WO2001059112A1 - Peptides intestinaux anti-angiogeniques, zdint5 - Google Patents

Peptides intestinaux anti-angiogeniques, zdint5 Download PDF

Info

Publication number
WO2001059112A1
WO2001059112A1 PCT/US2001/004198 US0104198W WO0159112A1 WO 2001059112 A1 WO2001059112 A1 WO 2001059112A1 US 0104198 W US0104198 W US 0104198W WO 0159112 A1 WO0159112 A1 WO 0159112A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
zdint5
residues
amino acid
Prior art date
Application number
PCT/US2001/004198
Other languages
English (en)
Inventor
James L. Holloway
Paul O. Sheppard
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 AU36816/01A priority Critical patent/AU3681601A/en
Priority to EP01909020A priority patent/EP1171602A1/fr
Priority to JP2001558250A priority patent/JP2004500087A/ja
Priority to CA002368681A priority patent/CA2368681A1/fr
Publication of WO2001059112A1 publication Critical patent/WO2001059112A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Proteins involved in extracellular matrix formation and degradation are critical in establishing tissue architecture during development and in tissue degradation in a variety of diseases including cancer, arthritis, Alzheimer's disease and a variety of inflammatory conditions.
  • extracelluar proteolysis are the zinc metalloproteases which have been identified in ADAMs (A Disintegrin and Me tallopro tease), MMPs (Matrix Metalloproteases), MDCs (Metalloprotease-Disintegrin-Cysteine-rich proteins), and SVMPs (Snake Venom Metalloprotease Proteins).
  • ADAMs Disintegrin and Me tallopro tease
  • MMPs Microx Metalloproteases
  • MDCs Metalloprotease-Disintegrin-Cysteine-rich proteins
  • SVMPs Senom Metalloprotease Proteins
  • Thrombospondin-1 is an extracellular matrix associated protein that has the ability to inhibit angiogenesis in vivo. TSPl blocks capillary-like tube formation and endothelial cell proliferation in vitro. The anti-angiogenic activity of TSPl has been mapped to a region which contains three type 1 repeats. Recombinant and proteolytic fragments of these repeats exhibited angio-inhibitory activity in the rabbit corneal pocket and chorioallantoic membrance assays. Peptides derived from the second and third type 1 repeats of TSPl inhibit endothelial cells and suppress tumor growth when injected systemically. See Vazquez, F. et al., J. Biol. Chem. 274: 23349- 23357, 1999.
  • ADAM-TS A Metalloprotease and Disintegrin with Thrombospondin-1 repeats
  • METHs Metalloprotease and Thrombospondin-1 repeat proteins
  • ADAMTS-1 is characterized as a disintegrin and metalloprotease with thrombospondin motifs and is an inflammation-associated gene that has also identified as a cachexia tumor slelective gene (Kuno, K. et al., J. Biol. Chem. 272: 556-562, 1997).
  • METH-1 is a combination of metalloprotease and thrombospondin domains and inhibits angiogenesis (Vasquez, ibid.)
  • ADAMTS protein family see Tang; B.L., et al., FEBS Letters 445: 223- 225, 1999.
  • An additional ADAMTS family member, ADAMTS, ADAMTS-2 has been implicated as a cartilage "aggrecanase"; see Flannery, C.R., et al.. Bioc. and Bioph. Res. Comm. 260:318-322. 1999.
  • ADAMs/ ADAMS-TS/ MDCs/ SVMPs/ METH family of proteins which have been shown to be therapeutically useful include eptifibatide (Integrilin®, made by COR Therapeutics, Inc. and Key Pharmaceuticals, Inc.) which is useful as an anti-clotting agent for acute coronary syndrome, and contortrostatin, which inhibits ilntegrin-mediated human metastatic melanoma cell adhesion and blocks experimental metastasis (Trikha, M. et at., Cancer Research 54: 4993-4998, 1994) and inhibits platelet aggregation (Clark, E.A. et al., J. Biol. Chem. 269 (35):21940-21943. 1994).
  • eptifibatide Integrilin®, made by COR Therapeutics, Inc. and Key Pharmaceuticals, Inc.
  • contortrostatin which inhibits ilntegrin-mediated human metastatic melanoma cell adhesion and blocks experimental
  • the present invention provides a novel member of the ADAMs/ ADAMS-TS/ MDCs/ SVMPs/ METH family and related compositions whose uses will be apparent to those skilled in the art from the teachings herein.
  • the invention provides an isolated polypeptide comprising the amino acid sequence as shown in SEQ LO NO:2.
  • the polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence as shown in SEQ ID NO:2.
  • the amino acid sequence is at least 90%, 95%, 98%, or 99% identical to the amino acid sequence as shown in SEQ ID NO:2.
  • the invention provides an isolated polypeptide comprising the amino acid sequence as shown in SEQ ID NO:5.
  • the polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence as shown in SEQ ID NO:5.
  • the amino acid sequence is at least 90%, 95%, 98%, or 99% identical to the amino acid sequence as shown in SEQ ID NO: 5.
  • the invention provides an isolated polypeptide comprising the amino acid sequence as shown in SEQ LO NO:8.
  • the polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence as shown in SEQ LO NO: 8.
  • the amino acid sequence is at least 90%, 95%, 98%, or 99% identical to the amino acid sequence as shown in SEQ ID NO:8.
  • the invention provides an isolated polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 11.
  • the polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence as shown in SEQ LD NO: 11.
  • the amino acid sequence is at least 90%, 95%, 98%, or 99% identical to the amino acid sequence as shown in SEQ LO NO:l 1.
  • the invention provides an isolated polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence as shown in SEQ LD NO:2; b) a polypeptide comprising the amino acid sequence as shown in SEQ LD NO: 5; c) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO8; and d) a polypeptide comprising the amino acid sequence as shown in SEQ LD NOl l; wherein the polypeptide is operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein, an immunoglobulin constant region, and a polyhistidine tag.
  • Wthin another aspect is provided an isolated polynucleotide encoding a fusion protein comprising a first polypeptide segment and a second polypeptide segment, wherein the first polypeptide segment comprises a protease domain and the second polypeptide segment comprises a polypeptide selected from the group consisting of: a polypeptide comprising residues 1 to 48 of SEQ LD NO:5; and a polypeptide comprising residues 1 to 59 of SEQ LD NO:8; wherein the first polypeptide segment is positioned amino-terminally to the second polypeptide segment.
  • the invention provides an isolated polynucleotide encoding a fusion protein comprising a first polypeptide segment and a second polypeptide segment, wherein the first polypeptide segment comprises residues 1 to 203 of SEQ LD NO:2, and the second polynucleotide segment encodes a second polypeptide that is a TSPl-like domain, and wherein the first polynucleotide segment is positioned amino-terminally to the second polynucleotide segment.
  • the invention provides an expression vector comprising the following operably linked elements: a) a transcription promoter; b)a DNA segment encoding a polypeptide, wherein the amino acid sequnce of the polypeptide comprises the amino acid sequence selected from the group consisting of: the amino acid sequence as shown in SEQ ED NO:2; the amino acid sequence as shown in SEQ LD NO:5; the amino acid sequence as shown in SEQ ID NO:8; and the amino acid sequence as shown in SEQ LD NO:2;and c) a transcription terminator.
  • a cultured cell into which has been introduced an expression vector, wherein said cell expresses the polypeptide encoded by the DNA segment.
  • the invention provides a method of producing a polypeptide comprising culturing the cell, whereby said cell expresses the polypeptide encoded by the DNA segment, and recovering the polypeptide.
  • the polypeptide made by the method
  • a method for modulating extracellular matrix interactions by combining a polypeptide with cells, wherein the polypeptide is selected from the group consisting of: a) a polypeptide comprising the amino acid sequence as shown in SEQ LD NO: 2; b) a polypeptide comprising the amino acid sequence as shown in SEQ LD NO:5; c) a polypeptide comprising the amino acid sequence as shown in SEQ LD NO8; and d) a polypeptide comprising the amino acid sequence as shown in SEQ LD NOl l.
  • the method for modulating extracellular matrix interactions whereby the cells are derived from tissues selected from the group consisting of: a) tissues from colon; b) tissues from small intestine; c) tissues from testes; and d) tissues from lung.
  • the invention provides a method of producing an antibody to the polypeptide made by the method of claim 31 comprising the following steps: inoculating an animal with the polypeptide such that the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal.
  • the antibody specifically binds to a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence as shown in SEQ ED NO:2; b) a polypeptide comprising the amino acid sequence as shown in SEQ ED NO: 5; c) a polypeptide comprising the amino acid sequence as shown in SEQ ED NO8; and d) a polypeptide comprising the amino acid sequence as shown in SEQ ED NO11.
  • the invention provides an isolated polypeptide comprising at least 11 contiguous amino acid residues of SEQ ED NO:l l.
  • the isolated polypeptide comprises at least 15 contiguous amino aicds of SEQ ED NO: 11.
  • the isolated polypeptide comprises at least 30 contiguous amino aicds of SEQ ED NO: 11.
  • the 11 contiguos amino acid residues are from the group consisting of: (a) SEQ ED NO:2; (b) SEQ ED NO:5; and (c)SEQ ED NO:8.
  • the isolated polypeptide is between 48 and 1120 amino acids in length.
  • At least nine of the contiguous amino acid residues are operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein, an immunoglobulin constant region, and a polyhistidine tag.
  • the invention provides an isolated polynucleotide encoding a polypeptide comprising at least 11 contiguous amino acid residues of SEQ ID NO: 11, wherein the contiguous sequence of 11 amino acids is selected from the group consisting of: (a) a polypeptide comprising the amino acids of SEQ ED NO:5; (b) a polypeptide comprising the amino acids of SEQ ED NO:8; and (c) a polypeptide comprising the amino acids of SEQ ED NO:l l.
  • the polypeptide molecule is between 48 and 1120 amino acids in length.
  • the invention provides an isolated polynucleotide encoding a fusion protein comprising a first polypeptide segment and a second polypeptide segment, wherein the first polypeptide segment comprises a protease domain and the second polypeptide segment comprises a polypeptide molecule selected from the group consisting of: (a) a polypeptide comprising residues 1 to 48 of SEQ ED NO:5; and (b) a polypeptide comprising residues 1 to 59 of SEQ ED NO:8; wherein the first polypeptide segment is positioned amino-terminally to the second polypeptide segment.
  • the invention provides an isolated polynucleotide encoding a fusion protein comprising a first polypeptide segment and a second polypeptide segment, wherein the first polypeptide segment comprises residues 1 to 203 of SEQ ED NO:2, and the second polynucleotide segment encodes a second polypeptide that is a TSPl-like domain, and wherein the first polynucleotide segment is positioned amino-terminally to the second polynucleotide segment.
  • the invention provides an expression vector comprising the following operably linked elements: a) a transcription promoter; b) a DNA segment encoding the polypeptide of comprising at least 11 contiguous amino acid residues of SEQ ED NO:l l; and c) a transcription terminator.
  • the DNA segment further encodes an affinity tag.
  • the invention provides a cultured cell into which has been introduced the expression vector, wherein said cell expresses the polypeptide encoded by the DNA segment.
  • a method of producing a polypeptide comprising culturing the cell, whereby said cell expresses the polypeptide encoded by the DNA segment, and recovering the polypeptide.
  • the invention provides the polypeptide made by the method.
  • the invention provides a method for modulating extracellular matrix interactions by combining the polypeptide comprising at least 11 contiguous amino acid residues of SEQ ED NO: 11 with cells in vivo or in vitro.
  • the cells are derived from tissues selected from the group consisting of: a) tissues from colon; b) tissues from small intestine; c) tissues from testes; and d) tissues from lung.
  • the invention provides a method of producing an antibody to the polypeptide comprising the following steps: inoculating an animal with the polypeptide of claim 15 such that the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal.
  • an antibody produced by the method which binds to a protein comprising the a polypeptide wherein the polypeptide is selected from the group consisting of: (a) SEQ ED NO:2; (b) SEQ ED NO:5; (c) SEQ ED NO:8; and (d) SEQ ED NO: 11.
