reporter gene. The reporter gene is linked to a promoter element that is responsive to the receptor-linked pathway, and the assay detects activation of transcription of the reporter gene.
  • a preferred promoter element in this regard is a serum response element, ST AT or SRE (see, for example, Shaw et al., Cell 56:563-572, 1989).
  • a preferred such reporter gene is a luciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, 1987). Expression of the luciferase gene is detected by luminescence using methods known in the art (e.g., Baumgartner et al., J. Biol. Chem. 269:19094-29101. 1994; Schenborn and Goiffin, Promega Notes 41: 11, 1993). Luciferase assay kits are commercially available from, for example, Promega Corp., Madison, WI.
  • zgpal may be involved in cellular apoptotic processes.
  • Polypeptides, such as zgpal that induce apoptosis can have use in killing cancer cells.
  • Apoptosis activity can be measured in a number of ways. For example cytotoxicity assays are well known and can be assessed in zgpal transfected cells in the presence of Trypan Blue.
  • apoptotic events can be assessed using target L929 cells in the presence of Actinomycin D that are subjected to zgpal polypeptides (purified or in conditioned medium).
  • Anti-zgpal antibodies can be used as a specificity control to block the effects of zgpal.
  • DNA fragmentation assays can also be employed to show zgpal -induced apoptosis. See Kiselev, SI et al., J. Biol. Chem. 273:18633-18639, 1998; Sambrook,. et al., (Eds.) Molecular Cloning; a Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1990; Ausubel et al, (eds.), ibid; Tian, Q. et al., Cell 67:629-639, 1991.
  • Zgpal mutant or variant polypeptides that antagonize that natural apoptotic activity of zgpal can be assayed for apoptosis inhibitory activity using the methods of Ambrosini, G. et al., Nature Med. 3:917-921, 1997.
  • cDNAs encoding Bcl-2 and zgpal mutant or variant polypeptide are cloned into mammalian expression vector pcDNA3 (Invitrogen) and transfected into the IL-3-dependant murine pre-B-cell line, BaF3, using standard molecular biology techniques (Ausubel et al., supra.; Palacios and Steinmetz, Cell 4 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986; Ascaso, R. et al., Eur. J. Immunol. 24:537-541, 1994).
  • Stable cell lines are selected and cloned by methods disclosed herein, for example by G418 selection.
  • survival of cells co-expressing Bcl-2 and zgpal mutant polypeptide is measured under conditions where apoptosis is normally induced, i.e., when IL-3 is withdrawn from the cell culture medium. Viability can be measured, for, example, by trypan blue staining. Wild-type Baf3 cells, and cells expressing only Bcl-2 are used as positive controls for apoptosis. In the presence of zgpal mutant or variant polypeptide, inhibition of apoptosis is shown as increased survival of cells expressing zgpal mutant polypeptide relative to the control cells.
  • a further aspect of the invention provides a method of studying cytocidal activity against mammalian cells, such as tumor cells, in cell culture, comprising incubating such cells in a culture medium comprising a zgpal polypeptide, fragment, fusion protein, antibody, agonist or antagonist at high test agent and low cell concentration to study or evaluate cytocidal activity.
  • a culture medium comprising a zgpal polypeptide, fragment, fusion protein, antibody, agonist or antagonist at high test agent and low cell concentration to study or evaluate cytocidal activity.
  • Such evaluation may be conducted using methods known in the art, such as those described by Lichtenstein et al., Blood 68: 1407-10, 1986 and Sheu et al., Antimicrob. Agents Chemother. 28: 626- 9, 1993.
  • the related mouse Tag7 polypeptide has been shown to have direct cytocidal activity on mallian cells (see, See Kiselev, SI et al., J. Biol. Chem. 273:18633-186
  • Another aspect of the present invention involves the use of zgpal polypeptides, fragments, fusion proteins or agonists 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.
  • An additional aspect of the present invention is to study epithelial cell defensin induction in cell culture.
  • epithelial cells are cultured and exposed to pathogenic stimuli. Induction of zgpal polypeptide production by the epithelial cells is then measured.
  • Another aspect of the present invention involves the detection of zgpal polypeptides in the serum or tissue biopsy of a patient undergoing evaluation for microbial infection, salivary gland function or dysfunction. Several antimicrobial polypeptides are secreted by the salivary gland. Such zgpal polypeptides can be detected using immunoassay techniques and antibodies capable of recognizing zgpal polypeptide epitopes.
  • the present invention contemplates methods for detecting zgpal polypeptide comprising: exposing a solution possibly containing zgpal polypeptide to an antibody attached to a solid support, wherein said antibody binds to a first epitope of a zgpal polypeptide; washing said immobilized antibody-polypeptide to remove unbound contaminants; exposing the immobilized antibody-polypeptide to a second antibody directed to a second epitope of a zgpal polypeptide, wherein the second antibody is associated with a detectable label; and detecting the detectable label.
  • Changes in serum or biopsy zgpal polypeptide concentration can be indicative of microbial infection or of salivary gland dysfunction.
  • test proteins can also be measured in animal models by administering the test protein, by itself or in combination with other agents, including other proteins. Using such models facilitates the assay of the test protein by itself or as an inhibitor or modulator of another agent, and also facilitates the measurement of combinatorial effects of bioactive compounds.
  • Zgpal polynucleotides in animals provides models for further study of the biological effects of overproduction or inhibition of protein activity in vivo.
  • Zgpal -encoding polynucleotides and antisense polynucleotides can be introduced into test animals, such as mice, using viral vectors or naked DNA, or transgenic animals can be produced.
  • viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV).
  • Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acids. For review, see Becker et al., Meth. Cell Biol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine 4:44-53, 1997.
  • the adenovirus system offers several advantages.
  • Adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co- transfected plasmid.
  • the essential El gene is deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (e.g., the human 293 cell line).
  • the host cell e.g., the human 293 cell line.
  • adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.
  • 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.
  • adenovirus system can also be used for protein production in vitro.
  • adenovirus-infected non-293 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. 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., Cvtotechnol. 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.