  • 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.
  • a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • the term "complements of a polynucleotide molecule” is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • 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. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
  • Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985).
  • An "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • isolated does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • “Operably linked” 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.
  • the term "ortholog” or "species homolog” 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.
  • polynucleotide is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt”), or kilobases ("kb”). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded.
  • a “polypeptide” is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides”.
  • promoter is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
  • a “protein” is a macromolecule comprising one or more polypeptide chains.
  • a protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • the term "receptor” denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell.
  • Membrane-bound receptors are characterized by a multi-domain or multi-peptide structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylati ⁇ n, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.
  • receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta- adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, EL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and EL-6 receptor).
  • secretory signal sequence denotes a DNA sequence that encodes a polypeptide (a "secretory peptide”) that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • secretory signal sequence denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • secretory signal sequence denotes a DNA sequence that encodes a polypeptid
  • 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 propeptide domain is usually amino-terminal to the metalloprotease domain and is can act as an inhibitor for the metalloprotease domain (presumably via a cysteine-switch mechanism), such that the metalloprotease domain is activated in certain circumstances. This inhibition can be by blocking the active site of the metalloprotease domain.
  • the protease domain may be active or inactive. Some members of the disintegrin family have "active" zinc catalytic sites, which may be regulated by a "cysteine-switch" in the cysteine-rich domain. An example of a family member which has an "active" protease domain is ADAM-TS 1, which is thought to be involved in the inflammatory process through a processing of proteins in the extracellular matrix. Members of this family, which do not have such a catalytic site, include, for example, ADAM 11, which may be involved in tumor suppression. Other protein families, which are known to have inactive protease domains, are the serine proteases.
  • the adhesion (disintegrin) domain binds integrins or cell surface receptors which can be located on the surface of a multitude of cells, depending on the specificity of the disintegrin.
  • the predicted binding site within this disintegrin domain is often an amino acid loop comprising about 13 to 14 amino acids. See Wolfsberg and White, supra) The conformation of this sequence upon folding results in a hairpin loop presenting an amino acid sequence at its tip. This sequence is often "RGD", but may be substituted by a variety of other amino acid residues (Wolfsberg and White, supra; and Jia, J. Biol. Chem. 272:13094-13102, 1997). Receptors for the specific classes of disintegrin domains can recognize a multitude of disintegrin binding loop sequences.
  • Disintegrin domains have been shown to be responsible for cell-cell interactions, including inhibition of platelet aggregation by binding GPLTb/LIIa (fibronectin receptor) and/or GPIa/EIa (collagen receptor).
  • the METH proteins have two disintegrin domains.
  • disintegrin family members have a fusion domain, a relatively hydrophobic domain of about 23 amino acids. This domain is present within some of the ADAM family members, and has been shown to be involved in cell-cell fusion, and particularly in sperm/egg fusion, and muscle fusion.
  • the cysteine-rich domain varies in the ADAMs/ ADAMS-TS/ MDCs/ SVMPs/ METH -like family members and is believed to be involved in structurally presenting the integrin-binding region to integrins.
  • the cysteine-rich domain may also be necessary for secondary structure conformation of the polypeptide, specifically, disulfide bonding between the disintegrin domain and the cysteine domain.
  • Many ADAMs/ MDCs/ SVMPs family members have a transmembrane domain, which acts to anchor the polypeptide to the cell membrane. In the case of the METH proteins, the polypeptide is thought to be anchored via the binding of the TSPl- like domains to the extracellular matrix.
  • METH-1 and METH-2 proteins have been shown to be effective inhibitors of angiogenesis.
  • Membrane-anchored ADAMs/MDCs/SVMPs family members can be involved in a process called "protein ectodomain shedding" wherein the metalloprotease domain cleaves extracellular domain(s) of another protein.
  • the metalloprotease can be active on the cell surface itself, as in the case of fertilin (ADAMs 1 and 2), or TACE (ADAM 17), or the metalloprotease can act intracellularly in the secretory pathway as has been described for KUZ and ADAM 10 (Blobel, C.P., supra; and Lammich, S. et al., Proc. Natl. Acad. Sci.
  • membrane-anchored metalloproteases are likely to be active in the tissues where their genes are transcribed, in which cases they can be acting in cis, on other proteins bound to the same cell surface, in trans, on proteins bound to other cell surfaces, or on other proteins which are not membrane bound. Additionally the membrane anchor itself can be cleaved resulting in a soluble form of the metalloprotease/disintegrin which can be active at other sites in the body.
  • the cytoplasmic, or signaling, domain of the ADAMs/ MDCs/ SVMPs family members tends to be conserved in length and sites for phosphorylation.
  • Some disintegrin family members may signal by binding to the SH3 domain of Abl, Src, and/or Src -related SH3 domains.
  • the thrombospondin-like (TSP-like ) domain is located at the carboxyl terminal of the protein. Multiple TSP-like domains can be present.
  • METH-1 has three TSP-like domains
  • another METH homolog METH-2 (Vasquez, ibid) has two TSP-like domains.
  • Thrombospondin-1 is a modular protein that associates with the extracellular matrix and has the ability to inhibit angiogenesis in vivo. Under culture conditions, thrombospondin-1 blocks capillary-like formation and endothelial cell proliferation. Both METH-1 and METH-2 have also been shown to inhibit angiogenesis in the cornea pocket and CAM assays (Vasquez, ibid).
  • the present invention is based upon the discovery of novel domains of a member of the METH subfamily of proteins designated zdint5.
  • Domains of zdint5 include: a metalloprotease domain, and two TSPl-like domains.
  • the polynucleotide and polypeptide sequences for the metalloprotease domain are shown in SEQ ED NOs: 1 and 2, respectively.
  • Within the metalloprotease domain is a zinc-binding motif from residue 151 to residue 161 of SEQ ED NO: 2
  • the polynucleotide and polypeptide sequences for the first TSPl-like domain are shown in SEQ ED NOs: 4 and 5, respectively.
  • polynucleotide and polypeptide sequences for the second TSPl-like domain are shown in SEQ ED NOs: 7and 8, respectively.
  • the degenerate polynucleotide sequences for the metalloprotease, and the first and second TSPl-like domains are shown in SEQ ED NOs: 3, 6, and 9, respectively.
  • An illustrative example of how these domains can be combined in a protein is shown in SEQ ED NO: 10 (polynucleotides), SEQ ED NO: 11 (polypeptides ), and SEQ ED NO: 12, (degenerate polynucleotides).
  • Amino acid residues 69 as shown in SEQ ED NO:2 and 172 as shown in SEQ ED NO: 11; 73 as shown in SEQ ED NO:2 and 176 as shown in SEQ ED NO: 11; and 485, 533, 560, 595, and 635 all as shown in SEQ ED NO: 11 are potential N-linked glycosylation sites.
  • Analysis of the tissue distribution of zdint5 can be performed by the Northern blotting technique using Human Multiple Tissue and Master Dot Blots. Such blots are commercially available (Clontech, Palo Alto, CA) and can be probed by methods known to one skilled in the art. Also see, for example, Wu W. et al., Methods in Gene Biotechnology, CRC Press LLC, 1997. Additionally, portions of the polynucleotides of the present invention can be identified by querying sequence databases and identifying the tissues form which the sequences are derived.
  • Portions of the polynucleotides of the present invention have been identified in a colon adenocarcinoma cDNA library, a small intestine cDNA library, and a cDNA library made from fetal lung, testis, and B-cells.
  • ADAM 12 also known as meltrin ⁇ .
  • meltrin ⁇ The truncated form of this molecule, which lacks the propeptide and metalloprotease domains, is associated with ectopic muscle formation in vivo, but not in vitro, indicating that cells expressing this gene produce a growth factor that acts on neighboring progenitor cells.
  • SEQ ED NO: 3 is a degenerate DNA sequence that encompasses all DNAs that encode the zdint5 polypeptide of SEQ ED NO:2.
  • SEQ LD NO:6 is a degenerate DNA sequence that encompasses all DNAs that encode the zdint5 polypeptide of SEQ ED NO:5.
  • SEQ ED NO:9 is a degenerate DNA sequence that encompasses all DNAs that encode the zdint5 polypeptide of SEQ ED NO:8.
  • SEQ ED NO: 12 is a degenerate DNA sequence that encompasses all DNAs that encode the zdint5 polypeptide of SEQ ED NO: 11.
  • the degenerate sequence of SEQ ED NOs:3, 6, 9 and 12 also provides all RNA sequences encoding SEQ LD NOs:2, 5, 8 and 11, respectively, by substituting U for T.
  • zdint5 polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 609 of SEQ LD NO: 3; comprising nucleotide 1 to nucleotide 144 of SEQ ED NO:6; comprising nucleotide 1 to nucleotide 177 of SEQ ED NO:9; and comprising nucleotide 1 to nucleotide 2379 of SEQ ED NO: 12 and their RNA equivalents are contemplated by the present invention.
  • Table 1 sets forth the one-letter codes used within SEQ ED NOs:3, 6, 9 and 12 to denote degenerate nucleotide positions.
  • “Resolutions” are the nucleotides denoted by a code letter. “Complement” indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C. TABLE 1
  • degenerate codons used in SEQ ED NOs:3, 6, 9 and 12, encompassing all possible codons for a given amino acid, are set forth in Table 2.
  • any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid.
  • the degenerate codon for serine can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY).
  • WSN can, in some circumstances, encode arginine
  • MGN degenerate codon for arginine
  • AGY serine
  • polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences of SEQ ED NOs:2, 5, 8 and 11. Variant sequences can be readily tested for functionality as described herein.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequences disclosed in SEQ LD NOs:3, 6, 9 and 12 serve as templates for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
  • the isolated polynucleotides will hybridize to similar sized regions of SEQ LD NOs:l, 3, 4, 6, 7, 9 10 and 12 or a sequence complementary thereto under stringent conditions.
  • Polynucleotide hybridization is well known in the art and widely used for many applications, see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987; Berger and Kimmel, eds., Guide to Molecular Cloning Techniques, Methods in Enzymology, volume 152, 1987 and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227-59, 1990.
  • Polynucleotide hybridization exploits the ability of single stranded complementary sequences to form a double helix hybrid.
  • Such hybrids include DNA-DNA, RNA-RNA and DNA-RNA.
  • a nucleic acid molecule encoding a variant zdint5 polypeptide can be hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ID NOs: 1, 3, 4, 6, 7, 9, 10 and 12 (or their complements) at 42°C overnight in a solution comprising 50% formamide, 5xSSC (lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution (lOOx Denhardt's solution: 2% (w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin), 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA.
  • 5xSSC lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate
  • 50 mM sodium phosphate pH 7.6
  • hybridization mixture can be incubated at a higher temperature, such as about 65°C, in a solution that does not contain formamide.
  • a higher temperature such as about 65°C
  • premixed hybridization solutions are available (e.g., ExpressHybTM Hybridization Holution from CLONTECH Laboratories, Inc., Palo Alto, CA) according to the manufacturer's instructions.
  • nucleic acid molecules can be washed to remove non-hybridized nucleic acid molecules under stringent conditions, or under highly stringent conditions.
  • Typical stringent washing conditions include washing in a solution of 0.5x - 2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 55 - 65°C.
  • nucleic acid molecules encoding a variant zdint5 polypeptide hybridize with a nucleic acid molecule having the nucleotide sequences of SEQ ID NOs: 1, 3, 4, 6, 7, 9, 10 and 12 (or their complements) under stringent washing conditions, in which the wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, including 0.5x SSC with 0.1% SDS at 55°C, or 2xSSC with 0.1% SDS at 65°C.
  • wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, including 0.5x SSC with 0.1% SDS at 55°C, or 2xSSC with 0.1% SDS at 65°C.
  • SSPE for SSC in the wash solution.
  • the present invention also contemplates zdint5 variant nucleic acid molecules that can be identified using two criteria: a determination of the similarity between the encoded polypeptides with the amino acid sequences of SEQ ED NOs:2 , 5, 8 and 11 (as described below), and a hybridization assay, as described above.
  • Such zdint5 variants include nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ED NOs: 1, 3, 4, 6, 7, 9, 10 and 12 (or their complements) under stringent washing conditions, in which the wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, and (2) that encode a polypeptide having at least 80%, preferably 90%, ⁇ nore preferably, 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ED NOs:2, 5, 8 or 11.
  • zdint5 variants can be characterized as nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ED NOs:l or 3 (or their complements) under highly stringent washing conditions, in which the wash stringency is equivalent to O.lx - 0.2x SSC with 0.1% SDS at 50 - 65°C, and (2) that encode a polypeptide having at least 80%, preferably 90%, more preferably 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ED NOs:2, 5, 8 or 11.
  • RT-PCR reverse transcription-polymerase chain reaction
  • highly degenerate primers designed from the zdint5 sequences are useful for this purpose.
  • 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 zdint5 RNA.
  • tissue and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include colon, small intestine, fetal lung, testis, and B-cells.
  • Total RNA can be prepared using guanidine isothiocyante extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al.,
  • 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 is prepared from poly(A) + RNA using known methods. In the alternative, genomic DNA can be isolated. Polynucleotides encoding zdint5 polypeptides are then identified and isolated by, for example, hybridization or PCR. A full-length clone encoding zdint5 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 zdint5 or other specific binding partners.
  • Zdint5 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a zdint5 gene.
  • This gene is expected to provide for specific expression in colon, small intestine, fetal lung, testis, and B-cells.
  • Promoter elements from a zdint5 gene could thus be used to direct the tissue-specific expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy.
  • Cloning of 5' flanking sequences also facilitates production of zdint5 proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670.
  • an endogenous zdint5 gene in a cell is altered by introducing into the zdint5 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 zdint5 5' non-coding sequence that permits homologous recombination of the construct with the endogenous zdint5 locus, whereby the sequences within the construct become operably linked with the endogenous zdint5 coding sequence.
  • an endogenous zdint5 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.
  • the polynucleotides of the present invention can also be synthesized using DNA synthesizers.
  • DNA synthesizers Currently the method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately.
  • the production of short genes 60 to 80 bp is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longer genes (>300 bp), however, special strategies must be invoked, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%.
  • the present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are zdint5 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human zdint5 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 zdint5 as disclosed herein.
  • tissue would include, for example, colon, small intestine, fetal lung, testis, and B-cells.
  • Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein.
  • a library is then prepared from mRNA of a positive tissue or cell line.
  • a zdint5-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences.
  • a cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human zdint5 sequences disclosed herein.
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to zdint5 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
  • SEQ ED NOs: l, 3, 4, 6, 7, 9, 10 and 12 represent a single allele of human zdint5 and that allelic variation and alternative splicing are expected to occur.
  • Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.
  • Allelic variants of the DNA sequences shown in SEQ ED NOs: 1, 3, 4, 6, 7, 9, 10 and 12, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ED NOs:2, 5, 8 and 11.
  • cDNAs generated from alternatively spliced mRNAs, which retain the properties of the zdint5 polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
  • Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.
  • the present invention also provides isolated zdint5 polypeptides that are substantially similar to the polypeptides of SEQ ED NOs:2, 5, 8 and 11 and their orthologs. Such polypeptides will more preferably be at least 90% identical, and more preferably 95% or more identical to SEQ ED NOs: 2, 5, 8 and 11 and their orthologs. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9, 1992.
  • the "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant zdint5.
  • the FASTA algorithm is described by Pearson and Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990).
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions.
  • FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from four to six.
  • the present invention includes nucleic acid molecules that encode a polypeptide having one or more conservative amino acid changes, compared with the amino acid sequences of SEQ ED NOs:2, 5, 8 and 11.
  • the BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915 (1992)).
  • 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.
  • conservative amino acid substitution refers to a substitution represented by a BLOSUM62 value of greater than -1.
  • an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
  • Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • Conservative amino acid changes in an zdint5 gene can be introduced by substituting nucleotides for the nucleotides recited in SEQ ED NOs:l, 3, 4, 6, 7, 9, 10 and 12.
  • Such "conservative amino acid” variants can be obtained, for example, by oligonucleotide-directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22; and McPherson (ed.), Directed Mutagenesis: A Practical Approach (LRL Press 1991)).
  • the ability of such variants to promote cell-cell interactions can be determined using a standard method, such as the assay described herein.
  • a variant zdint5 polypeptide can be identified by the ability to specifically bind anti-zdint5 antibodies.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-502, 1991).
  • site-directed mutagenesis or alanine-scanning mutagenesis Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-502, 1991.
  • single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699-708, 1996.
  • Sites of disintegrin-integrin, protease, or extracellular matrix interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992.
  • the identities of essential amino acids can also be inferred from analysis of homologies with related metalloprotease/disintegrin/thrombospondin-1 like molecules.
  • variants of the disclosed zdint5 DNA and polypeptide sequences can be generated through DNA shuffling, as disclosed by Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91_:10747-51, 1994 and WEPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process.
  • Mutagenesis methods as disclosed herein can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
  • Mutagenized DNA molecules that encode active polypeptides e.g., protease activity, or angiogenesis inhibition
  • variant zdint5 encodes a polypeptide that is characterized by its protease activity, or angiogenesis inhibition, or by the ability to bind specifically to an anti-zdint5 antibody. More specifically, variant zdint5 genes encode polypeptides which exhibit at least 50%, and preferably, greater than 70, 80, or 90%, of the activity of polypeptide encoded by the human zdint5 gene described herein.
  • Variant zdint5 polypeptides or substantially homologous zdint5 polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
  • the present invention thus includes polypeptides of from 18 to 2000 amino acid residues that comprise a sequence that is at least 85%, preferably at least 90%, and more preferably 95% or more identical to the corresponding region of SEQ ED NOs:2, 5, 8 or 11.
  • Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zdint5 polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.
  • the present invention includes a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ ED NOs: l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.
  • Suitable forms of computer-readable media include magnetic media and optically-readable media.
  • magnétique media examples include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT), a disk cache, and a ZEP 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).
  • compact discs e.g., CD-read only memory (ROM), CD-rewritable (RW), and CD- recordable
  • DVD digital versatile/video discs
  • the present invention further provides a variety of other polypeptide fusions and related multimeric proteins comprising one or more polypeptide fusions.
  • a metalloprotease of TSPl-like polypeptide domain 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 other disintegrin polypeptide domains, TSPl-like domains, disintegrin polypeptide domain fragments, or polypeptides comprising other members of the Disintegrin Protease family of proteins, such as, for example, members of the MDCs, SVMPs, METHs and ADAMs.
  • These disintegrin polypeptide domain fusions, disintegrin polypeptide domain fragment fusions, or fusions with other Disintegrin Proteases can be expressed in genetically engineered cells to produce a variety of multimeric disintegrin-like analogs.
  • 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(s) conferring a biological function may be swapped between zdint5 of the present invention with the functionally equivalent domain(s) from another family member, such as ADAM, MDC, SVMP, ADAM-TS, and METH.
  • Such domains include, but are not limited to, conserved motifs such as the secretory signal sequence, propeptide, protease, disintegrin and disintegrin loop domains, including the "RGD"-like sequence, the cysteine, transmembrane, TSPl-like and signaling domains.
  • Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known disintegrin-like family proteins (e.g. ADAMs, MDCs, SVMPs, ADAM-TS, and METH), depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein.
  • polypeptide fusions, or hybrid zdint5 proteins are constructed using regions or domains of the inventive zdint5 in combination with those of other disintegrin and disintegrin-like molecules, (e.g. ADAM, MDC, and SVMP), 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.
  • Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure.
  • Auxiliary domains can be fused to zdint5 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., colon, small intestine, fetal lung, testis, and B-cells).
  • a protease polypeptide domain, or protease polypeptide fragment or protein could be targeted to a predetermined cell type by fusing it to a disintegrin polypeptide domain or fragment that specifically binds to an integrin polypeptide or integrin-like polypeptide on the surface of the target cell.
  • Such disintegrins or protease polypeptide domains or fragments can be fused to two or more moieties, such as an affinity tag for purification and a targeting-disintegrin domain.