  • Polynucleotides encoding zgpal polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zgpal activity. If a mammal has a mutated or absent zgpal gene, the zgpal gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zgpal 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 infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest.
  • HSV1 herpes simplex virus 1
  • a zgpal gene can be introduced in a retro viral 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. 62:1120, 1988; Temin et al., U.S. Patent No. 5,124,263; International Patent Publication 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. 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
  • non- peptide molecules can be coupled to liposomes chemically.
  • An alternative method of gene delivery comprises removing cells from the body and introducing a vector into the cells as a naked DNA plasmid. The transformed cells are then re-implanted in the body.
  • Naked DNA vectors are introduced into host cells by methods known in the art, including transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter. See, Wu et al., J. Biol Chem. 263:14621-14624, 1988; Wu et al., J. Biol. Chem. 267:963- 967, 1992; and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994.
  • Antisense methodology can be used to inhibit zgpal gene transcription to examine the effects of such inhibition in vivo.
  • Polynucleotides that are complementary to a segment of a zgpal -encoding polynucleotide e.g., a polynucleotide as set forth in SEQ ID NO:l, SEQ ID NO:4 or SEQ ID NO:7 are designed to bind to zgpal -encoding mRNA and to inhibit translation of such mRNA.
  • Such antisense oligonucleotides can also be used to inhibit expression of zgpal polypeptide-encoding genes in cell culture.
  • mice engineered to express the zgpal gene referred to as "transgenic mice,” and mice that exhibit a complete absence of zgpal 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 zgpal 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 zgpal polypeptide, polypeptide fragment or a mutant thereof may alter normal cellular processes, resulting in a phenotype that identifies a tissue in which zgpal expression is functionally relevant and may indicate a therapeutic target for the zgpal, its agonists or antagonists.
  • a preferred transgenic mouse to engineer is one that over-expresses the zgpal mature polypeptide (residue 18 (Asp) to residue 375 (His) of SEQ ID NO:5; or residue 18 (Asp) to residue 369 (His) of SEQ ID NO:8).
  • Transgenic mice engineered to over- expresses zgpal N-terminal polypeptide, domain 1 and domain 2 together or individually, or the C-terminal polypeptide, can also be used. Moreover, such over- expression may result in a phenotype that shows similarity with human diseases.
  • knockout zgpal mice can be used to determine where zgpal is absolutely required in vivo. The phenotype of knockout mice is predictive of the in vivo effects of that a zgpal antagonist, such as those described herein, may have.
  • the human zgpal cDNA can be used to isolate murine zgpal mRNA, cDNA and genomic DNA (Genbank Accession No. ACO 11666), which are subsequently used to generate knockout mice.
  • Transgenic mice engineered to over-expresses human polypeptides or mouse polypeptides corresponding to the human N-terminal polypeptide, domain 1 and domain 2 together or individually, or the C-terminal polypeptide can also be used.
  • These transgenic or knockout mice may be employed to study the zgpal 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 over-expressing zgpal can serve as animal tumor models.
  • transgenic mice expression of zgpal antisense polynucleotides or ribozymes directed against zgpal, described herein can be used analogously to transgenic mice described above.
  • the proteins of the present invention are formulated according to conventional methods. Routes of delivery include topical, mucosal, and parenteral, the latter including intravenous and subcutaneous delivery. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours.
  • pharmaceutical formulations will include a protein of the present invention 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, diluents, fillers, emulsifiers, preservatives, solubilizers, buffering agents, wetting agents, stabilizers, colorings, penetration enhancers, albumin to prevent protein loss on vial surfaces, etc.
  • Topical formulations are typically provided as liquids, ointments, salves, gels, emulsions, and the like. 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 be determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. 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, and the like. 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. In general, a therapeutically effective amount is an amount sufficient to produce a clinically significant change in the targeted condition.
  • zgpal proteins can also be used as molecular weight standards or as reagents in assays for determining circulating levels of the protein, such as in the diagnosis of disorders characterized by over- or underproduction of zgpal protein or in the analysis of cell phenotype.
  • the activity of zgpal proteins can also be measured using reporter assay systems that measure signal transduction. Such reporter assay systems can also be used to identify inhibitors of zgpal activity. For example, test compounds are added to the assays disclosed above to identify compounds that inhibit the activity of zgpal protein. In addition to those assays disclosed above, samples can be tested for zgpal activity or inhibition of zgpal activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of zgpal -dependent cellular responses. For example, zgpal -responsive cell lines can be transfected with a reporter gene construct that is responsive to a zgpal -stimulated cellular pathway.
  • Reporter gene constructs of this type are known in the art, and will generally comprise a zgpal -activated serum response element (SRE) operably linked to a gene encoding an assayable protein, such as luciferase.
  • SRE serum response element
  • Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of zgpal on the target cells as evidenced by a decrease in zgpal stimulation of reporter gene expression.
  • Assays of this type will detect zgpal directly, as well as compounds that directly block zgpal binding to cell-surface receptors (in a competition-type assay), 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 zgpal binding to receptor using zgpal 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.
  • 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.
  • the present invention includes stimulating or inhibiting the proliferation of lymphoid cells, hematopoietic cells and endothelial cells.
  • the molecules of the present invention are expressed in fetal tissue.
  • cancer markers are of fetal origin and not normally expressed in full-differentiated tissues.
  • molecules of the present invention may have use in identifying or inhibiting tumor cells.
  • 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).
  • zgpal polypeptide itself can serve as an additional cell-surface or secreted marker associated with stage- specific expression of a tissue.