  • protease polypeptide domain or protease polypeptide fragment or protein, could be targeted to the extracellular matrix by fusing it to a TSPl-like polypeptide domain or fragment that specifically binds to the extracellular matrix.
  • polypeptides, polypeptide fragments and proteins can be targeted for therapeutic or diagnostic purposes.
  • Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34:1-9, 1996.
  • Polypeptide fusions of the present invention will generally contain not more than about 2,000 amino acid residues, preferably not more than about 1,700 residues, more preferably not more than about 1,500 residues, and will in many cases be considerably smaller.
  • residues of zdint5 polypeptide can be fused to E. coli j8-galactosidase (1,021 residues; see Casadaban et al., J. Bacteriol. 143:971-980, 1980), a 10-residue spacer, and a 4-residue factor Xa cleavage site.
  • residues of zdint5 polypeptide can be fused to maltose binding protein (approximately 370 residues), a 4-residue cleavage site, and a 6-residue polyhistidine tag.
  • the zdint5 DNA is linked to a second DNA segment encoding a secretory peptide, such as a t-PA secretory peptide or a zdint5 secretory peptide.
  • a secretory peptide such as a t-PA secretory peptide or a zdint5 secretory peptide.
  • a C-terminal extension such as a poly-histidine tag, substance P, Flag peptide (Hopp et al., Bio/Technology 6:1204-1210, 1988; available from Eastman Kodak Co., New Haven, CT), maltose binding protein, or another polypeptide or protein for which an antibody or other specific binding agent is available, can be fused to the zdint5 polypeptide.
  • the present invention also includes "functional fragments" of zdint5 polypeptides and nucleic acid molecules encoding such functional fragments.
  • Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes an zdint5 polypeptide.
  • DNA molecules having the nucleotide sequence of SEQ ED NOs:l, 3, 4, 6, 7, 9, 10 or 12 can be digested with J5 ⁇ /31 nuclease to obtain a series of nested deletions.
  • the fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for protease activity, or angiogenesis inhibition, or for the ability to bind anti-zdint5 antibodies.
  • exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired fragment.
  • particular fragments of an zdint5 gene can be synthesized using the polymerase chain reaction.
  • the present invention also contemplates functional fragments of an zdint5 gene that has amino acid changes, compared with the amino acid sequence of SEQ ED NOs:2, 5, 8 and 11.
  • a variant zdint5 gene can be identified on the basis of structure by determining the level of identity with nucleotide and amino acid sequences of SEQ ED NOs:l, 2, 3, 4, 5, 6, 7, 8 and 9, as discussed above.
  • An alternative approach to identifying a variant gene on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant zdint5 gene can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ED NOs:l, 2, 4, 6, 7, 9, 10 and 12, as discussed above.
  • polypeptide fragments or variants of SEQ ED NOs: 2, 5, 8, and 11 that retain the metalloprotease, TSPl-like, and/or disintegrin activity of the wild-type zdint5 protein.
  • polypeptides may include additional amino acids from, for example, a secretory domain, a propeptide domain, a protease domain, a disintegrin domain, a TSPl-like domain, a disintegrin loop (native or synthetic), part or all of a transmembrane and intracellular domains, including amino acids responsible for intracellular signaling; fusion domains; affinity tags; and the like.
  • the present invention also provides polypeptide fragments or peptides comprising an epi tope-bearing portion of an zdint5 polypeptide described herein.
  • Such fragments or peptides may comprise an "immunogenic epitope," which is a part of a protein that elicits an antibody response when the entire protein is used as an immunogen.
  • Lmmunogenic epitope-bearing peptides can be identified using standard methods (see, for example, Geysen et al., Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
  • polypeptide fragments or peptides may comprise an "antigenic epitope," which is a region of a protein molecule to which an antibody can specifically bind.
  • Certain epitopes consist of a linear or contiguous stretch of amino acids, and the antigenicity of such an epitope is not disrupted by denaturing agents. It is known in the art that relatively short synthetic peptides that can mimic epitopes of a protein can be used to stimulate the production of antibodies against the protein (see, for example, Sutcliffe et ai. Science 219:660 (1983)). Accordingly, antigenic epitope- bearing peptides and polypeptides of the present invention are useful to raise antibodies that bind with the polypeptides described herein.
  • Antigenic epitope-bearing peptides include, for example, residues 727 to 732 of SEQ LD NO:l 1; residues 99 to 104 of SEQ ED NO:l l; residues 726 to 731 of SEQ ED NO: 11; and residues 127 to 132 of SEQ ED NO:l l.
  • Antigenic epitope-bearing peptides and polypeptides contain at least four to ten amino acids, preferably at least ten to fifteen amino acids, more preferably 15 to 30 amino acids of SEQ ED NOs:2, 5, 8 and 11.
  • Such epitope-bearing peptides and polypeptides can be produced by fragmenting a zdint5 polypeptide, or by chemical peptide synthesis, as described herein.
  • epitopes can be selected by phage display of random peptide libraries (see, for example, Lane and Stephen. Curr. Opin. Immunol. 5:268 (1993), and Cortese et al., Curr. Opin. Biotechnol. 7:616 (1996)).
  • potential antigenic sites in zdint5 SEQ ED NOs: 2, 5, 8, and 11 were identified using the Jameson-Wolf method, Jameson and Wolf, CABIOS 4:181, (1988), as implemented by the PROTEAN program (version 3.14) of LASERGENE (DNASTAR; Madison, WI). Default parameters were used in this analysis.
  • Suitable antigens of zdint5 include: amino acid residues 23 to 29 as shown in SEQ ED NO:2; residues 40 to 49 as shown in SEQ ED NO:2; residues 62 to 70 as shown in SEQ ED NO:2; residues 87 to 98 as shown in SEQ ED NO:2; residues 108 to 120 as shown in SEQ LD NO:2; residues 137 to 144 as shown in SEQ ED NO:2; residues 157 to 172 as shown in SEQ ED NO:2; residues 177 to 183 as shown in SEQ LD NO:2; residues 190 to 197 as shown in SEQ LD NO:2; residues 23 to 49 as shown in SEQ ED NO:2; residues 40 to 70 as shown in SEQ ED NO:2; residues 62 to 98 as shown in SEQ ED NO:2; residues 87 to 120 as shown in SEQ ED NO:2; residues 108 to 144 as shown in SEQ ED NO:
  • Zdint5 polypeptides can also be used to prepare antibodies that specifically bind to zdint5 epitopes, peptides or polypeptides.
  • the zdint5 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 zdint5 polypeptide (e.g., SEQ ED NOs:2, 5, 8 and 11).
  • Polypeptides comprising a larger portion of a zdint5 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 zdint5 polypeptides encoded by SEQ ED NOs:2, 5, 8 or 11 from amino acid number 1 to amino acid number 1120, or a contiguous 9 to 1170 amino acid fragment thereof.
  • Preferred peptides to use as antigens are hydrophilic peptides such as those predicted by one of skill in the art from a hydrophobicity plot.
  • zdint5 hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: residues 20 to 32 of SEQ ED NO:2; residues 64 to 69 of SEQ ED NO:2; residues 86 to 98 of SEQ ED NO:2; residues 110 to 121 of SEQ ED NO:2; residues 154 to 171 of SEQ ED NO:2; residues 190 to 199 of SEQ ED NO:2; residues 20 to 69 of SEQ ED NO:2; residues 64 to 98 of SEQ ED NO:2; residues 86 to 121 of SEQ ED NO:2; residues 110 to 171 of SEQ ED NO:2; residues 154 to 199 of SEQ ED NO:2; residues 1 to 13 of SEQ ED NO:5; residues 15 to 31 of SEQ ED NO:5; residues 1 to 31 of SEQ ED NO:5; residues 25 to 36 of SEQ ED NO:
  • antigens can be generated to portions of the polypeptide which are likely to be on the surface of the folded protein. These antigens include: residues 22 to 31 of SEQ ED NO:2; residues 87 to 97 of SEQ ED NO:2; residues 113 to 118 of SEQ ED NO:2; residues 26 to 32 of SEQ ED NO:8; residues 44 to 50 of SEQ ED NO:8; residues 31 to 39 of SEQ ED NO: 11; residues 86 to 104 of SEQ ED NO: 11; residues 125 to 134 of SEQ ED NO:l 1; residues 190 to 200 of residues 216 to 221 of SEQ ED NO:l 1; SEQ ED NO:l l; residues 492 to 498 of SEQ ED residues 516 to 522 of SEQ ED NO: 11; NO:l l; residues 546 to 551 of SEQ ED NO: 11; residues 593 to 598 of SEQ ED NO: 11;
  • 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. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982.
  • 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 zdint5 polypeptide or a fragment thereof.
  • the immunogenicity of a zdint5 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 zdint5 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
  • antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included.
  • Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non- human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody). In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
  • Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zdint5 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zdint5 protein or peptide).
  • Genes encoding polypeptides having potential zdint5 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 zdint5 sequences disclosed herein to identify proteins which bind to zdint5. These "binding proteins" which interact with zdint5 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 proteins can also be used in analytical methods such as for screening expression libraries and neutralizing activity.
  • the binding proteins can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease.
  • These binding proteins can also act as zdint5 "antagonists" to block zdint5 binding and signal transduction in vitro and in vivo.
  • These anti-zdint5 binding proteins would be useful for modulating, for example, protease activity, or angiogenesis inhibition, in general.
  • Antibodies are determined to be specifically binding if they exhibit a threshold level of binding activity (to a zdint5 polypeptide, peptide or epitope) of at least 10-fold greater than the binding affinity to a control (non-zdint5) polypeptide.
  • the binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51: 660- 672, 1949).
  • assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to zdint5 proteins or peptides. 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 zdint5 protein or polypeptide.
  • Antibodies to zdint5 may be used for tagging cells that express zdint5; for isolating zdint5 by affinity purification; for diagnostic assays for determining circulating levels of zdint5 polypeptides; for detecting or quantitating soluble zdint5 as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block ⁇ dint5 in vitro and in vivo.
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti- complement pairs as intermediates.
  • Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • antibodies to zdint5 or fragments thereof may be used in vitro to detect denatured zdint5 or fragments thereof in assays, for example, Western Blots or other assays known in the art.
  • Antibodies or polypeptides herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (integrin or antigen, respectively, for instance).
  • zdint5 polypeptides or anti-zdint5 antibodies, or bioactive fragments or portions thereof can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti- complementary molecule.
  • Suitable detectable molecules may be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like.
  • Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine- 131, rhenium- 188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance).
  • Polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin.
  • cytotoxic drugs such as adriamycin.
  • the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion.
  • biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
  • polypeptide-toxin fusion proteins or antibody- toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues).
  • a fusion protein including only the TSPl-like domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest (i.e., extracellular matrix).
  • the corresponding binding partner to zdint5 can be conjugated to a detectable or cytotoxic molecule and provide a generic targeting vehicle for cell/tissue-specific delivery of generic anti-complementary-detectable/ cytotoxic molecule conjugates.
  • zdint5 -cytokine fusion proteins or antibody- cytokine fusion proteins can be used for enhancing in vivo killing of target tissues (for example, colon, small intestine, fetal lung, testis, and B-cells), if the zdint5 polypeptide or anti-zdint5 antibody targets hyperproliferative tissues from these organs.
  • target tissues for example, colon, small intestine, fetal lung, testis, and B-cells
  • zdint5 polypeptide or anti-zdint5 antibody targets hyperproliferative tissues from these organs.
  • Suitable zdint5 polypeptides or anti-zdint5 antibodies target an undesirable cell or tissue (i.e., a tumor or a leukemia), and the fused cytokine mediates 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.
  • 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.
  • the zdint5 polypeptides of the present invention can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred.
  • a DNA sequence encoding a zdint5 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
  • the secretory signal sequence may be that of zdint5, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo.
  • the secretory signal sequence is operably linked to the zdint5 DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).
  • the native secretory signal sequence of the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway.
  • the present invention provides for such fusion polypeptides.
  • a signal fusion polypeptide can be made wherein a secretory signal sequence derived from a zdint5 polypeptide is operably linked to another polypeptide using methods known in the art and disclosed herein.
  • the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
  • Such constructs have numerous applications known in the art.
  • these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non- secreted protein, such as a receptor.
  • a normally non- secreted protein such as a receptor.
  • Such fusions may be used in vivo or in vitro to direct peptides through the secretory pathway.
  • the protease domain of zdint5 can be substituted by a heterologous sequence providing a different protease domain.
  • the fusion product can be secreted, and the TSPl-like domain of zdint5 can direct the substituted protease domain to a specific tissue described above.
  • This substituted protease domain can be chosen from the protease domains represented by the ADAMs/MDCs/SVMPs/ADAM-TS/METH like family members, or domains from other known proteases.
  • the TSPl-like domain of zdint5 protein can be substituted by a heterlogous sequence providing a different TSPl-like domain.
  • the fusion product can be secreted and the substituted TSPl-like domain can target the protease domain of zdint5 to a specific tissue.
  • the substituted TSPl-like domain can be chosen from the TSPl-like domains of the ADAMs/MDCs/SVMPs/METH-like family members.
  • the fusion products can be soluble or membrane- anchored proteins.
  • Cultured mammalian cells are suitable hosts within the present invention.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981 : Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines.
  • COS-1 ATCC No. CRL 1650
  • COS-7 ATCC No. CRL 1651
  • BHK ATCC No. CRL 1632
  • BHK 570 ATCC No. CRL 10314
  • 293 ATCC No. CRL 1573
  • Graham et al. J. Gen. Virol. 36:59-72, 1977
  • Chinese hamster ovary e.g. CHO-K1; ATCC No. CCL 61
  • Suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection (Manasas, VA).
  • strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288.
  • Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants”. 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
  • drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • Alternative markers that introduce an altered phenotype such as green fluorescent protein, or cell surface proteins, such as CD4, CD8, Class I MHC, or placental alkaline phosphatase, may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
  • Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells.
  • 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.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV).
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • a second method of making recombinant zdint5 baculovirus utilizes a transposon-based system described by Luckow (Luckow, V.A, et al., J Virol 67:4566-79, 1993). This system, which utilizes transfer vectors, is sold in the Bac-to- BacTM kit (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBaclTM (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zdint5 polypeptide into a baculovirus genome maintained in E.
  • the pFastBaclTM transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case zdint5.
  • pFastBaclTM can be modified to a considerable degree.
  • the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins, M.S. and Possee, R.D., J. Gen. Virol.
  • transfer vector constructs a short or long version of the basic protein promoter can be used.
  • transfer vectors can be constructed which replace the native zdint5 secretory signal sequences with secretory signal sequences derived from insect proteins.
  • a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen, Carlsbad, CA), or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native zdint5 secretory signal sequence.
  • transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed zdint5 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al, Proc. Natl. Acad. Sci. 82:7952-4, 1985).
  • a transfer vector containing zdint5 is transformed into E. coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells.
  • Recombinant virus that expresses zdint5 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. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994.
  • Another suitable cell line is the High FiveOTM cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent #5,300,435).
  • Commercially available serum-free media are used to grow and maintain the cells.
  • Suitable media are Sf900 DTM (Life Technologies) or ESF 921TM (Expression Systems) for the Sf9 cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, KS) or Express FiveOTM (Life Technologies) for the T. ni cells.
  • the cells are grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
  • MOI multiplicity of infection
  • Fungal cells including yeast cells, can also be used within the present invention.
  • Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
  • Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
  • a preferred vector system for use in Saccharomyces cerevisiae is the POTl vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No.
  • Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533. The use of Pichia methanolica as host for the production of recombinant proteins is disclosed in U.S. patents 5,716,808, 5,736,383, 5,854,039, and 5,888,768.
  • Prokaryotic host cells including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.).
  • the polypeptide When expressing a zdint5 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.
  • P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25°C to 35°C.
  • Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors.
  • a preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% BactoTM Peptone (Difco Laboratories, Detroit, ML), 1% BactoTM yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, tr «s-3-methylproline, 2,4-methanoproline, cw-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, ⁇ /7othreonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine.
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
  • the non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for zdint5 amino acid residues.
  • polypeptides of the present invention it is preferred to purify the polypeptides of the present invention to >80% purity, more preferably to >90% purity, even more preferably >95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
  • a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • Expressed recombinant zdint5 proteins are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See, in general, Affinity Chromatography: Principles & Methods. Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice, Springer- Verlag, New York, 1994. Proteins comprising a polyhistidine affinity tag (typically about 6 histidine residues) are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bio/Technol. 6: 1321-1325, 1988.
  • Proteins comprising a Glu-Glu tag can be purified by immunoaffinity chromatography according to conventional procedures. See, for example, Grussenmeyer et al., ibid. Maltose binding protein fusions are purified on an amylose column according to methods known in the art.
  • polypeptides of the present invention can be isolated by a combination of procedures including, but not limited to, anion and cation exchange chromatography, size exclusion, and affinity chromatography.
  • immobilized metal ion adsorption (LMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • LMAC immobilized metal ion adsorption
  • Zdint5 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, /. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, EL, 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, ERL Press, Oxford, 1989. In vitro synthesis is particularly advantageous for the preparation of smaller polypeptides.
  • zdint5 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.
  • the metalloprotease (SEQ ED NO:2) and TSPl-like domains are of particular interest for use in assays and treatment of disorders of the colon, small intestine, fetal lung, testis, and B-cells.
  • the metalloprotease domain may be involved in activating the host defense system against infection.
  • One such metalloprotease is matrilysin (Wilson, C. et al., Science 286: 113-117, 1999). Matrilysin has been shown to be involved in the host defense to bacterial pathogens in the small intestine. Wilson et al.
  • the zdint5 metalloprotease domain (SEQ LD NO:2) alone, or in conjunction with other domains (i.e., the TSPl-like domains of zdint5, SEQ ED NOs: 5 and 8, or SEQ ED NO: 11, ot other TSPl-like domains from the ADAM-TS and METH prtoein families) may be involved in the body's response to pathogenic bacterial invading the epithelium of colon, small intestine, and lung, for example.
  • the metalloprotease domain (SEQ ED NO: 2) can be used as an enzymatic detergent for use in industrial applications.
  • One skilled in the art can readily identify assays to measure the proteolytic function of zdint5 molecules.
  • An exemplary assay to measure the activity of the metalloprotease domain may be by measuring its ability to bind a plasma proteolytic enzyme inhibitor, oc2M.
  • This protein contains a bait region that provides a target for the proteases including metalloproteases. Cleavage of the bait region triggers conformational changes in the ⁇ 2M subunits that cause an encapsulation of the protease and activation of the internal thioesters of cc2M, resulting in a covalent cross-linking of the active protease. See also, Nagase, H. et al., Ann. N. Y. Acad. Sci. 732: 294-302, 1994.
  • Heparin and heparin sulfate are molecules which facilitate the binding of secreted growth factors and other proteins to the extracellular matrix. Thus molecules which bind heparin and heparin sulfate are useful to modulate the effects of these growth factors, etc. Heparin and heparin binding motifs have been identified in thrombospondin repeats in the METH and ADAM-TS subgroups of proteins (see Kuno, 1998, ibid). Thus, the TSPl-like domains (SEQ ED NOs: 5 and/or 8) may be useful in modulating the effects of growth factors by binding to heparin and heparin sulfate.
  • the activity of zdint5 polypeptides can be measured using a variety of assays that measure, for example, protease activity, angiogenesis inhibition; extracellular matrix formation or remodeling; metastasis, and other biological functions associated with ADAM/MDC/SVMP/ADAM-TS/METH family members or with integrin/disintegrin interactions, such as, apoptosis; or differentiation, for example.
  • assays that measure, for example, protease activity, angiogenesis inhibition; extracellular matrix formation or remodeling; metastasis, and other biological functions associated with ADAM/MDC/SVMP/ADAM-TS/METH family members or with integrin/disintegrin interactions, such as, apoptosis; or differentiation, for example.
  • integrin/disintegrin interactions such as, apoptosis
  • differentiation for example.
  • a change in tumor suppression is a change in tumor suppression.
  • Proteins, including alternatively spliced peptides, of the present invention are useful for tumor suppression, gamete maturation, immunologic recognition, and growth and differentiation either working in isolation, or in conjunction with other molecules (growth factors, cytokines, etc.) in colon, small intestine, fetal lung, testis, and B-cells.
  • Alternative splicing of zdint5 may cell-type specific and confer activity to specific tissues.
  • Another assay of interest measures or detects changes in proliferation, differentiation, and/or development of intestinal, colon or lung tissues. Additionally, the effects of zdint5 polypeptides on protease activity, or angiogenesis inhibition of endothelial cells, in general, and tumor cells would be of interest to measure. Yet other assays examine changes in protease activity and apoptosis.