  • direct measurement of zgpal polypeptide, or its loss of expression in a tissue as it differentiates can serve as a marker for differentiation of tissues.
  • mouse Tag7 polypeptide may be associated with tumor growth (see, See Kiselev, SI et al., J. Biol. Chem. 273:18633- 18639, 1998).
  • zgpal may serve as a cancer marker whose presence or absence is indicative of disease.
  • zgpal is expressed in specific human tissues, such as salivary gland, keratinocytes, cervix, fetal skin, prostate, and trachea, and hence can be used as a stage-specific marker in these tissues as described above, and as a marker for detecting the presence these tissues as described below.
  • zgpal is expressed in a variety of epithelial tumors including ovarian, lung, esophagus, and rectal tumors it can be used as a tumor marker for such tumors. Because zgpal is not expressed in normal lung tissue, in a preferred embodiment, the polypeptides, antibodies and polynucleotides of the present invention can be used to detect zgpal expression in lung cancer tissue compared to a normal control as a diagnostic for human lung cancer.
  • direct measurement of zgpal 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 zgpal 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 zgpal 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).
  • zgpal gain or loss of expression may serve as a diagnostic for testicular, lymphoid, B-cell, endothelial, hematopoietic and other cancers.
  • PSA prostate specific antigen
  • zgpal polypeptides, or anti-zgpal antibodies in a patient serum or tissue sample relative to a normal control can be indicative of disease, such as cancer (See, e.g., Mulders, TMT, et al., Eur. J. Surgical Oncol. 16:37-41, 1990).
  • zgpal expression appears to be restricted to normal human tissues
  • lack of zgpal expression in cervical, salivary gland, prostate, skin, and trachea or strong zgpal expression in other tissues would serve as a diagnostic of an abnormality in the cell or tissue type, of invasion or metastasis of cancerous tissues into non-cervical, salivary gland, keratinocyte, prostate, skin, and trachea tissue, and could aid a physician in directing further testing or investigation, or aid in directing therapy.
  • zgpal is expressed in specific cancerous tissues, such as lung, ovarian, esophageal and rectal tumors
  • detection of zgpal polynucleotides or polypeptides in those tissues serves as a diagnostic for those cancers.
  • tissue-specific, polynucleotide probes, anti-zgpal antibodies, and detection the presence of zgpal polypeptides in tissue can be used to assess whether cervical, salivary gland, keratinocyte, prostate, skin, trachea, lung tumor, ovarian tumor, esophageal tumor and rectal tumor tissue is present, for example, after surgery involving the excision of a diseased or cancerous tissue of that type.
  • the polynucleotides, polypeptides, and antibodies of the present invention can be used as an aid to determine whether all cervical, salivary gland, keratinocyte, prostate, skin, trachea, lung tumor, ovarian tumor, esophageal tumor and rectal tumor tissue is excised after surgery, for example, after surgery for cancer. In such instances, it is especially important to remove all potentially diseased tissue to maximize recovery from the cancer, and to minimize recurrence.
  • Preferred embodiments include fluorescent, radiolabeled, or calorimetrically labeled anti-zgpal antibodies and zgpal polypeptide binding partners, that can be used histologically or in situ.
  • tumor cells passaged in culture are implanted into mice of the same strain as the tumor donor.
  • the cells will develop into tumors having similar characteristics in the recipient mice, and metastasis will also occur in some of the models.
  • Appropriate tumor models for our studies include the Lewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No. CRL-6323), amongst others. These are both commonly used tumor lines, syngeneic to the 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. Cell 79: 315-328,1994).
  • C57BL6/J mice are treated with an experimental agent either through daily injection of recombinant protein, agonist or antagonist or a one-time injection of recombinant adenovirus. Three days following this treatment, 10 to 10 cells are implanted under the dorsal skin.
  • the cells themselves may be infected with recombinant adenovirus, such as one expressing zgpal, before implantation so that the protein is synthesized at the tumor site or intracellularly, rather than systemically.
  • adenovirus such as one expressing zgpal
  • the mice normally develop visible tumors within 5 days. The tumors are allowed to grow for a
  • the implanted cells can be transiently transfected with zgpal.
  • Use of stable zgpal transfectants as well as use of induceable promoters to activate zgpal expression in vivo are known in the art and can be used in this system to assess zgpal induction of metastasis.
  • purified zgpal or zgpal conditioned media can be directly injected in to this mouse model, and hence be used in this system.
  • O'Reilly MS et al. Cell 79:315-328, 1994
  • Rusciano D et al. Murine Models of Liver Metastasis. Invasion Metastasis 14:349-361, 1995.
  • zgpal and its derivatives conjugates
  • xenograft models Several mouse models have been developed in which human tumor cells are implanted into immunodeficient mice, collectively referred to as xenograft models. See Cattan, AR and Douglas, E Leuk. Res. 18:513-22, 1994; and Flavell, DJ, Hematological Oncology 14:67-82, 1996.
  • the characteristics of the disease model vary with the type and quantity of cells delivered to the mouse. Typically, the tumor cells will proliferate rapidly and can be found circulating in the blood and populating numerous organ systems.
  • Therapeutic strategies appropriate for testing in such a model include antibody induced toxicity, ligand-toxin conjugates or cell-based therapies.
  • the latter method commonly referred to adoptive immunotherapy, involves treatment of the animal with components of the human immune system (i.e. lymphocytes, NK cells) and may include ex vivo incubation of cells with zgpal or other immunomodulatory agents.
  • Zgpal polypeptides can also be used to prepare antibodies that bind to zgpal epitopes, peptides or polypeptides.
  • the zgpal 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 zgpal polypeptide (e.g., SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8).
  • Polypeptides comprising a larger portion of a zgpal 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 zgpal polypeptide encoded by SEQ ID NO:2 from amino acid number 18 (Asp) to amino acid number 114 (Lys), or a contiguous 9 to 97 amino acid fragment thereof; the zgpal polypeptide encoded by SEQ ID NO:5 from amino acid number 18 (Asp) to amino acid number 375 (His) or a contiguous 9 to 358 amino acid fragment thereof; or the zgpal polypeptide encoded by SEQ ID NO: 8 from amino acid number 18 (Asp) to amino acid number 369 (His) or a contiguous 9 to 352 amino acid fragment thereof.
  • antigens include the zgpal N-terminal polypeptide, domain 1 and domain 2 together or individually, the linker domain, or the C-terminal polypeptide, 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.