  • Proliferation can be measured using cultured cells or in vivo by administering molecules of the claimed invention to an appropriate animal model. Generally, proliferative effects are observed as an increase in cell number and therefore, may include inhibition of apoptosis, as well as mitogenesis.
  • Cultured cells include colon adenocarcinoma, testis, fetal and adult lung, B cells, melanoma and human umbilical vein endothelial cells from primary cultures. Assays measuring cell proliferation are well known in the art.
  • assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990), incorporation of radiolabelled nucleotides (Cook et al., Analytical Biochem. 179: 1-7, 1989), incorporation of 5-bromo-2'- deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J. Immunol. Methods 82:169-179, 1985), and use of tetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley et al., Cancer Res.
  • assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990), incorporation of radiolabelled nucleotides (Cook et al., Analytical Biochem. 179: 1
  • zdint5 polypeptides may play a role cell proliferation, migration, and angiogenesis by mediating cell adhesion.
  • zdint5 can be given by intradermal or intraperitoneal injection. Characterization of the accumulated leukocytes at the site of injection can be determined using lineage specific cell surface markers and fluorescence immunocytometry or by immunohistochemistry (Jose, J. Exp. Med. 179:881-87, 1994). Release of specific leukocyte cell populations from bone marrow into peripheral blood can also be measured after zdint5 injection.
  • 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 and receptor-like complementary molecules. 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. Symp. 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.
  • zdint5 polypeptides may stimulate inhibition or proliferation of endocrine and exocrine cells of the colon, small intestine, fetal lung, testis, and B-cells.
  • Assays measuring differentiation include, for example, measuring cell- surface markers associated with stage-specific- expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989).0
  • the zdint5 polypeptides of the present invention can be used to study proliferation or differentiation in colon, small intestine, fetal lung, testis, and B-cells.
  • Such methods of the present invention generally comprise incubating cells derived from these tissues in the presence and absence of zdint5 polypeptide, monoclonal antibody, agonist or antagonist thereof and observing changes in cell proliferation or differentiation.
  • Cell lines from these tissues are commercially available from, for example, American Type Culture Collection (Manasas, VA).
  • Proteins, including alternatively spliced peptides, and fragments, of the present invention are useful for studying protease activity, or angiogenesis inhibition.
  • zdint5 molecules, variants, and fragments can be applied in isolation, or in conjunction with other molecules (growth factors, cytokines, etc.) in colon, small intestine, fetal lung, testis, and B-cells.
  • Proteins of the present invention are useful for delivery of therapeutic agents such as, but not limited to, proteases, radionuclides, chemotherapy agents, and small molecules. Effects of these therapeutic agents can be measured in vitro using cultured cells, ex vivo on tissue slices, or in vivo by administering molecules of the claimed invention to the appropriate animal model.
  • An alternative in vivo approach for assaying proteins of the present invention involves viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, lentivirus, vaccinia virus and adeno-associated virus (AAV).
  • Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see T.C.
  • 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 a large number of available vectors containing different promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
  • adenovirus 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 has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary).
  • the host cell the human 293 cell line is exemplary.
  • adenovirus primarily targets the liver. Lf the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells.
  • the host's tissue e.g., liver
  • the host's tissue will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein.
  • Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.
  • 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., X Virol. 72:2022-2032, 1998; Raper, S.E. et al., Human Gene Therapy 9:671- 679, 1998).
  • deletion of E2b is reported to reduce immune responses (Amalfitano, A. et al., J. Virol. 72:926-933, 1998).
  • by deleting the entire adenovirus genome very large inserts of heterologous DNA can be accommodated.
  • the adenovirus system can also be used for protein production in vitro.
  • the cells By culturing adenovirus-infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division.
  • adenovirus vector infected 293S cells can be grown in suspension culture at relatively high cell density to produce significant amounts of protein (see Gamier et al., Cvtotechnol. 15:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant. Within the infected 293S cell production protocol, non- secreted proteins may also be effectively obtained.
  • the activity of zdint5 polypeptide or a peptide to which zdint5 binds can be measured by a silicon-based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with cell-surface protein interactions and subsequent physiologic cellular responses.
  • An exemplary device is the CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
  • CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
  • a variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell, H.M. et al.. Science 257: 1906-1912, 1992; Pitchford, S.
  • the microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including zdint5 proteins, their agonists, and antagonists.
  • the microphysiometer is used to measure responses of a zdint5-responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to zdint5 polypeptide.
  • zdint5-responsive eukaryotic cells comprise cells into which a polynucleotide for a binding partner for zdint5 has been transfected creating a cell that is responsive to zdint5; or cells naturally responsive to zdint5. Differences, measured by a change in the response of cells exposed to zdint5 polypeptide, relative to a control not exposed to zdint5, are a direct measurement of zdint5-modulated cellular responses.
  • the present invention provides a method of identifying agonists and antagonists of zdint5 protein, comprising providing cells responsive to a zdint5 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 measurable change in extracellular acidification rate of the second portion of the cells as compared to the first portion of the cells.
  • culturing a third portion of the cells in the presence of zdint5 polypeptide and the absence of a test compound provides a positive control for the zdint5 -responsive cells, and a control to compare the agonist activity of a test compound with that of the zdint5 polypeptide.
  • Antagonists of zdint5 can be identified by exposing the cells to zdint5 protein in the presence and absence of the test compound, whereby a reduction in zdint5-modulated activity is indicative of agonist activity in the test compound.
  • zdint5 can be used to identify cells, tissues, or cell lines which respond to a zdint5-modulated pathway.
  • the microphysiometer, described above, can be used to rapidly identify cells expressing a zdint5 binding partner, such as cells responsive to zdint5 of the present invention.
  • Cells can be cultured in the presence or absence of zdint5 polypeptide. Those cells which elicit a measurable change in extracellular acidification in the presence of zdint5 are responsive to zdint5.
  • Such cell lines can be used to identify, antagonists and agonists of zdint5 polypeptide as described above.
  • cells expressing zdint5 can be used to identify cells which stimulate a zdint5-signalling pathway.
  • agonists including the native protease and TSPl-like domains
  • antagonists have enormous potential in both in vitro and in vivo applications.
  • Compounds identified as zdint5 agonists and antagonists are useful for studying protease activity, angiogenesis inhibition, extracellular matrix proteins, repair and remodeling of ischemia reperfusion and inflammation in vitro and in vivo.
  • zdint5 and agonist compounds are useful as components of defined cell culture media, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture.
  • Agonists are thus useful in specifically promoting the growth and/or development of cells of the myeloid and lymphoid lineages in culture.
  • zdint5 polypeptides and zdint5 agonists including small molecules are useful as a research reagent, such as for the expansion, differentiation, and/or protease activity, or angiogenesis inhibition of colon, small intestine, fetal lung, testis, and B-cells.
  • Zdint5 polypeptides are added to tissue culture media for these cell types.
  • Antagonists are also useful as research reagents for characterizing sites of interactions between members of complement/anti-complement pairs as well as sites of protease activity, or angiogenesis inhibition.
  • Inhibitors of zdint5 activity include anti-zdint5 antibodies and soluble zdint5 polypeptides (such as in SEQ ED NOs:2, 5, 8 and 11), as well as other peptidic and non-peptidic agents (including ribozymes).
  • Zdint5 can also be used to identify inhibitors (antagonists) of its activity. Test compounds are added to the assays disclosed herein to identify compounds that inhibit the activity of zdint5. In addition to those assays disclosed herein, samples can be tested for inhibition of zdint5 activity within a variety of assays designed to measure TSPl-like domain/extracellular matrix binding or the stimulation/inhibition of zdint5- dependent cellular responses. For example, zdint5-modulated cell lines can be transfected with a reporter gene construct that is responsive to a zdint5-stimulated cellular pathway.
  • Reporter gene constructs of this type are known in the art, and will generally comprise a DNA response element operably linked to a gene encoding an assayable protein, such as luciferase, or a metabolite, such as cyclic AMP.
  • DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE), insulin response element (ERE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) and serum response elements (SRE) (Shaw et al. Cell 56: 563-72, 1989). Cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem.
  • compounds or other samples can be tested for direct blocking of zdint5 binding using zdint5 tagged with a detectable label (e.g., I, biotin, horseradish peroxidase, FLTC, and the like).
  • a detectable label e.g., I, biotin, horseradish peroxidase, FLTC, and the like.
  • zdint5 polypeptides, agonists or antagonists thereof may be therapeutically useful for promoting wound healing, for example, in colon, small intestine, fetal lung, testis, and B-cells tissues.
  • agonists or antagonists of the present invention such zdint5 polypeptides, agonists or antagonists are evaluated with respect to their ability to facilitate wound healing according to procedures known in the art.
  • zdint5 polypeptide performance in this regard can be compared to growth factors, such as EGF, NGF, TGF- ⁇ , TGF- ⁇ , insulin, IGF-I, IGF-EI, fibroblast growth factor (FGF) and the like.
  • zdint5 polypeptides or agonists or antagonists thereof may be evaluated in combination with one or more growth factors to identify synergistic effects.
  • a zdint5 polypeptide can also be used for purification of its binding partner(s).
  • the polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • a solid support such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation.
  • the resulting medium will generally be configured in the form of a column, and fluids containing the binding partners are passed through the column one or more times to allow binding partners to bind to the zdint5 polypeptide.
  • the binding partner is then eluted using changes in salt concentration, chaotropic agents (guanidine HC1), or pH to disrupt zdint5/binding partner binding.
  • An assay system that uses a ligand-binding receptor (or an antibody, one member of a complementary/ anti-complementary pair or other cell-surface binding protein) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed.
  • a ligand-binding receptor or an antibody, one member of a complementary/ anti-complementary pair or other cell-surface binding protein
  • a commercially available biosensor instrument (BIAcore, 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, disintegrin 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 an integrin, epitope, or opposite member of the complementary/anti- complementary pair is present in the sample, it will bind to the immobilized disintegrin, 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 .
  • Protease substrate polypeptides and TSPl-like binding polypeptides which bind proteases or TSPl-like polypeptides can also be used within other assay systems known in the art.
  • extracellular matrix polypeptides which bind to the TSPl-like polypeptides of zdint5 can also be used with other assay systems.
  • 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).
  • a "soluble protein” is a protein that is not bound to a cell membrane. Soluble proteins are most commonly ligand-binding receptor polypeptides that lack transmembrane and cytoplasmic domains. Soluble proteins can comprise additional amino acid residues, such as affinity tags that provide for purification of the polypeptide or provide sites for attachment of the polypeptide to a substrate, or immunoglobulin constant region sequences. Many cell-surface proteins have naturally occurring, soluble counterparts that are produced by proteolysis or translated from alternatively spliced mRNAs. Proteins are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction, respectively.
  • Soluble forms of zdint5 polypeptides may act as antagonsits to or agonists of zdint5 polypeptides, and would be useful to modulate the effects of zdint5 in colon, small intestine, fetal lung, testis, and B-cells.
  • Molecules of the present invention can be used to identify and isolate extracellular matrix proteins, or members of complement/anti-complement pairs involved in protease activity, or angiogenesis inhibition.
  • 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.
  • polypeptides, nucleic acid and/or antibodies of the present invention can be used in treatment of disorders associated with recovery after gastrointestinal irradiation, chemotherapy, or antibody use. Additionally, molecules of the present invention may be useful as anti-infectives, and/or extracellular matrix repair and remodeling.
  • the molecules of the present invention can be used to modulate proteolysis, apoptosis, angiogenesis, infection, cell adhesion, cell fusion, and signaling or to treat or prevent development of pathological conditions in such diverse tissue as colon, small intestine, fetal lung, testis, and B-cells. In particular, certain diseases may be amenable to such diagnosis, treatment or prevention.
  • the molecules of the present invention can be used to modulate inhibition and proliferation of endothelium in colon, small intestine, fetal lung, testis, and B-cells.
  • Disorders which may be amenable to diagnosis, treatment or prevention with zdint5 polypeptides include, for example, tumor formation, Crohn's Disease, inflammatory Bowel Disease, food poisoning, melanoma, and degenerative diseases.
  • Polynucleotides encoding zdint5 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zdint5 activity. If a mammal has a mutated or absent zdint5 gene, the zdint5 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zdint5 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 zdint5 gene can be introduced in a retroviral vector, e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., J. Virol. 62:1120, 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, or non- peptide molecules can be coupled to liposomes chemically.
  • the zdint5 polynucleotides can be used to target specific tissues such as colon, small intestine, fetal lung, testis, and B-cells. It is possible to remove the target cells from the body; to introduce the vector as a naked DNA plasmid; and then to re-implant the transformed cells into the body. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem. 267:963-7, 1992; Wu et al.. J. Biol. Chem. 263:14621-4, 1988.
  • Various techniques can be used to inhibit zdint5 gene transcription and translation, such as to inhibit cell proliferation in vivo.
  • Polynucleotides that are complementary to a segment of a zdint5- encoding polynucleotide e.g., a polynucleotide as set forth in SEQ ED NOs:l, 3, 4, 6, 7, 9, 10 or 12
  • a polynucleotide as set forth in SEQ ED NOs:l, 3, 4, 6, 7, 9, 10 or 12 are designed to bind to zdint5 -encoding mRNA and to inhibit translation of such mRNA.
  • antisense polynucleotides are used to inhibit expression of zdint5 polypeptide-encoding genes in cell culture or in a subject.
  • mice engineered to express the zdint5 gene referred to as "transgenic mice,” and mice that exhibit a complete absence of zdint5 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 zdint5, 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 zdint5 polypeptide, polypeptide fragment or a mutant thereof may alter normal cellular processes, resulting in a phenotype that identifies a tissue in which zdint5 expression is functionally relevant and may indicate a therapeutic target for the zdint5, its agonists or antagonists.
  • a transgenic mouse to engineer is one that over-expresses the soluble zdint5 polypeptide (approximately amino acids 104 to 306 of SEQ ED NO: 11).
  • knockout zdint5 mice can be used to determine where zdint5 is absolutely required in vivo.
  • the phenotype of knockout mice is predictive of the in vivo effects of that a zdint5 antagonist, such as those described herein, may have.
  • the human zdint5 cDNA can be used to isolate murine zdint5 mRNA, cDNA and genomic DNA, which are subsequently used to generate knockout mice. These mice may be employed to study the zdint5 gene and the protein encoded thereby in an in vivo system, and can be used as in vivo models for corresponding human diseases.
  • transgenic mice expression of zdint5 antisense polynucleotides or ribozymes directed against zdint5, described herein can be used analogously to transgenic mice described above.
  • Zdint5 polypeptides, variants, and fragments thereof may be useful as replacement therapy for disorders associated with protease activity, or angiogenesis inhibition, including disorders related to, for example, immuntiy, inflammation, fertility, gamete maturation, immunology, trauma, and epithelial disorders, in general.
  • tissue morphogenesis A less widely appreciated determinant of tissue morphogenesis is the process of cell rearrangement: Both cell motility and cell-cell adhesion are likely to play central roles in morphogenetic cell rearrangements. Cells need to be able to rapidly break and probably simultaneously remake contacts with neighboring cells. See Gumbiner, B.M., Cell 69:385-387, 1992. As a secreted protein in colon, small intestine, fetal lung, testis, and B-cells, zdint5 can play a role in intercellular rearrangement in these and other tissues.
  • the zdint5 polypeptide is expressed in tissues of the colon, small intestine, testis, lung and B cells.
  • the polypeptides of the present invention are useful in studying cell adhesion and the role thereof in metastasis and may be useful in preventing metastasis, in particular metastasis in tumors of the colon, small intestine, testis, lung and B cells.
  • polynucleotides and polypeptides of zdint5 may be used to replace their defective counterparts in tumor or diseased tissues.
  • zdint5 polypeptide pharmaceutical compositions of the present invention may be useful in prevention or treatment of disorders associated with pathological regulation or the expansion of these tissues.
  • the polynucleotides of the present invention may also be used in conjunction with a regulatable promoter, thus allowing the dosage of delivered protein to be regulated.
  • 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.
  • Tumor models 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 zdint5, before implantation so that the protein is synthesized at the tumor site or intracellularly, rather than systemically.
  • adenovirus such as one expressing zdint5
  • the mice normally develop visible tumors within 5 days. The tumors are allowed to grow for a period of up to 3 weeks, during which time they may
  • the implanted cells can be transiently transfected with zdint5.
  • purified zdint5 or zdint5-conditioned media can be directly injected in to this mouse model, and hence be used in this system.
  • Use of stable zdint5 transfectants as well as use of induceable promoters to activate zdint5 expression in vivo are known in the art and can be used in this system to assess zdint5 induction of metastasis. For general reference see, O'Reilly MS, et al. Cell 79:315-328, 1994; and Rusciano D, et al. Murine Models of Liver Metastasis. Invasion Metastasis 14:349-361, 1995.
  • Zdint5 gene may be useful to as a probe to identify humans who have a defective zdint5 gene.
  • the strong expression of zdint5 in colon, small intestine, fetal lung, testis, and B-cells suggests that zdint5 polynucleotides or polypeptides can be used as measured as an indication of aberrant growth in these tissues.
  • polynucleotides and polypeptides of zdint5, and mutations to them can be used a diagnostic indicators of cancer in these tissues.
  • polypeptides of the present invention are useful in studying cell adhesion and the role thereof in metastasis and may be useful in preventing metastasis, in particular metastasis in tumors of the colon, small intestine, fetal lung, testis, and B- cells.
  • polynucleotides and polypeptides of zdint5 may be used to replace their defective counterparts in tumor or malignant tissues.
  • zdint5 polypeptide is expressed in the colon, small intestine, fetal lung, testis, and B-cells.
  • zdint5 polypeptide pharmaceutical compositions of the present invention may be useful in prevention or treatment of disorders associated with pathological regulation or the expansion of colon, small intestine, fetal lung, testis, and B-cells.
  • the zdint5 polynucleotides of SEQ ED NOs:2, 5, 8, and 11 have been mapped to chromosome 9q34.
  • the present invention also provides reagents which will find use in diagnostic applications.
  • the zdint5 gene, a probe comprising zdint5 DNA or RNA or a subsequence thereof can be used to determine if the zdint5 gene is present on chromosome 9q34 or if a mutation has occurred.
  • Detectable chromosomal aberrations at the zdint5 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
  • Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).
  • molecular genetic techniques such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).
  • the proteins of the present invention can be administered orally, rectally, parenterally (particularly intravenous or subcutaneous), intracisternally, intravaginally, intraperitoneally, topically (as powders, ointments, drops or transdermal patch) bucally, in utero or as a pulmonary or nasal inhalant.
  • Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours.
  • pharmaceutical formulations will include a zdint5 protein, alone, or in conjunction with a dimeric partner, in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like.
  • Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
  • Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995.
  • Therapeutic doses will generally be in the range of 0.1 to 100 ⁇ g/kg of patient weight per day, preferably 0.5-20 mg/kg per day, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art.
  • the proteins may be administered for acute treatment, over one week or less, often over a period of one to three days or may be used in chronic treatment, over several months or years.
  • a therapeutically effective amount of zdint5 is an amount sufficient to produce a clinically significant change in extracellular matrix remodeling, scar tissue formation, tumor suppression, platelet aggregation, apoptosis, myogenesis, colon, small intestine, fetal lung, testis, and B-cells tissues.
  • a therapeutically effective amount of zdint5 is an amount sufficient to produce a clinically significant change in disorders associated with colon, small intestine, lung, testis, and B-cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne des molécules polynucléotides et polypeptides, et des variantes de celles-ci, destinées à des zdint5, un nouveau membre des désintégrines protéases. Les polypeptides et les polynucléotides codant ces désintégrines protéases exercent un effet modulateur sur l'interaction intercellulaire et peuvent être mis en application sur le plan médical et thérapeutique. La présente invention concerne également des anticorps de polypeptides de zdint5.
PCT/US2001/004198 2000-02-10 2001-02-09 Peptides intestinaux anti-angiogeniques, zdint5 WO2001059112A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU36816/01A AU3681601A (en) 2000-02-10 2001-02-09 Anti-angiogenic intestinal peptides, zdint5
EP01909020A EP1171602A1 (fr) 2000-02-10 2001-02-09 Peptides intestinaux anti-angiogeniques, zdint5
JP2001558250A JP2004500087A (ja) 2000-02-10 2001-02-09 抗−脈管形成性腸ペプチド、ｚｄｉｎｔ５
CA002368681A CA2368681A1 (fr) 2000-02-10 2001-02-09 Peptides intestinaux anti-angiogeniques, zdint5