  • Zgpal hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: (1) amino acid number 92 (Ser) to amino acid number 97 (Glu) of SEQ ID NO:8 (98 (Lys) to amino acid number 103 (Glu) of SEQ ID NO:5); (2) amino acid number 117 (Val) to amino acid number 122 (Arg) of SEQ ID NO: 8 (123 (Val) to amino acid number 128 (Arg) of SEQ ID NO:5); (3) amino acid number 150 (Gly) to amino acid number 155 (His) of SEQ ID NO:8 (156 (Gly) to amino acid number 161 (His) of SEQ ID NO:5); (4) amino acid number 151 (Thr) to amino acid number 156 (Ser) of SEQ ID NO:8 (157 (Thr) to amino acid number 162 (Ser) of SEQ ID NO:5); and (5) amino acid 152 (Lys) to amino acid number 157 (Pro) of SEQ ID NO:8
  • polypeptides that comprise residues predicted from a Jameson-Wolf plot are also preferred antigens with reference to SEQ ID NO: 8: (1) amino acid number 20 (Ser) to amino acid number 29 (Val); (2) amino acid number 84 (Glu) to amino acid number 90 (Val); (3) amino acid number 105 (Asn) to amino acid number 110 (Asp); (4) amino acid number 150
  • preferred polypeptide antigens comprise amino acid number 47 (Gly) to amino acid number 52 (Asp) of SEQ ID NO:5; and more preferably polypeptide antigens that comprise in reference to SEQ ID NO:5, up to 5 additional amino acids directly N-terminal and/or C-terminal to amino acid number 47 (Gly) to amino acid number 52 (Asp) of SEQ ID NO:5.
  • preferred polypeptide antigens comprise amino acid number 205 (Ala) to amino acid number 223 (Pro) of SEQ ID NO: 8; and more preferably polypeptide antigens that comprise in reference to SEQ ID NO: 8, up to 5 additional amino acids directly N-terminal and/or C-terminal to amino acid number 205 (Ala) to amino acid number 223 (Pro) of SEQ ID NO: 8.
  • Antibodies from an immune response generated by inoculation of an animal 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.
  • polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a zgpal polypeptide or a fragment thereof.
  • the immunogenicity of a zgpal 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 zgpal 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.
  • 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 related polypeptide molecules.
  • a threshold level of binding is determined if anti-zgpal antibodies herein bind to a zgpal polypeptide, peptide or epitope with an affinity at least 10-fold greater than the binding affinity to control (non-zgpal) polypeptide. It is preferred that the antibodies exhibit a binding affinity (K a ) of 10 M or greater,
  • 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. 5L 660-672, 1949).
  • anti-zgpal antibodies do not significantly cross-react with related polypeptide molecules is shown, for example, by the antibody detecting zgpal polypeptide but not known related polypeptides using a standard Western blot analysis
  • 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 zgpal, and zgpal mutant polypeptides.
  • antibodies can be "screened against" known related polypeptides, to isolate a population that specifically binds to the zgpal polypeptides. For example, antibodies raised to zgpal are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to zgpal will flow through the matrix under the proper buffer conditions.
  • assays known to those skilled in the art can be utilized to detect antibodies which bind to zgpal 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 zgpal protein or polypeptide.
  • Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zgpal protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zgpal protein or peptide).
  • Genes encoding polypeptides having potential zgpal 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 zgpal sequences disclosed herein to identify proteins which bind to zgpal. These "binding polypeptides" which interact with zgpal 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 zgpal polypeptides; for detecting or quantitating soluble zgpal polypeptides as marker of underlying pathology or disease.
  • These binding polypeptides can also act as zgpal "antagonists" to block zgpal binding and signal transduction in vitro and in vivo. These anti-zgpal binding polypeptides would be useful for inhibiting zgpal activity or protein-binding.
  • Antibodies to zgpal may be used for tagging cells that express zgpal; for isolating zgpal polypeptides by affinity purification; for diagnostic assays for determining circulating levels of zgpal polypeptides; for detecting or quantitating soluble zgpal as marker of underlying pathology or disease; for immunolocalization within whole animals or tissue sections, including immunodiagnostic applications; for immunohistochemistry; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zgpal activity in vitro and in vivo.
  • Antibodies can be linked to other compounds, including therapeutic and diagnostic agents, using known methods to provide for targetting of those compounds to cells expressing receptors for zgpal.
  • 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 zgpal or fragments thereof may be used in vitro to detect denatured zgpal or fragments thereof in assays, for example, Western Blots or other assays known in the art.
  • the present invention also provides reagents that will find use in diagnostic applications.
  • the zgpal gene a probe comprising zgpal DNA or RNA or a subsequence thereof can be used to determine if the zgpal gene is present on human chromosome 1 or if a mutation has occurred.
  • Zgpal is located at the lq21 region of human chromosome 1.
  • Detectable chromosomal aberrations at the zgpal 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, fluorescence in situ hybridization methods, 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, fluorescence in situ hybridization methods, 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).
  • 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.
  • the zgpal gene is located at the lq21 region of chromosome 1 (Example 2).
  • chromosome 1 Several genes of known function or correlated with human disease map to this region.
  • the autosomal dominant papillary renal carcinoma maps to the lq21 region, and is associated with translocation at the lq21 locus (Meloni, AM et al., Cancer Genet. Cvtogenet. 65: 1-6, 1993; Sidhar, SK et al satisfy Hum. Molec. Genet. 5:1333- 1338, 1996).
  • zgpal polynucleotide probes can be used to detect abnormalities or genotypes associated with papillary renal carcinoma.
  • zgpal polynucleotide probes can be used to detect abnormalities or genotypes associated with some genetically linked forms of hyperparathyroidism, wherein one of the major variants of the disease is linked to a loss of heterogeneity (LOH) in the lq region between lq21- q32.
  • LOH heterogeneity
  • a chromosomal translocation breakpoint at lq21 seen in translocations associated with precursor acute lymphoblastic leukemia.
  • Jumping translocation breakpoint (JTB) (lq21) that can be associated with cancers. Research has shown that translocations producing partial duplication of lq can involve variable segments, but always include lq21, has been reported for the Ewing family of tumors, renal cell carcinoma, and in liposarcoma. (Hatakeyama, S. Oncogene 18:2085-2090, 1999).