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US50180600A 2000-02-10 2000-02-10
US09/501,806 2000-02-10

Publications (1)

Publication Number Publication Date
WO2001059112A1 true WO2001059112A1 (fr) 2001-08-16

Family

ID=23995100

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/004198 WO2001059112A1 (fr) 2000-02-10 2001-02-09 Peptides intestinaux anti-angiogeniques, zdint5

Country Status (5)

Country Link
EP (1) EP1171602A1 (fr)
JP (1) JP2004500087A (fr)
AU (1) AU3681601A (fr)
CA (1) CA2368681A1 (fr)
WO (1) WO2001059112A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002026999A2 (fr) * 2000-09-28 2002-04-04 Bayer Aktiengesellschaft Regulation de la proteine humaine de type adam-ts

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999029855A1 (fr) * 1997-12-08 1999-06-17 Beth Israel Deaconess Medical Center Mutants d'endostatine 'em 1' presentant une activite anti-angiogenique et leurs methodes d'utilisation
WO1999065940A1 (fr) * 1998-06-17 1999-12-23 Beth Israel Deaconess Medical Center Proteines anti-angiogeniques et methodes d'utilisation de ces proteines
WO2000003726A1 (fr) * 1998-07-14 2000-01-27 Bristol-Myers Squibb Company Fragments de l'angiostatine se liant a la lysine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999029855A1 (fr) * 1997-12-08 1999-06-17 Beth Israel Deaconess Medical Center Mutants d'endostatine 'em 1' presentant une activite anti-angiogenique et leurs methodes d'utilisation
WO1999065940A1 (fr) * 1998-06-17 1999-12-23 Beth Israel Deaconess Medical Center Proteines anti-angiogeniques et methodes d'utilisation de ces proteines
WO2000003726A1 (fr) * 1998-07-14 2000-01-27 Bristol-Myers Squibb Company Fragments de l'angiostatine se liant a la lysine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PARRY T J ET AL: "Bioactivity of anti-angiogenic ribozymes targeting Flt-1 and KDR mRNA.", NUCLEIC ACIDS RESEARCH, (1999 JUL 1) 27 (13) 2569-77., XP002168772 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002026999A2 (fr) * 2000-09-28 2002-04-04 Bayer Aktiengesellschaft Regulation de la proteine humaine de type adam-ts
WO2002026999A3 (fr) * 2000-09-28 2003-01-23 Bayer Ag Regulation de la proteine humaine de type adam-ts

Also Published As

Publication number Publication date
AU3681601A (en) 2001-08-20
EP1171602A1 (fr) 2002-01-16
CA2368681A1 (fr) 2001-08-16
JP2004500087A (ja) 2004-01-08

Similar Documents

Publication Publication Date Title
US20060024805A1 (en) Disintegrin homologs
US6762044B2 (en) Mammalian adhesion protease peptides
US6416988B1 (en) Beta-1,3-galactosyltransferase homologs
EP1198560B1 (fr) Proteases a adhesion mammalienne (mapp)
US6361985B1 (en) Beta-1,3-galactosyltransferase homolog, ZNSSP6
US20020081685A1 (en) Disintegrin homologs, ZSNK10, ZSNK11, and ZSNK12
EP1171602A1 (fr) Peptides intestinaux anti-angiogeniques, zdint5
US20020142439A1 (en) Anti-angiogenic intestinal peptides, zdint5
US20050239166A1 (en) Polynucleotides encoding human secreted protein, ZZP1
US20030096393A1 (en) Disintegrin homolog, ZSNK16
WO2001044479A1 (fr) Homologue de galactosyltransferase, znssp8
ZA200007766B (en) Disintegrin homologs.
US20040259163A1 (en) Antibodies to mammalian adhesion protease peptides
JP2004500087A5 (fr)
US6514741B1 (en) Tryptase-like polypeptide ztryp1
US20030153064A1 (en) Disintegrin homologue, MAHBP
US20030104398A1 (en) Galactosyltransferase homolog, ZNSSP11
US20020042095A1 (en) Secreted protein, ZSIG89
WO2001038505A2 (fr) Proteine secretee, zalpha37
US20030119035A1 (en) Tryptase-like polypeptide ztryp1
EP1206552A2 (fr) Polypeptide ztryp1 de type tryptase
WO2000050594A2 (fr) Proteine mammalienne en helice alpha, zsig83

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ 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 MZ 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: A1

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 TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

ENP Entry into the national phase

Ref document number: 2368681

Country of ref document: CA

Ref country code: CA

Ref document number: 2368681

Kind code of ref document: A

Format of ref document f/p: F

121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref country code: JP

Ref document number: 2001 558250

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 36816/01

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2001909020

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2001909020

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 2001909020

Country of ref document: EP