  • zgpal polynucleotide probes are particularly useful for diagnosis of gross chromosomal abnormalities associated with loss of heterogeneity (LOH), chromosome gain, translocation, DNA amplification, and the like within the zgpal chromosomal locus, lq21.
  • LOH loss of heterogeneity
  • Translocations within chromosomal locus lq21 wherein the zgpal gene is located are known to be associated with human disease. For example, lq21 deletions, LOH, and translocations are associated with several cancers and carcinomas as described above.
  • zgpal polynucleotide probes of the present invention can be used to detect abnormalities or genotypes associated with lq21 translocation and LOH described above, and as such serve as a diagnostic.
  • zgpal is expressed in human lung, ovarian, esophagus and rectal tumor tissue.
  • defects in the zgpal locus itself may result in a heritable human disease state.
  • an antimicrobial polypeptide or signaling polypeptide involved in cell chemotaxis, migration, or apoptosis, deletions, rearrangements, or other defects in the zgpal gene itself can cause susceptibility to microbial infection or other immune dysfunction.
  • zgpal is expressed in human lung, ovarian, esophagus and rectal tumor tissue, and hence may be a involved in susceptibility or contribute to the pathology of these diseases.
  • defects in the zgpal gene may fully or in part cause known human diseases for which the genetic defects are not known, for example, those diseases associated with psoriasis susceptibility (lcen-lq21), the human homolog of Loop-tail mouse (Iq21-q23), schizophrenia (SCZD) (Iq21-q22), and familial hemiplegic migraine 2 (MHP2) (Iq21-q23).
  • 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 zgpal genetic defect.
  • 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 zgpal genetic defect.
  • a diagnostic could assist physicians in determining the type of disease and appropriate associated therapy, or assistance in genetic counseling.
  • the inventive anti-zgpal antibodies, polynucleotides, and polypeptides can be used for the detection of zgpal polypeptide, mRNA or anti-zgpal antibodies, thus serving as markers and be directly used for detecting or genetic diseases or cancers, as described herein, using methods known in the art and described herein.
  • zgpal polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome lq21 deletions and translocations associated with human diseases, other translocations involved with malignant progression of tumors or other lq21 mutations, which are expected to be involved in chromosome rearrangements in malignancy; or in other cancers, or in spontaneous abortion.
  • zgpal polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome lq21 trisomy and chromosome loss associated with human diseases or spontaneous abortion.
  • zgpal polynucleotide probes can be used to detect abnormalities or genotypes associated with these defects.
  • zgpal polynucleotide probes can be used to detect allelic differences between diseased or non-diseased individuals at the zgpal chromosomal locus. As such, the zgpal sequences can be used as diagnostics in forensic DNA profiling.
  • Analytical probes will be generally at least 20 nt in length, although somewhat shorter probes can be used (e.g., 14-17 nt).
  • PCR primers are at least 5 nt in length, preferably 15 or more, more preferably 20-30 nt.
  • a zgpal polynucleotide probe may comprise an entire exon or more. Exons are readily determined by one of skill in the art by comparing zgpal sequences (SEQ ID NO: l,
  • 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 zgpal 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 zgpal 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 zgpal 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:l, 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. 1991), White (ed.), PCR Protocols: Current Methods and Applications (Humana Press,
  • Aberrations associated with the zgpal 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-as
  • Polypeptides and proteins of the present invention can 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 may be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance).
  • zgpal polypeptides, anti-zgpal antibodies, other polypeptides and proteins, and bioactive fragments or portions thereof can be directly or indirectly 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 include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles, and the like.
  • Suitable cytotoxic molecules include bacterial and plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin, saporin, and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium-188, and yttrium-90. These can be either directly attached to the polypeptide or antibody, or indirectly attached according to known methods, such as through a chelating moiety.
  • Polypeptides and proteins can also be conjugated to cytotoxic drugs, such as adriamycin.
  • cytotoxic drugs such as adriamycin.
  • the detectable or cytotoxic molecule may be conjugated with a member of a complementary/anticomplementary pair, where the other member is bound to the polypeptide or protein portion.
  • biotin/streptavidin is an exemplary complementary/anticomplementary pair.
  • Polypeptide-toxin fusion proteins or antibody/fragment-toxin fusion proteins may be used for targeted cell or tissue inhibition or ablation, such as in cancers or microbial infection.
  • conjugates of a zgpal polypeptide and a cytotoxin which can be used to target the cytotoxin to microbes or tumors.
  • Target cells bind the zgpal -toxin conjugate, which is then internalized, killing the cell.
  • receptor-specific cell killing targets ablation
  • ligand- dependent, receptor-directed cyotoxicity can be used to enhance understanding of the physiological significance of a protein ligand.
  • a preferred toxin is saporin. Mammalian cells have no receptor for saporin, which is non-toxic when it remains extracellular.
  • zgpal -cytokine fusion proteins or antibody/fragment-cytokine fusion proteins may be used for enhancing in vitro cytotoxicity (for instance, that mediated by monoclonal antibodies against tumor targets) and for enhancing in vivo killing of target tissues (for example, epithelial cancers, and more specifically lung, ovarian, esophageal, and rectal cancers). See, generally, Hornick et al., Blood 89:4437-4447, 1997).
  • target tissues for example, epithelial cancers, and more specifically lung, ovarian, esophageal, and rectal cancers.
  • target tissues for example, epithelial cancers, and more specifically lung, ovarian, esophageal, and rectal cancers.
  • target tissues for example, epithelial cancers, and more specifically lung, ovarian, esophageal, and rectal cancers.
  • target tissues for example, epithelial cancers, and more specifically lung, ovarian,
  • Suitable cytokines for this purpose include, for example, interleukin-2 and granulocyte-macrophage colony- stimulating factor (GM-CSF).
  • GM-CSF granulocyte-macrophage colony- stimulating factor
  • Such fusion proteins may be used to cause cytokine- induced killing of tumors and other tissues undergoing angiogenesis or neovascularization.
  • zgpal expression is tissue-specific, such uses in detection and targeting of cytotoxic molecules to lung, ovarian, esophageal, and rectal cancers, and use of such anti-complementary molecules in identification of lung, ovarian, esophageal, and rectal cancer tissues, and use of such anti-complementary molecules in identification of cervical, prostate, salivary gland, skin, and tracheal tissues are evident.
  • bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intra-arterially or intraductally, or may be introduced locally at the intended site of action.
  • Polynucleotides and polypeptides of the present invention will additionally find use as educational tools as a laboratory practicum kits for courses related to genetics and molecular biology, protein chemistry and antibody production and analysis. Due to its unique polynucleotide and polypeptide sequence molecules of zgpal can be used as standards or as "unknowns" for testing purposes.
  • zgpal polynucleotides can be used as an aid, such as, for example, to teach a student how to prepare expression constructs for bacterial, viral, and/or mammalian expression, including fusion constructs, wherein zgpal is the gene to be expressed; for determining the restriction endonuclease cleavage sites of the polynucleotides; determining mRNA and DNA localization of zgpal polynucleotides in tissues (i.e., by Northern and Southern blotting as well as polymerase chain reaction); and for identifying related polynucleotides and polypeptides by nucleic acid hybridization.
  • Zgpal polypeptides can be used educationally as an aid to teach preparation of antibodies; identifying proteins by Western blotting; protein purification; determining the weight of expressed zgpal polypeptides as a ratio to total protein expressed; identifying peptide cleavage sites; coupling amino and carboxyl terminal tags; amino acid sequence analysis, as well as, but not limited to monitoring biological activities of both the native and tagged protein (i.e., receptor binding, signal transduction, proliferation, and differentiation) in vitro and in vivo.
  • native and tagged protein i.e., receptor binding, signal transduction, proliferation, and differentiation
  • Zgpal polypeptides can also be used to teach analytical skills such as mass spectrometry, circular dichroism to determine conformation, especially of the four alpha helices, x-ray crystallography to determine the three-dimensional structure in atomic detail, nuclear magnetic resonance spectroscopy to reveal the structure of proteins in solution.
  • analytical skills such as mass spectrometry, circular dichroism to determine conformation, especially of the four alpha helices, x-ray crystallography to determine the three-dimensional structure in atomic detail, nuclear magnetic resonance spectroscopy to reveal the structure of proteins in solution.
  • a kit containing the zgpal can be given to the student to analyze. Since the amino acid sequence would be known by the professor, the specific protein can be given to the student as a test to determine the skills or develop the skills of the student, the teacher would then know whether or not the student has correctly analyzed the polypeptide. Since every polypeptide is unique, the educational utility of zgpal would be unique unto itself.
  • zgpal expression is tissue-specific, and is a molecule with a granulocyte peptide protein structure that has a distinct chromosomal localization.
  • expression of zgpal polynucleotides and polypeptides in these specific tissues can be analyzed in order to train students in the use of diagnostic and tissue-specific identification and methods.
  • zgpal polynucleotides can be used to train students on the use of chromosomal detection and diagnostic methods, since it's locus is known.
  • students can be specifically trained and educated about human chromosome 1, and more specifically the lp21 locus.
  • Such assays are well known in the art, and can be used in an educational setting to teach students about cytokines and examine different properties, such as cellular effects on cells, enzyme kinetics, varying antibody binding affinities, tissue specificity, and the like, between zgpal and other proteins in the art.
  • the antibodies which bind specifically to zgpal can be used as a teaching aid to instruct students how to prepare affinity chromatography columns to purify zgpal, cloning and sequencing the polynucleotide that encodes an antibody and thus as a practicum for teaching a student how to design humanized antibodies.
  • antibodies that bind specifically to zgpal can be used as a teaching aid for use in detection of cervix, fetal skin, prostate, salivary gland, trachea, lung tumor, ovarian tumor, esophageal tumor, and rectal tumor tissue using histological, and in situ methods amongst others known in the art.
  • the zgpal gene, polypeptide or antibody would then be packaged by reagent companies and sold to universities so that the students gain skill in art of molecular biology. Because each gene and protein is unique, each gene and protein creates unique challenges and learning experiences for students in a lab practicum.
  • Such educational kits containing the zgpal gene, polypeptide or antibody are considered within the scope of the present invention.
  • Example 2 Chromosomal Assignment and Placement of Zgpal Zgpal was mapped to chromosome 1 using the commercially available "GeneBridge 4 Radiation Hybrid (RH) Mapping Panel"(Research Genetics, Inc., Huntsville, AL).
  • the GeneBridge 4 panel contains DNA from each of 93 radiation hybrid clones, plus two control DNAs (the HFL donor and the A23 recipient).
  • a publicly available WWW server http://www-genome.wi.mit.edu/cgi- bin/contig/rhmapper.pl) allows mapping relative to the Whitehead Institute/MIT Center for Genome Research's radiation hybrid map of the human genome (the "WICGR” radiation hybrid map) that was constructed with the GeneBridge 4 panel.
  • 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 66°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 (EM Science, Gibbstown, NJ) and visualized by staining with ethidium bromide.
  • the marathon cDNAs were made using the marathon-ReadyTM kit (Clontech, Palo Alto, CA) and QC tested with clathrin primers ZC21195 (SEQ ID NO: 13) and ZC21196 (SEQ ID NO: 14) and then diluted based on the intensity of the clathrin band.
  • the PCR reactions were set up using oligos ZC29627 (SEQ ID NO: 18) and ZC29625 (SEQ ID NO: 19), TaKaRa Ex TaqTM (TAKARA Shuzo Co LTD, Biomedicals Group, Japan), and Rediload dye (Research Genetics, Inc., Huntsville, AL).
  • the amplification was carried out as follows: 1 cycle at 94°C for 5 minutes; 5 cycles of 94°C for 30 seconds and 70°C for 30 seconds; 35 cycles of 94°C for 30 seconds, 62°C for 30 seconds and 72°C for 30 seconds; followed by 1 cycle at 72°C for 5 minutes.
  • the DNA fragment for cervix and tumor esophagus were excised and purified using a Gel Extraction Kit (Qiagen, Chatsworth, CA) according to manufacturer's instructions. Fragments were confirmed by sequencing to show that they were indeed zgpal.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

On décrit de nouveaux polypeptides zgpa1 homologues de peptides de granulocytes, de nouveaux matériaux et de nouveaux procédés de préparation et d'utilisation de ces derniers. Les polypeptides comprennent au moins neuf restes d'acides aminés contigus de la SEQ ID NO:5 ou de la SEQ ID NO:8, et peuvent être préparés sous forme de fusions de polypeptides comprenant des séquences hétérologues telles que des polypeptides chimériques et des marqueurs d'affinité. Les polypeptides et les polynucléotides codant ces derniers, ainsi que leurs anticorps peuvent être utilisés dans une grande diversité d'applications dans les domaines de la thérapie, du diagnostic et de la recherche.
PCT/US2000/029177 1999-10-20 2000-10-20 Homologue zgpa1 de peptide de granulocyte WO2001029224A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU15744/01A AU1574401A (en) 1999-10-20 2000-10-20 Granulocyte peptide homolog zgpa1

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US16071299P 1999-10-20 1999-10-20
US60/160,712 1999-10-20
US21807000P 2000-07-12 2000-07-12
US60/218,070 2000-07-12

Publications (2)

Publication Number Publication Date
WO2001029224A2 true WO2001029224A2 (fr) 2001-04-26
WO2001029224A3 WO2001029224A3 (fr) 2002-02-07

Family

ID=26857154

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/029177 WO2001029224A2 (fr) 1999-10-20 2000-10-20 Homologue zgpa1 de peptide de granulocyte

Country Status (2)

Country Link
AU (1) AU1574401A (fr)
WO (1) WO2001029224A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009100309A2 (fr) 2008-02-08 2009-08-13 Medimmune, Llc Anticorps anti-ifnar1 ayant une affinité réduite pour le ligand fc
WO2012019061A2 (fr) 2010-08-05 2012-02-09 Stem Centrx, Inc. Nouveaux effecteurs et leurs procédés d'utilisation
WO2012078813A2 (fr) 2010-12-08 2012-06-14 Stem Centrx, Inc. Nouveaux modulateurs et procédés d'utilisation
EP2604628A2 (fr) 2007-12-21 2013-06-19 Medimmune Limited Éléments de liaison pour le récepteur alpha interleukin-4 (IL-4R) - 173
EP2737907A2 (fr) 2007-05-07 2014-06-04 MedImmune, LLC Anticorps anti-ICOS et leur utilisation dans le traitement de l'oncologie, la transplantation et les maladies auto-immunes

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8986691B1 (en) 2014-07-15 2015-03-24 Kymab Limited Method of treating atopic dermatitis or asthma using antibody to IL4RA
US8980273B1 (en) 2014-07-15 2015-03-17 Kymab Limited Method of treating atopic dermatitis or asthma using antibody to IL4RA

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997029765A1 (fr) * 1996-02-16 1997-08-21 The Regents Of The University Of California Peptides antimicrobiens et procedes d'utilisation associes
WO1999002686A1 (fr) * 1997-07-11 1999-01-21 Boehringer Ingelheim International Gmbh Genes et polypeptides associes a l'inhibition de la croissance tumorale et a l'apoptose
WO2000039327A1 (fr) * 1998-12-23 2000-07-06 Human Genome Sciences, Inc. Proteines de reconnaissance de peptidoglycane

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997029765A1 (fr) * 1996-02-16 1997-08-21 The Regents Of The University Of California Peptides antimicrobiens et procedes d'utilisation associes
WO1999002686A1 (fr) * 1997-07-11 1999-01-21 Boehringer Ingelheim International Gmbh Genes et polypeptides associes a l'inhibition de la croissance tumorale et a l'apoptose
WO2000039327A1 (fr) * 1998-12-23 2000-07-06 Human Genome Sciences, Inc. Proteines de reconnaissance de peptidoglycane

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [Online] AF242518, accession number AF242518, 16 August 2000 (2000-08-16) W. ZHANG ET AL: "Homo sapens hypothetical protein SBBI67 mRNA, complete cds" XP002166773 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2737907A2 (fr) 2007-05-07 2014-06-04 MedImmune, LLC Anticorps anti-ICOS et leur utilisation dans le traitement de l'oncologie, la transplantation et les maladies auto-immunes
EP2604628A2 (fr) 2007-12-21 2013-06-19 Medimmune Limited Éléments de liaison pour le récepteur alpha interleukin-4 (IL-4R) - 173
WO2009100309A2 (fr) 2008-02-08 2009-08-13 Medimmune, Llc Anticorps anti-ifnar1 ayant une affinité réduite pour le ligand fc
WO2012019061A2 (fr) 2010-08-05 2012-02-09 Stem Centrx, Inc. Nouveaux effecteurs et leurs procédés d'utilisation
WO2012078813A2 (fr) 2010-12-08 2012-06-14 Stem Centrx, Inc. Nouveaux modulateurs et procédés d'utilisation

Also Published As

Publication number Publication date
AU1574401A (en) 2001-04-30
WO2001029224A3 (fr) 2002-02-07

Similar Documents

Publication Publication Date Title
US6436400B1 (en) Protease-activated receptor PAR4 ZCHEMR2
CA2343001A1 (fr) Polypeptide stomacal zsig28
US7087407B2 (en) Beta-defensins
AU744152B2 (en) Beta-defensins
WO2001029224A2 (fr) Homologue zgpa1 de peptide de granulocyte
AU746956B2 (en) Novel tumor antigens
US6828419B2 (en) Secreted salivary zsig63 polypeptide
WO1998041627A1 (fr) Polypeptides secretes presentant une homologie avec les proteines de la glande cementaire du xenopus
US20020042094A1 (en) RING finger protein zapop2
US6440697B1 (en) Ring finger protein zapop3
US7122342B1 (en) Protease-activated receptor PAR4 (ZCHEMR2)
US20010049432A1 (en) Human semaphorin ZSMF-16
CA2350603A1 (fr) Proteine a doigt ring zapop3
EP1241180A2 (fr) Protéine semblable à l'antigène A33 humaine et acides nucléiques le codant
US20040058354A1 (en) Mammalian alpha-helical protein-53
US20030171542A1 (en) Mammalian secretory peptide - 9
WO2001040278A2 (fr) Semaphorine humaine zsmf-16
CA2429265A1 (fr) Proteine 53 alpha helicoidale secretee par des mammiferes
AU5448700A (en) Secreted alpha-helical protein-31
JP2003500054A (ja) 分泌型アルファヘリックスタンパク質−３２
EP1224219A1 (fr) Beta-defensines

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP