WO2001030831A1 - Secreted proteins and uses thereof - Google Patents

Secreted proteins and uses thereof Download PDF

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Publication number
WO2001030831A1
WO2001030831A1 PCT/US2000/029797 US0029797W WO0130831A1 WO 2001030831 A1 WO2001030831 A1 WO 2001030831A1 US 0029797 W US0029797 W US 0029797W WO 0130831 A1 WO0130831 A1 WO 0130831A1
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Prior art keywords
polypeptide
nucleic acid
seq
amino acid
tango
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PCT/US2000/029797
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French (fr)
Inventor
Christopher C. Fraser
Martin R. Hodge
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Millennium Pharmaceuticals, Inc.
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Priority to AU13517/01A priority Critical patent/AU1351701A/en
Publication of WO2001030831A1 publication Critical patent/WO2001030831A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • secreted proteins for example, cytokines and cytokine receptors
  • cytokines and cytokine receptors play a vital role in the regulation of cell growth, cell differentiation, and a variety of specific cellular responses.
  • a number of medically useful proteins including erythropoietin, granulocyte-macrophage colony stimulating factor, human growth hormone, and various interleukins, are secreted proteins.
  • erythropoietin granulocyte-macrophage colony stimulating factor
  • human growth hormone and various interleukins
  • the identification and characterization of such a receptor enables one to identify both the ligands which bind to the receptor and the intracellular molecules and signal transduction pathways associated with the receptor, permitting one to identify or design modulators of receptor activity, e.g., receptor agonists or antagonists and modulators of signal transduction.
  • the present invention is based, at least in part, on the discovery of cDNA molecules which encode the TANGO 269 and 298 proteins, both of which are wholly secreted or transmembrane proteins.
  • the TANGO 269 proteins share homology to lectin-like oxidized low-density lipoprotein receptor (LOX-1), a cell surface receptor and membrane protein that belongs structurally to the C-type lectin family, and is expressed in vivo in vascular endothelium and vascular-rich organs.
  • the TANGO 298 proteins share homology to adipsin, a serine protease homolog that is synthesized and secreted by adipose cells and which has been proven to be the same as complement factor D.
  • complement factor D is known to play an important role in obesity and energy regulation pathways.
  • TANGO 269 and TANGO 298 proteins, fragments, derivatives, and variants thereof are collectively referred to as "polypeptides of the invention” or “proteins of the invention.”
  • Nucleic acid molecules encoding the polypeptides or proteins of the invention are collectively referred to as “nucleic acids of the invention.”
  • nucleic acids and polypeptides of the present invention are useful as modulating agents in regulating a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding a polypeptide of the invention or a biologically active portion thereof. The present invention also provides nucleic acid molecules which are suitable for use as primers or hybridization probes for the detection of nucleic acids encoding a polypeptide of the invention.
  • the invention features nucleic acid molecules which are at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:l 1, the nucleotide sequence of the cDNA insert of a clone deposited with ATCC as Accession Number 207218, or a complement thereof.
  • the invention features nucleic acid molecules which are at least 37% (or 40%, 45%, 50%,
  • nucleotide sequence of SEQ ID NO:24 55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequence of SEQ ID NO:24, the nucleotide sequence of the cDNA insert of a clone deposited with ATCC as Accession Number 207216, or a complement thereof.
  • the invention also features nucleic acid molecules which are at least 25% (or 30%, 35%, 40%, 45%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%) identical to the nucleotide sequence of SEQ ID NO: 1 , 11 , or 24, the nucleotide sequence of the cDNA insert of a clone deposited with ATCC as Accession Number 207218 or 207216, or a complement thereof, wherein such nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or functional feature of a polypeptide of the invention.
  • the invention features nucleic acid molecules which include a fragment of at least 500
  • nucleotides of the nucleotide sequence of SEQ ID NO:l the nucleotide sequence of the cDNA of ATCC Accession Number 207218, or a complement thereof.
  • the invention features nucleic acid molecules which include a fragment of at least 400 (425, 475, 500, 525, 550, 575, 600, 650, 700, 750, 800, 850, 900, or 930) nucleotides of the nucleotide sequence of SEQ ID NO: 11 or a complement thereof.
  • the invention features nucleic acid molecules which include a fragment of at least 400 (425, 475, 500, 525, 550, 575, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, or 1070) nucleotides of the nucleotide sequence of SEQ ID NO:24, the nucleotide sequence of the cDNA of ATCC Accession Number 207216, or a complement thereof.
  • the invention also features nucleic acid molecules comprising at least 50, 75, 100, 125, 150, 75, 200, 225, 250, 275, 300, or 325 nucleotides of the nucleotide sequence from nucleotide 475 to 800 of SEQ ID NO:24, or a complement thereof.
  • the invention also features nucleic acid molecules which include a fragment of at least 50
  • nucleotides of the nucleotide sequence of SEQ ID NO:l, 11, or 24, or a complement thereof, wherein such nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or functional feature of a polypeptide of the invention.
  • the invention also features nucleic acid molecules which include a nucleotide sequence encoding a protein having an amino acid sequence that is at least 25% (or 35%, 45%, 55%, 65%, 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NO:3 or 13, the amino acid sequence encoded by the cDNA of ATCC Accession Number 207218, or a complement thereof.
  • the invention also features nucleic acid molecules which include a nucleotide sequence encoding a protein having an amino acid sequence that is at least 40% (or 45%, 55%, 65%, 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NO:26, the amino acid sequence encoded by the cDNA of ATCC Accession Number 207216, or a complement thereof.
  • the nucleic acid molecules have the nucleotide sequence of
  • nucleic acid molecules which encode a fragment (e.g., a biologically active fragment) of a polypeptide having the amino acid sequence of SEQ ID NO: 3 or 13, or a fragment including at least 15 (25, 30, 50, 75, 100, 125, 150, 175, 200, or 225) contiguous amino acids of SEQ ID NO:3 or 13, or the amino acid sequence encoded by the cDNA of ATCC Accession Number 207218.
  • nucleic acid molecules which encode a fragment of a polypeptide having the amino acid sequence of SEQ ID NO:26, or a fragment including at least 15 (25, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, or 280) contiguous amino acids of SEQ ID NO:26, or the amino acid sequence encoded by the cDNA of ATCC Accession Number 207216.
  • the invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 13, or 26, or the amino acid sequence encoded by the cDNA of ATCC Accession Number 207218 or 207216, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule consisting of a nucleic acid sequence encoding SEQ ID NO:3, 13, or 26, the amino acid sequence encoded by the cDNA of ATCC Accession Number 207218 or 207216, or a complement thereof under stringent conditions.
  • isolated polypeptides or proteins having an amino acid sequence that is at least about 25%, preferably 35%, 45%, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 3 or 13, or the amino acid sequence encoded by the cDNA of ATCC Accession Number 207218.
  • isolated polypeptides or proteins having an amino acid sequence that is at least about 40%, preferably 45%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:26 or the amino acid sequence encoded by the cDNA of ATCC Accession Number 207216.
  • isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 90%, preferably 92%, 94%, 96%, or 98% identical to the nucleic acid sequence encoding SEQ ID NO:3, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 or 2, a complement thereof, or the non-coding strand of the cDNA of ATCC Accession Number 207218.
  • isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to the nucleic acid sequence encoding SEQ ID NO: 13, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l 1 or 12, a complement thereof, or the non-coding strand of the cDNA of ATCC Accession Number 207216.
  • isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 30%, preferably 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% identical to the nucleic acid sequence encoding SEQ ID NO:26, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:24 or 25, a complement thereof, or the non-coding strand of the cDNA of ATCC Accession Number 207216.
  • polypeptides which are naturally occurring allelic variants of a polypeptide that includes the amino acid sequence of SEQ ID NO:3, 13, or 26, or the amino acid sequence encoded by the cDNA of ATCC Accession Number 207218 or 207216, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule having the sequence of SEQ ID NO:l, 2, 11, 12, 24, or 25, or a complement thereof under stringent conditions.
  • the invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, the cDNA of ATCC Accession Number 207218, or a complement thereof.
  • the nucleic acid molecules are at least 500 (525, 550, 575, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, or 1080) nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, the cDNA of ATCC Accession Number 207218, or a complement thereof.
  • the invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l 1 or a complement thereof.
  • the nucleic acid molecules are at least 400 (425, 475, 500, 525, 550, 575, 600, 650, 700, 750, 800, 850, 900, or 930) nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 11 or a complement thereof.
  • the invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:24, the cDNA of ATCC Accession Number 207216, or a complement thereof.
  • the nucleic acid molecules are at least 400 (425, 475, 500, 525, 550, 575, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, or 1070) nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:24, the cDNA of ATCC Accession Number 207216, or a complement thereof.
  • the invention also features nucleic acid molecules at least 15, preferably 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or more nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, 11, or 24, the cDNA of ATCC Accession Number 207216 or 207218, or a complement thereof, wherein such nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or functional feature of a polypeptide of the invention.
  • the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a nucleic acid of the invention.
  • Another aspect of the invention provides vectors, e.g., recombinant expression vectors, comprising a nucleic acid molecule of the invention.
  • the invention provides host cells containing such a vector or a nucleic acid molecule of the invention.
  • the invention also provides methods for producing a polypeptide of the invention by culturing, in a suitable medium, a host cell of the invention containing a recombinant expression vector such that a polypeptide is produced.
  • Another aspect of this invention features isolated or recombinant proteins and polypeptides of the invention.
  • Preferred proteins and polypeptides possess at least one biological activity possessed by the corresponding naturally-occurring human polypeptide.
  • An activity, a biological activity, or a functional activity of a polypeptide or nucleic acid of the invention refers to an activity exerted by a protein, polypeptide or nucleic acid molecule of the invention on a responsive cell as determined in vivo, or in vitro, according to standard techniques.
  • activities can be a direct activity, such as an association with or an enzymatic activity on a second protein, or an indirect activity, such as a cellular signaling activity mediated by interaction of the protein with a second protein.
  • biological activities can include, e.g., (1) the ability to form, e.g., stabilize, promote, inhibit, or disrupt, protein-protein interactions (e.g., homophilic and/or heterophilic) with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; (3) the ability to interact with a TANGO 269 receptor; and (4) the ability to modulate the function, migration, proliferation (e.g., suppress cell growth), and/or differentiation of cells, e.g., cells in tissues in which it is expressed (see description of expression data below) and, in particular, hepatocytes.
  • protein-protein interactions e.g., homophilic and/or heterophilic
  • TANGO 269 biological activities can include, e.g., (1) the ability to bind proteins, e.g., Hpoproteins, e.g., low-density Hpoproteins, e.g., oxidatively modified low-density Hpoproteins; (2) the ability to modulate internalization of proteins, e.g., Hpoproteins, e.g., low- density Hpoproteins, e.g., oxidatively modified low-density Hpoproteins; (3) the ability to modulate degradation, e.g., proteolytic degradation, of proteins, e.g., Hpoproteins, e.g., low- density Hpoproteins, e.g., oxidatively modified low-density Hpoproteins; (4) the ability to modulate, e.g., increase, uptake of proteins, e.g., Hpoproteins, e.g., low-density Hpoprotein
  • TANGO 269 biological activities can include, e.g., (1) the ability to modulate, e.g., prevent, lipid deposition, e.g., in arteries; (2) the ability to modulate, e.g., induce or prevent, changes in cells, e.g., transformation of cells (e.g., macrophages and smooth muscle cells) into foam cells and functional alteration of cells (e.g., endothelial cells, e.g., intimal neovascular endothelial cells); (3) the ability to bind and phagocytose cells, e.g., aged and apoptotic cells; (4) the ability to modulate, e.g., prevent, intimal thickening, e.g., by preventing deposit and buildup of substances (e.g., lipids) on blood vessel walls; and (5) the ability to remove debris, e.g., aged and apoptotic (dead) cells, from blood vessel walls.
  • cells
  • Still other TANGO 269 biological activities can include, e.g., (1) the ability to modulate homeostasis, e.g., vascular homeostasis, e.g., by modulating, e.g., preventing the impairment of, nitric oxide production; (2) the ability to modulate, e.g., inhibit, the expression of molecules, e.g., adhesion molecules (e.g., leukocyte adhesion molecules) and growth factors (e.g., smooth-muscle growth factors); (3) the ability to alter, e.g., increase, expression in response to stimuli, e.g., TNF- ⁇ , shear stress, and pathophysiological stimuli relevant to disorders (e.g., atherosclerosis and inflammation).
  • homeostasis e.g., vascular homeostasis
  • Still other TANGO 269 biological activities can include, e.g., (1) the ability to form, e.g., stabilize, promote, facilitate, inhibit, or disrupt, cell-extracellular matrix interactions, e.g., adhesion between cells and extracellular matrix; (2) the ability to form, e.g., stabilize, promote, facilitate, inhibit, or disrupt, cell to cell and cell to blood product interaction, e.g., between leukocytes and platelets or leukocytes and vascular endothelial cells; and (3) the ability to recognize large molecules, e.g., carbohydrates.
  • TANGO 269 biological activities can also include, e.g., the ability to perform one or more of the functions of LOX-1 described, for example, in the following: Sawamura et al. (1997) Nature 386:73-77; Kataoka et al. (1999) Circulation 99:3110-3117; and Kita (1999) Circulation Research 84 : 1113 - 1115 , the contents of all of which are incorporated herein by reference.
  • biological activities can include, e.g., (1) the ability to form, e.g., stabilize, promote, facilitate, inhibit, or disrupt, protein-protein interactions (e.g., homophilic and/or heterophilic) with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; and (3) the ability to interact with a TANGO 298 receptor.
  • protein-protein interactions e.g., homophilic and/or heterophilic
  • TANGO 298, biological activities can include, e.g., (1) the ability to alter e.g., enzymatically cleave (e.g., by hydrolysis), proteins, e.g., alternative complement factor B; (2) the ability to act as a proteolytic enzyme cleaving either itself or other substrates; (3) the ability to modulate, e.g., activate, the alternative complement cascade; (4) the ability to alter, e.g., lyse (e.g., via activation of the alternative complement cascade), cells, e.g., red blood cells; (5) the ability to modulate systemic energy balance, e.g., via the alternative complement cascade; and (6) the ability to modulate host defense, e.g., by suppressing infection or invasion by foreign agents, e.g., bacteria, viruses, parasites, neoplastic cells, e.g., via the alternative complement cascade.
  • host defense e.g., by suppressing infection or invasion by foreign agents,
  • Still other TANGO 298 biological activities can include, e.g., (1) the ability to modulate, e.g., inhibit or activate, the immune response, e.g., by modulating inflammation; (2) the ability to modulate, e.g., inhibit, cellular (e.g., immune cells, e.g., neutrophils, monocytes, lymphocytes) and tissue destruction; and (3) the ability to modulate adipocyte proliferation, differentiation, and/or function.
  • cellular e.g., immune cells, e.g., neutrophils, monocytes, lymphocytes
  • TANGO 298 biological activities can include, e.g., (1) the ability to modulate cyclic hematopoiesis, e.g., the ability to modulate the cyclic change in numbers of blood cells (e.g., neutrophils, monocytes, platelets, lymphocytes, and erythrocytes) in circulation.
  • blood cells e.g., neutrophils, monocytes, platelets, lymphocytes, and erythrocytes
  • the various biological activities of TANGO 298 described herein can be modulated by serpins, a gene family that encompasses a wide variety of protein molecules, such as protease inhibitors.
  • other TANGO 298 modulators e.g., antisense molecules and antibodies, can also be used to modulate, e.g., inhibit, these TANGO 298 biological activities.
  • TANGO 298 biological activities can include, e.g., the ability to perform one or more of the functions of adipsin described, for example, in the following: White et al. (1992) J Biol Chem. 13:9210-9213; Spiegelman et al. (1989) J Biol Chem. 264:1811-1815; Yamauchi et al. (1994) J mmunol. 152:3645-3653; Meri et al. (1998) Vox Sang. 74 Suppl 2:291-302; and PCT Publication Number WO 90/06365, the contents of all of which are incorporated herein by reference.
  • a polypeptide of the invention has an amino acid sequence sufficiently identical to an identified domain of a polypeptide of the invention.
  • the term "sufficiently identical" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity.
  • amino acid or nucleotide sequences which contain a common structural domain having about 60% identity, preferably 65% identity, more preferably 75%, 85%, 95%, 98% or more identity are defined herein as sufficiently identical.
  • a TANGO 269 protein includes at least one of the following domains: a signal sequence, an extracellular link domain, and a C-type lectin domain. In another embodiment, a TANGO 269 protein includes two or more of the following domains: a signal sequence, an extracellular link domain, and a C-type lectin domain, and is a type II transmembrane protein.
  • a TANGO 298 protein includes at least one of the following domains: a signal sequence and a trypsin domain.
  • a nucleic acid molecule of the present invention encodes a TANGO 269 protein which includes at least one of the following domains: a signal sequence, an extracellular link domain, and a C-type lectin domain.
  • a nucleic acid molecule of the present invention encodes a TANGO 269 protein which includes two or more of the following domains: a signal sequence, an extracellular link domain, and a C-type lectin domain.
  • a nucleic acid molecule of the present invention encodes a TANGO 298 protein which includes at least one of the following domains: a signal sequence and a trypsin domain.
  • the polypeptides of the present invention, or biologically active portions thereof, can be operably linked to a heterologous amino acid sequence to form fusion proteins.
  • the invention further features antibodies, such as monoclonal or polyclonal antibodies or fragments thereof, that specifically bind to a polypeptide of the invention.
  • the antibodies of the invention can be conjugated antibodies comprising, for example, therapeutic or diagnostic agents.
  • the antibodies can be conjugated to a therapeutic moiety such as a chemotherapeutic cytotoxin, e.g., a cytostatic or cytocidal agent (e.g., paclitaxol, cytochalasin B or diphtheria toxin), a thrombotic or anti-angiogenic agent or a radioactive or fluorescent label.
  • a chemotherapeutic cytotoxin e.g., a cytostatic or cytocidal agent (e.g., paclitaxol, cytochalasin B or diphtheria toxin), a thrombotic or anti-angiogenic agent or a radioactive or fluorescent label.
  • a chemotherapeutic cytotoxin e.g., a cytostatic or cytocidal agent (e.g., paclitaxol, cytochalasin B or diphtheria toxin)
  • a thrombotic or anti-angiogenic agent e.g.
  • polypeptides of the invention or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
  • the present invention provides methods for detecting the presence of the activity or expression of a polypeptide of the invention in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of activity such that the presence of activity is detected in the biological sample.
  • the invention provides methods for modulating activity of a polypeptide of the invention comprising contacting a cell with an agent that modulates (inhibits or stimulates) the activity or expression of a polypeptide of the invention such that activity or expression in the cell is modulated.
  • the agent is an antibody that specifically binds to a polypeptide of the invention.
  • the agent modulates expression of a polypeptide of the invention by modulating transcription, splicing, or translation of an mRNA encoding a polypeptide of the invention.
  • the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an mRNA encoding a polypeptide of the invention.
  • the present invention also provides methods to treat a subject having a disorder characterized by aberrant activity of a polypeptide of the invention or aberrant expression of a nucleic acid of the invention by administering an agent which is a modulator of the activity of a polypeptide of the invention or a modulator of the expression of a nucleic acid of the invention to the subject.
  • the modulator is a protein of the invention.
  • the modulator is a nucleic acid of the invention.
  • the modulator is a peptide, peptidomimetic, or other small molecule.
  • the present invention also provides diagnostic assays for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of: (i) aberrant modification or mutation of a gene encoding a polypeptide of the invention, (ii) mis-regulation of a gene encoding a polypeptide of the invention, and (iii) aberrant post-translational modification of the invention wherein a wild-type form of the gene encodes a protein having the activity of the polypeptide of the invention.
  • the invention provides a method for identifying a compound that binds to or modulates the activity of a polypeptide of the invention.
  • such methods entail measuring a biological activity of the polypeptide in the presence and absence of a test compound and identifying those compounds which alter the activity of the polypeptide.
  • the invention also features methods for identifying a compound which modulates the expression of a polypeptide or nucleic acid of the invention by measuring the expression of the polypeptide or nucleic acid in the presence and absence of the compound.
  • Figure 1 depicts the cDNA sequence of human TANGO 269 (SEQ ID NO: 1) and the predicted amino acid sequence of TANGO 269 (SEQ ID NO:3).
  • the open reading frame of SEQ ID NO:l extends from nucleotide 66 to nucleotide 749 of SEQ ID NO:l (SEQ ID NO:2).
  • Figure 2 depicts a hydropathy plot of human TANGO 269. Relatively hydrophobic regions of the protein are above the dashed horizontal line, and relatively hydrophilic regions of the protein are below the dashed horizontal line.
  • the cysteine residues (cys) and potential N- glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace.
  • the dashed vertical line separates the signal sequence (amino acids 1 to 47 of SEQ ID NO:3; SEQ ID NO:5) on the left from the mature protein (amino acids 48 to 228 of SEQ ID NO:3; SEQ ID NO:4) on the right. Thicker gray horizontal bars below the dashed horizontal line indicate extracellular ("out"), transmembrane ("TM”), and intracellular (“in”) regions of the molecule.
  • Figures 3A-3B depict a local alignment of the nucleotide sequence of human LOX-1 (SEQ ID NO:21; GenBank Accession Number ABO 10710) and the nucleotide sequence of human TANGO 269 (SEQ ID NO:l).
  • the nucleotide sequences of human LOX-1 and human TANGO 269 are about 46% identical over a 1084 base pair overlap. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
  • Figures 4A-4B depict an alignment of the nucleotide sequence of the open reading frames of human LOX-1 (nucleotides 62 to 880 of SEQ ID NO:22 (GenBank Accession Number AB010710)) and human TANGO 269 (SEQ ID NO:2).
  • the nucleotide sequences of the open reading frames of human LOX-1 and human TANGO 269 (SEQ ID NO:l) are 44.9 % identical. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
  • Figure 5 depicts an alignment of the amino acid sequence of human LOX-1 (SEQ ID NO:23 (GenBank Accession Number AB010710)) and the amino acid sequence of human TANGO 269 (SEQ ID NO:3).
  • the amino acid sequences of human LOX-1 and human TANGO 269 are 23.8% identical. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
  • Figure 6 depicts the cDNA sequence of murine TANGO 269 (SEQ ID NO: 11 ) and the predicted amino acid sequence of murine TANGO 269 (SEQ ID NO: 13).
  • the open reading frame of SEQ ID NO: l 1 extends from nucleotide 106 to nucleotide 792 of SEQ ID NO: l 1 (SEQ ID NO: 12).
  • Figure 7 depicts a hydropathy plot of murine TANGO 269. Relatively hydrophobic regions of the protein are above the dashed horizontal line, and relatively hydrophilic regions of the protein are below the dashed horizontal line.
  • the cysteine residues (cys) and potential N- glycosylation sites (Ngty) are indicated by short vertical lines just below the hydropathy trace.
  • the dashed vertical line separates the signal sequence (amino acids 1 to 46 of SEQ ID NO: 13; SEQ ID NO: 15) on the left from the mature protein (amino acids 47 to 229 of SEQ ID NO: 13; SEQ ID NO: 14) on the right. Thicker gray horizontal bars below the dashed horizontal line indicate extracellular ("out"), transmembrane ("TM”), and intracellular (“in”) regions of the molecule.
  • Figures 8A-8B depict an alignment of the open reading frames of the nucleotide sequence of murine TANGO 269 (SEQ ID NO: 11) and the nucleotide sequence of human TANGO 269 (SEQ ID NO: 1).
  • the open reading frames of murine TANGO 269 and human TANGO 269 are 74.1% identical.
  • This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
  • This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
  • Figure 9 depicts an alignment of the amino acid sequence of murine TANGO 269 (SEQ ID NO:13) and the amino acid sequence of human TANGO 269 (SEQ ID NO:3).
  • the amino acid sequences of murine TANGO 269 and human TANGO 269 are 63.0 % identical. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
  • Figure 10 depicts the cDNA sequence of human TANGO 298 (SEQ ID NO:25) and the predicted amino acid sequence of TANGO 298 (SEQ ID NO:27).
  • the open reading frame of SEQ ID NO:25 extends from nucleotide 31 to nucleotide 879 of SEQ ID NO:25 (SEQ ID NO:26).
  • Figure 11 depicts a hydropathy plot of human TANGO 298. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) and potential N- glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1 to 31 of SEQ ID NO:27; SEQ ID NO:29) on the left from the mature protein (amino acids 32 to 283 of SEQ ID NO:27; SEQ ID NO:28) on the right.
  • FIGS. 12A-12B depict an alignment of the nucleotide sequence of human adipsin (SEQ
  • FIG. 13A-13B depict an alignment of the nucleotide sequence of the open reading frames of human adipsin (nucleotides 55 to 738 of SEQ ID NO:32 (SEQ ID NO:33); GenBank Accession Number NM_001928) and human TANGO 298 (SEQ ID NO:26).
  • nucleotide sequences of the open reading frames of human adipsin and human TANGO 298 are 50.8% identical. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
  • Figure 14 depicts an alignment of the amino acid sequence of human adipsin (SEQ ID NO: 34; GenBank Accession Number NM_001919) and the amino acid sequence of human TANGO 298 (SEQ ID NO:27).
  • the amino acid sequences of human adipsin and human TANGO 298 are 34.0% identical. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
  • Figure 15 shows a TaqMan analysis of human TANGO 298 expression in various tissues and cell types relative to expression in tonsil.
  • Figures 16A-16B show a TaqMan analysis of human TANGO 298 expression in various tissues and cell types relative to expression in CDl 5 CDl lb + cells from mobilized bone marrow (mBMCF157CDl lb + ).
  • Figure 77 shows an alignment between TANGO 298 and HUMGS00642, an EST (accession number D19687) which maps to chromosome 19, between markers D19S886 and D19S216. In the region between D19S886 and D19S216 lies 19pl3.3, the area which is the location for cyclic hematopoiesis (also called cyclic neutropenia).
  • TANGO 269 proteins and nucleic acid molecules comprise a family of molecules having certain conserved structural and functional features.
  • TANGO 298 proteins and nucleic acid molecules comprise a family of molecules having conserved functional and structural features.
  • family is intended to mean two or more proteins or nucleic acid molecules having a common structural domain and having sufficient amino acid or nucleotide sequence identity as defined herein. Family members can be from either the same or different species. For example, a family can comprises two or more proteins of human origin, or can comprise one or more proteins of human origin and one or more of non-human origin. Members of the same family may also have common structural domains.
  • TANGO 269 proteins and TANGO 298 proteins of the invention have signal sequences.
  • a "signal sequence" includes a peptide of at least about 15 or 20 amino acid residues in length which occurs at the N-terminus of secretory and membrane- bound proteins and which contains at least about 70% hydrophobic amino acid residues such as alanine, leucine, isoleucine, phenylalanine, proline, tyrosine, tryptophan, or valine.
  • a signal sequence contains at least about 15 to 25 amino acid residues, preferably about 18 to 22 amino acid residues, and has at least about 60-80%, more preferably 65-75%, and more preferably at least about 70% hydrophobic residues.
  • a signal sequence serves to direct a protein containing such a sequence to a lipid bilayer.
  • a human TANGO 269 protein contains a signal sequence of about amino acids 1 to 44, 1 to 45, 1 to 46, 1 to 47, 1 to 48, or 1 to 49 of SEQ ID NO:3 (SEQ ID NO:5).
  • the domains and mature protein resulting from cleavage of such signal peptides are also included herein.
  • the cleavage of a signal sequence consisting of amino acids 1 to 47 results in a mature human TANGO 269 protein corresponding to amino acids 48 to 228.
  • a murine TANGO 269 protein contains a signal sequence of about amino acids 1 to 44, 1 to 45, 1 to 46, 1 to 47, or 1 to 48 of SEQ ID NO: 13 (SEQ ID NO: 15).
  • the domains and mature protein resulting from cleavage of such signal peptides are also included herein.
  • the cleavage of a signal sequence consisting of amino acids 1 to 46 results in a mature murine TANGO 269 protein corresponding to amino acids 47 to 229.
  • a TANGO 298 protein contains a signal sequence of about amino acids 1 to 28, 1 to 29, 1 to 30, 1 to 31, 1 to 32, or 1 to 33 of SEQ ID NO:27 (SEQ ID NO:29).
  • the domains and mature protein resulting from cleavage of such signal peptides are also included herein.
  • the cleavage of a signal sequence consisting of amino acids 1 to 31 results in a mature TANGO 298 protein corresponding to amino acids 32 to 283.
  • TANGO 269 family members can also include a C-type lectin domain.
  • C-type lectin domains are found in a variety of families, including type-II membrane proteins, large proteoglycans, proteins collectively known as collectins, and proteins collectively known as selectins, which are cell adhesion molecules implicated in the interaction between leukocytes and platelets or leukocytes and vascular endothelium.
  • C-type lectin domain refers to a calcium-dependent carbohydrate-recognition domain that includes about 60 to 150 amino acid residues, preferably about 70 to 140 amino acid residues, and more preferably about 80 to 130 amino acid residues, and contains about 2 to 10, preferably 3 to 8, still more preferably 4, cysteine residues. Typically, the four cysteines are conserved, and involved in two disulfide bonds.
  • a C-type lectin domain includes at least the following consensus sequence: C-[LIVMFATG]-Xaa(nl)-[WLF]-Xaa-[DNSR]-Xaa(n2)-C-Xaa(n3)-[FYWLIVSTA]- [LIVSTAM]-C, wherein C is a cysteine residue, brackets ([]) indicate one of the group of amino acids contained therein, L is a leucine residue, I is an isoleucine residue, V is a valine residue, M is a methionine residue, F is a phenylalanine residue, A is an alanine residue, T is a threonine residue, G is a glycine residue, Xaa is any amino acid, nl is about 1 to 20 amino acid residues, more preferably about 1 to 15 amino acid residues, and more preferably 2 to 12 amino acid residues in length, W is a tryptophan residue, D is an as
  • a TANGO 269 family member includes an C-type lectin domain having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 127 to 215 of SEQ ID NO:3 or 128 to 216 of SEQ ID NO: 13, which are the C-type lectin domains of human and murine TANGO 269, respectively (the C-type lectin domains are also represented as SEQ ID NO:8 and 19).
  • a TANGO 269 family member includes a C-type lectin domain having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 127 to 215 of SEQ ID NO:3 or 128 to 216 of SEQ ID NO:13 (SEQ ID NO:8 and 19), includes 4 conserved cysteine residues, and a C-type lectin domain consensus sequence as described herein.
  • a TANGO 269 family member includes a C-type lectin domain having an amino acid sequence that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 127 to 215 of SEQ ID NO :3 or 128 to 216 of SEQ ID NO: 13 (SEQ ID NO:8 and 19), includes 4 conserved cysteine residues, a C-type lectin domain consensus sequence as described herein, and has at least one TANGO 269 biological activity as described herein.
  • a TANGO 269 family member has the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 13 wherein the 4 conserved cysteine residues are located at amino acid positions 130, 195, 208, and 216 of the human protein (SEQ ID NO:3), and amino acid positions 129, 194, 207, and 215 of the mouse protein (SEQ ID NO: 13), and the C-type lectin domain consensus sequence is located at amino acid residues 127 to 215 of the human protein and amino acids 128 to 216 of the mouse protein.
  • a TANGO 269 family member can also include an extracellular link domain.
  • extracellular link domain refers to a protein domain that includes about 10-40 amino acid residues, preferably about 15-35 amino acid residues, more preferably about 18-30 amino acid residues, and most preferably about 20-28 amino acid residues.
  • an extracellular link domain includes the following consensus sequence: R-Xaa(2)-L-T-Xaa-E-E- Xaa(4)-C-Xaa(4)-A, wherein R is an arginine residue, Xaa is any amino acid, L is a leucine residue, T is a threonine residue, E is a glutamic acid residue, C is a cysteine residue, and A is an alanine residue.
  • a TANGO 269 family member includes an extracellular link domain having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 117 to 136 of SEQ ID NO:3 or 110 to 137 of SEQ ID NO: 13, which are the extracellular link domains of human and murine TANGO 269, respectively (the extracellular link domains are also represented as SEQ ID NO:7 and 17).
  • a TANGO 269 family member includes an extracellular link domain having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 117 to 136 of SEQ ID NO:3 or 110 to 137 of SEQ ID NO:13 (SEQ ID NO:7 and 17), includes one conserved cysteine residue and two conserved glutamic acid residues, and an extracellular link domain consensus sequence as described herein.
  • a TANGO 269 family member includes an extracellular link domain having an amino acid sequence that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 117 to 136 of SEQ ID NO:3 or 110 to 137 of SEQ ID NO: 13 (SEQ ID NO:7 and 17), includes one conserved cysteine residue and two conserved glutamic acid residues, an extracellular link domain consensus sequence as described herein, and has at least one TANGO 269 biological activity as described herein.
  • a TANGO 269 family member has the amino acid sequence of SEQ
  • SEQ ID NO:3 or SEQ ID NO: 13 wherein the conserved cysteine residue is located at amino acid position 129 of the human protein (SEQ ID NO:3), and amino acid position 130 of the mouse protein (SEQ ID NO: 13), the conserved glutamic acid residues are located at amino acid positions 123 and 124 of the human protein (SEQ ID NO:3), and amino acid positions 124 and 125 of the mouse protein (SEQ ID NO: 13), and the extracellular link domain consensus sequence is located at amino acid residues 117 to 136 of the human protein and amino acids 110 to 137 of the mouse protein.
  • a TANGO 298 family member can include a trypsin domain. Trypsin domains are typically found in serine proteases, and can be found in, among other proteins, blood coagulation factors VII, XI, and X, thrombin, plasminogen, tryptases, mast cell proteases, and members of the complement system, which is known for regulation of energy balance and suppression of infectious agents. As used herein, the term "trypsin domain” refers to a protein domain that can be found in serine proteases and other proteins with catalytic activity, and includes about 100-400 amino acid residues, preferably about 150-350 amino acid residues, more preferably about 200- 300 amino acid residues, and most preferably about 225-260 amino acid residues.
  • a trypsin domain has about 2 to 15, preferably about 5 to 10, more preferably about 8 conserved cysteine residues, about 2 to 25, preferably about 5 to 15, more preferably about 13 conserved glycine residues, and about 2 to 15, preferably about 5 to 12, more preferably about 9 conserved valine residues.
  • a trypsin domain also typically has at least one of the following two consensus sequences: [LV]-[ST]-A-A-H-C, wherein L is a leucine residue, V is a valine residue, S is a serine residue, T is a threonine residue, A is an alanine residue, H is a histidine residue, and C is a cysteine residue; and [DG]-[AF]-C-Xaa-[GA]-D-S-G-G-P-L-V-C, wherein D is a aspartic acid residue, G is a glycine residue, F is a phenylalanine residue, Xaa is any amino acid, and P is a proline residue.
  • the histidine residue serves as the active site, to which an aspartic acid residue binds in order to confer upon the domain its catalytic activity.
  • a TANGO 298 family member includes a trypsin domain having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 34 to 258 of SEQ ID NO:27, which is the trypsin domain of human TANGO 298 (the trypsin domain is also represented as SEQ ID NO:31).
  • a TANGO 298 family member includes a trypsin domain having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 34 to 258 of SEQ ID NO:27 (SEQ ID NO:31), includes 8 conserved cysteine residues, 13 conserved glycine residues, and 9 valine residues, and a trypsin domain consensus sequence as described herein.
  • a TANGO 298 family member includes a trypsin domain having an amino acid sequence that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 34 to 258 of SEQ ID NO:27 (SEQ ID NO:31), includes 8 conserved cysteine residues, 13 conserved glycine residues, and 9 valine residues, a trypsin domain consensus sequence as described herein, and has at least one TANGO 298 biological activity as described herein.
  • a TANGO 298 family member has the amino acid sequence of SEQ
  • a cDNA encoding human TANGO 269 was identified by analyzing the sequences of clones present in a human adrenal gland cDNA library.
  • the signal peptide prediction program SIGNALP (Nielsen et al. (1997) Protein Engineering 10:1-6) predicted that human TANGO 269 includes a 47 amino acid signal peptide (amino acid 1 to about amino acid 47 of SEQ ID NO:3)(SEQ ID NO:5) preceding the mature TANGO 269 protein (corresponding to about amino acid 48 to amino acid 228 of SEQ ID NO:3)(SEQ ID NO:4).
  • the TANGO 269 protein molecular weight is 26.6 kDa prior to the cleavage of the signal peptide, 21.4 kDa after cleavage of the signal peptide.
  • An N-glycosylation site having the sequence NRTG is found from amino acids 68 to 71 of SEQ ID NO:3.
  • a second N-glycosylation site having the sequence NLTW is found from amino acids 119 to 122.
  • a third N-glycosylation site having the sequence NATL is found from amino acids 133 to 136.
  • a protein kinase C phosphorylation site having the sequence TRK is found from amino acids 14 to 16.
  • a second protein kinase C phosphorylation site having the sequence SHK is found from amino acids 95 to 97.
  • a casein kinase II phosphorylation site having the sequence SPCD is found from amino acids 99 to 102.
  • a second casein kinase II phosphorylation site having the sequence TWEE is found from amino acids 121 to 124.
  • a third casein kinase II phosphorylation site having the sequence TFCE is found from amino acids 205 to 208.
  • a fourth casein kinase II phosphorylation site having the sequence TKVD is found from amino acids 222 to 225.
  • RYYGDSCY is found from amino acids 106 to 113.
  • An N-myristoylation site having the sequence GMVVGL is found from amino acids 43 to 48.
  • a second N-myristoylation site having the sequence GSVISE is found from amino acids 174 to 179.
  • a third N-myristoylation site having the sequence GNMNCA is found from amino acids 190 to 195.
  • Chromosomal mapping was performed by computerized comparison of human TANGO 269 cDNA sequences against a chromosomal mapping database in order to identify the approximate location of the gene encoding human TANGO 269 protein. This analysis showed that the gene was located on chromosome 12 between markers D12S98 and D12S358.
  • LOX-1 is also mapped to chromosome 12, as seen in Aoyama et al. (1999) Biochem J. 339: 177-184, the contents of which are incorporated herein by reference.
  • Human TANGO 269 includes an extracellular link domain (about amino acids 117 to 136 of SEQ ID NO:3; SEQ ID NO:7) and a C-type lectin domain (about amino acids 127 to 215 of SEQ ID NO:3; SEQ ID NO:8).
  • Figures 3A-3B show a local alignment of the human TANGO 269 full length nucleic acid sequence (SEQ ID NO: 1) with the human LOX-1 full length nucleic acid sequence (SEQ ID NO:21).
  • Figures 4A-4B show an alignment of the human TANGO 269 nucleotide coding region (SEQ ID NO:2) with the human LOX-1 nucleotide coding region (SEQ ID NO:22).
  • Figure 5 shows an alignment of the human TANGO 269 protein sequence (SEQ ID NO:3) with the human LOX-1 protein sequence (SEQ ID NO:23).
  • the human TANGO 269 signal sequence (SEQ ID NO:5) is represented by amino acids 1-47 (and encoded by nucleotides 66 to 206 of SEQ ID NO:l (SEQ ID NO:6)), and the human LOX-1 signal sequence is represented by amino acids 1 to 61 (and encoded by nucleotides 62-244 of SEQ ID NO:21).
  • the human TANGO 269 extracellular link domain sequence (SEQ ID NO: 7) is represented by amino acids 117 to 136 (and encoded by nucleotides 414-473 of SEQ ID NO:l (SEQ ID NO:9)).
  • the human TANGO 269 C-type lectin domain-type domain sequence (SEQ ID NO: 8) is represented by amino acids 127 to 215 (and encoded by nucleotides 444-710 of SEQ ID NO:l (SEQ ID NO: 10)), and the human LOX-1 C-type lectin domain-type domain sequence is represented by amino acids 170 to 264 (and encoded by nucleotides 563 to 857 of SEQ ID NO:21).
  • Figures 3A-3B and Figures 4A-4B show that there is about a 46% identity over 1084 base pairs between the human TANGO 269 nucleic acid molecule and the human LOX-1 nucleic acid molecule, and an overall 44.9% identity between the open reading frame of the human TANGO 269 nucleic acid molecule and the open reading frame of the human LOX-1 nucleic acid molecule, respectively.
  • the amino acid alignment in Figure 5 shows a 23.8% overall amino acid sequence identity between human TANGO 269 and human LOX-1.
  • Human TANGO 269 is homologous to murine TANGO 269.
  • Figures 8A-8B depict an alignment of the open reading frame of murine TANGO 269 (SEQ ID NO: 12) with the open reading frame of human TANGO 269 (SEQ ID NO:2).
  • Figure 9 depicts an alignment of the amino acid sequence of murine TANGO 269 (SEQ ID NO: 13) with the amino acid sequence of human TANGO 269 (SEQ ID NO:3).
  • the human TANGO 269 signal sequence (SEQ ID NO:5) is represented by amino acids 1 to 47 (and encoded by nucleotides 66 to 206 of SEQ ID NO: 1 (SEQ ID NO:6)).
  • the murine TANGO 269 signal sequence (SEQ ID NO: 15) is represented by amino acids 1 to 46 (and encoded by nucleotides 106 to 243 of SEQ ID NO: 11 (SEQ ID NO: 16))
  • the human TANGO 269 extracellular link domain sequence (SEQ ID NO:7) is represented by amino acids 117 to 136 (and encoded by nucleotides 414 to 473 of SEQ ID NO:l (SEQ ID NO:9)
  • the murine TANGO 269 extracellular link domain sequence (SEQ ID NO: 19) is represented by amino acids 110 to 137 (and encoded by nucleotides 433 to 516 of SEQ ID NO: 11 (SEQ ID NO: 19)).
  • the human TANGO 269 C-type lectin domain-type domain sequence (SEQ ID NO:8) is represented by amino acids 127 to 215 (and encoded by nucleotides 444 to 713 of SEQ ID NO:l (SEQ ID NO: 10)), and the murine TANGO 269 C-type lectin domain-type domain sequence is represented by amino acids 128 to 216 (and encoded by nucleotides 487 to 753 of SEQ ID NO: 11 (SEQ ID NO:20)).
  • Figures 8A-8B and 9 show that there is an 74.1% identity between the human and murine TANGO 269 open reading frames, and a 63.0% identity between the TANGO 269 amino acid sequence and the murine TANGO 269 amino acid sequence. There is also about a 57% identity between the full length human TANGO 269 nucleic acid molecule and the full length murine TANGO 269 molecule.
  • cysteine residues are indicated by short vertical lines just below the hydropathy trace.
  • the dashed vertical line separates the signal sequence (amino acids 1 to 47 of SEQ ID NO:3; SEQ ID NO:5) on the left from the mature protein (amino acids 48 to 228 of SEQ ID NO:3; SEQ ID NO:4) on the right.
  • Thicker gray horizontal bars below the dashed horizontal line indicate extracellular ("out"), transmembrane ("TM”), and intracellular (“in”) regions of the molecule.
  • This hydropathy plot indicates that TANGO 269 is a type II transmembrane protein.
  • Northern analysis of human TANGO 269 expression in human tissues showed that an approximately 1.0 kB transcript is highly expressed in human fetal liver and is weakly expressed in bone marrow and peripheral blood leukocytes. There was no expression seen in spleen, lymph node, and thymus.
  • a second Northern analysis of different tissues showed an approximately 1.0 kB transcript highly expressed in adult liver and no expression in heart, brain, placenta, lung, skeletal muscle, kidney, and pancreas.
  • a cDNA encoding murine TANGO 269 was identified by analyzing the sequences of clones present in a mouse megakaryocyte cDNA library. This analysis led to the identification of a clone, jtmea040e07, encoding full-length murine TANGO 269.
  • the murine TANGO 269 cDNA of this clone is 932 nucleotides long ( Figure 6; SEQ ID NO:l 1).
  • the signal peptide prediction program SIGNALP Nielsen et al. (1997) Protein
  • murine TANGO 269 includes a 46 amino acid signal peptide (amino acid 1 to about amino acid 46 of SEQ ID NO:13)(SEQ ID NO: 15) preceding the mature murine TANGO 269 protein (corresponding to about amino acid 47 to amino acid 229 of SEQ ID NO: 13)(SEQ ID NO: 14).
  • the TANGO 269 protein molecular weight is 26.1 kDa prior to the cleavage of the signal peptide, 21.2 kDa after cleavage of the signal peptide.
  • N-glycosylation site having the sequence NLSA is found from amino acids 67 to 70 of SEQ ID NO: 13.
  • a second N-glycosylation site having the sequence NLTW is found from amino acids 120 to 123.
  • a third N-glycosylation site having the sequence NATL is found from amino acids 134 to 137.
  • a fourth N-glycosylation site having the sequence NLSG is found from amino acids 184 to 187.
  • a protein kinase C phosphorylation site having the sequence SVR is found from amino acids 155 to 157.
  • a second protein kinase C phosphorylation site having the sequence SKK is found from amino acids 166 to 168.
  • a third protein kinase C phosphorylation site having the sequence SCK is found from amino acids 207 to 209.
  • a casein kinase II phosphorylation site having the sequence SSAE is found from amino acids 20 to 23.
  • a second casein kinase II phosphorylation site having the sequence SCQE is found from amino acids 79 to 82.
  • a third casein kinase II phosphorylation site having the sequence STFE is found from amino acids 93 to 96.
  • a fourth casein kinase II phosphorylation site having the sequence TWEE is found from amino acids 122 to 125.
  • a fifth casein kinase II phosphorylation site having the sequence SLTD is found from amino acids 144 to 147.
  • a sixth casein kinase II phosphorylation site having the sequence SKKD is found from amino acids 166 to 169.
  • a seventh casein kinase II phosphorylation site having the sequence SCKE is found from amino acids 207 to 210.
  • An eighth casein kinase II phosphorylation site having the sequence TRVD is found from amino acids 223 to 226.
  • a tyrosine kinase phosphorylation site having the sequence RYHGDSCY is found from amino acids 107 to 114.
  • An N-myristoylation site having the sequence GLWGL is found from amino acids 42 to 47.
  • a second N-myristoylation site having the sequence GINLSG is found from amino acids 182 to 187.
  • Murine TANGO 269 includes an extracellular link domain (about amino acids 110 to 137 of SEQ ID NO:13; SEQ ID NO: 17) and a C-type lectin domain (about amino acids 128 to 216 of SEQ ID NO:13; SEQ ID NO:18).
  • Figure 7 depicts a hydropathy plot of murine TANGO 269. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace.
  • the dashed vertical line separates the signal sequence (amino acids 1 to 46 of SEQ ID NO:13; SEQ ID NO:15) on the left from the mature protein (amino acids 47 to 229 of SEQ ID NO:13; SEQ ID NO:14) on the right. Thicker gray horizontal bars below the dashed horizontal line indicate extracellular ("out"), transmembrane ("TM”), and intracellular (“in”) regions of the molecule.
  • This hydropathy plot indicates that TANGO 269 is a type II transmembrane protein.
  • TANGO 269 expression in murine tissues showed that an approximately 1.0 kB transcript is highly expressed in liver, moderately expressed in spleen, and weakly expressed in heart. There was no expression seen in brain, lung, skeletal muscle, kidney, and testis.
  • a murine in situ expression analysis of TANGO 269 revealed expression only in the developing fetal liver, with a multifocal signal pattern suggestive of a scattered cell population. No signal was detected in adult liver or spleen. The signal pattern is suggestive of megakaryocyte expression.
  • Gata-1 is a transcription factor involved in the development of hematopoietic cell lineages: gata-1 expression is required for proper development of erythrocytes and megakaryocytes.
  • deletion of the gata-1 gene is lethal at the embryonic stage due to a failure to form red blood cells
  • deletion of only the element of the gata-1 gene responsible for megakaryocyte-specific expression is not lethal and results in a reduction in gata-1 expression in the megakaryocyte without affecting gata-1 expression in red blood cells.
  • the megakaryocytes of mice with this element of the gata-1 gene knocked out fail to develop mature platelets, and the mice experience abnormal bleeding due to their profound thrombocytopenia.
  • TANGO 269 was found to be present in the wild type animals but its expression was greatly reduced when the megakaryocyte-specific element was knocked out.
  • TANGO 269 has been found to be upregulated in megakaryocytes and downregulated in immature megakaryocytes. Uses of TANGO 269 Nucleic acids, Polypeptides, and Modulators Thereof
  • TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat adrenal disorders, such as hypoadrenalism (e.g., primary chronic or acute adrenocortical insufficiency, and secondary adrenocortical insufficiency), hyperadrenalism (Cushing's syndrome, primary hyperaldosteronism, adrenal virilism, and adrenal hyperplasia), or neoplasia (e.g., adrenal adenoma and cortical carcinoma).
  • hypoadrenalism e.g., primary chronic or acute adrenocortical insufficiency, and secondary adrenocortical insufficiency
  • hyperadrenalism Cushing's syndrome, primary hyperaldosteronism, adrenal virilism, and adrenal hyperplasia
  • neoplasia e.g., adrenal adenoma and cortical carcinoma
  • TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat hepatic (liver) disorders, such as jaundice, hepatic failure, hereditary hyperbiliruinemias (e.g., Gilbert's syndrome, Crigler-Naijar syndromes and Dubin- Johnson and Rotor's syndromes), hepatic circulatory disorders (e.g., hepatic vein thrombosis and portal vein obstruction and thrombosis), hepatitis (e.g., chronic active hepatitis, acute viral hepatitis, and toxic and drug-induced hepatitis), cirrhosis (e.g., alcoholic cirrhosis, biliary cirrhosis, and hemochromatosis), and malignant tumors (e.g., primary carcinoma, hepatoblastoma, and angiosar
  • TANGO 269 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, differentiation, and/or function of cells that appear in the bloodstream and in the bone marrow, e.g., stem cells (e.g., hematopoietic stem cells), and blood cells, e.g., erythrocytes, platelets, and leukocytes.
  • stem cells e.g., hematopoietic stem cells
  • blood cells e.g., erythrocytes, platelets, and leukocytes.
  • TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat bone marrow, blood, and hematopoietic associated diseases and disorders, e.g., acute myeloid leukemia, hemophilia, leukemia, anemia (e.g., sickle cell anemia), and thalassemia.
  • diseases and disorders e.g., acute myeloid leukemia, hemophilia, leukemia, anemia (e.g., sickle cell anemia), and thalassemia.
  • TANGO 269 nucleic acids, proteins, and modulators thereof can be used to can be used to modulate the proliferation, differentiation, and/or function of cells that form the spleen, e.g., cells of the splenic connective tissue, e.g., splenic smooth muscle cells and/or endothelial cells of the splenic blood vessels.
  • TANGO 269 nucleic acids, proteins, and modulators thereof can also be used to modulate the proliferation, differentiation, and/or function of cells that are processed, e.g., regenerated or phagocytized within the spleen, e.g., erythrocytes and/or B and T lymphocytes and macrophages.
  • TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat spleen, e.g., the fetal spleen, associated diseases and disorders.
  • splenic diseases and disorders include e.g., splenic lymphoma and/or splenomegaly, phagocytotic disorders, e.g., those inhibiting macrophage engulfment of bacteria and viruses in the bloodstream, and/or diseases and disorders associated with cells processed in the spleen, e.g., erythrocytes (e.g., leukemia and hemophilia).
  • erythrocytes e.g., leukemia and hemophilia
  • TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat cardiovascular disorders, such as ischemic heart disease (e.g., angina pectoris, myocardial infarction, and chronic ischemic heart disease), hypertensive heart disease, pulmonary heart disease, valvular heart disease (e.g., rheumatic fever and rheumatic heart disease, endocarditis, mitral valve prolapse, and aortic valve stenosis), congenital heart disease (e.g., valvular and vascular obstructive lesions, atrial or ventricular septal defect, and patent ductus arteriosus), and myocardial disease (e.g., myocarditis, congestive cardiomyopathy, and hypertrophic cariomyopathy).
  • ischemic heart disease e.g., angina pectoris, myocardial infarction, and chronic ischemic heart disease
  • hypertensive heart disease e.g., pulmonary heart disease
  • TANGO 269 nucleic acids, proteins, and modulators thereof can play a role in treating disorders in which LOX-1 plays a role, some of which are described in the following references: Sawamura et al. (1997) Nature 386:73-77; Kataoka et al. (1999) Circulation 99:3110-3117; and Kita (1999) Circulation Research 84:1113-1115, the contents of all of which are incorporated herein by reference.
  • TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat atherosclerosis, e.g., by binding Ox-LDL (oxidatively modified low density lipoprotein) and its lipid constituents, thus preventing lipid deposition and intimal thickening in the arteries, and thus induction of endothelial expression of leukocyte adhesion molecules and smooth-muscle growth factors (both which are implicated in atherogenesis).
  • Ox-LDL oxidatively modified low density lipoprotein
  • TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat immune related diseases and disorders, as LOX-1 is implicated in inflammation, and as LOX-1 has highest homology with the NKR-P1 family of proteins, which are involved in target-cell recognition and natural killer cell activation.
  • Such immune disorders include, e.g., autoimmune disorders (e.g., arthritis, graft rejection (e.g., allograft rejection), and T cell autoimmune disorders (e.g., AIDS)) and inflammatory disorders (e.g., bacterial infection, psoriasis, septicemia, cerebral malaria, inflammatory bowel disease, multiple sclerosis, arthritis (e.g., rheumatoid arthritis, osteoarthritis), and allergic inflammatory disorders (e.g., asthma, psoriasis)).
  • autoimmune disorders e.g., arthritis, graft rejection (e.g., allograft rejection)
  • T cell autoimmune disorders e.g., AIDS
  • inflammatory disorders e.g., bacterial infection, psoriasis, septicemia, cerebral malaria, inflammatory bowel disease, multiple sclerosis, arthritis (e.g., rheumatoid arthritis, osteoarthritis), and allergic inflammatory disorders (e
  • TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat TNF- related disorders, as LOX-1 expression is induced by tumor necrosis factor- ⁇ .
  • TNF-related disorders include, e.g., acute myocarditis, myocardial infarction, congestive heart failure, T cell disorders (e.g., dermatitis, fibrosis)), differentiative and apoptotic disorders, and disorders related to angiogenesis (e.g., tumor formation and/or metastasis).
  • TANGO 269 can also can be used to treat hypertension and congestive heart failure.
  • TANGO 269 and LOX-1 have C-type lectin domains, and are similar in that respect to the selectins, which are implicated in cell-cell recognition (including endothelial- leukocyte adhesion), TANGO 269 nucleic acids, proteins, and modulators thereof, and LOX-1, can be used to treat cell adhesion and cell migration/motility related disorders.
  • disorders associated with adhesion and migration of cells include, e.g., disorders associated with adhesion and migration of cells, e.g., platelet aggregation disorders (e.g., Glanzmann's thromboasthemia, which is a bleeding disorders characterized by failure of platelet aggregation in response to cell stimuli), inflammatory disorders (e.g., leukocyte adhesion deficiency, which is a disorder associated with impaired migration of neutrophils to sites of extravascular inflammation), disorders associated with abnormal tissue migration during embryo development, and tumor metastasis.
  • platelet aggregation disorders e.g., Glanzmann's thromboasthemia, which is a bleeding disorders characterized by failure of platelet aggregation in response to cell stimuli
  • inflammatory disorders e.g., leukocyte adhesion deficiency, which is a disorder associated with impaired migration of neutrophils to sites of extravascular inflammation
  • disorders associated with abnormal tissue migration during embryo development e.g., tumor
  • TANGO 269 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, morphology, migration, differentiation, and/or function of megakaryocytes and platelets, including during development, e.g., embryogenesis.
  • TANGO 269 nucleic acids, proteins, and modulators thereof can also be used to modulate leukocyte-platelet and platelet-endothelium interactions in inflammation and/or thrombosis.
  • TANGO 269 nucleic acids, proteins, and modulators thereof can be used to modulate platelet aggregation and degranulation.
  • TANGO 269 nucleic acids, proteins, and modulators thereof can also be used to modulate disorders associated with abnormal or aberrant megakaryocyte and/or platelet proliferation, differentiation, morphology, migration, aggregation, degranulation and/or function. Examples of these disorders include, but are not limited to, bleeding disorders (e.g., bleeding tendency and/or prolonged bleeding time) such as thrombocytopenia (e.g., idiopathic thrombocytopenic purpura (ITP) or immune thrombocytopenia or thrombocytopenia induced by chemotherapy or radiation therapy).
  • TANGO 269 nucleic acids, proteins, and modulators thereof can also be used to modulate thrombotic or hemorrhagic disorders and diseases exhibiting quantitative or qualitative platelet dysfunction.
  • TANGO 269 nucleic acids, proteins, and modulators thereof can be used to modulate symptoms associated with platelet disorders and/or diseases (e.g., bleeding disorders, e.g., purpura and severe bleeding problems).
  • bleeding disorders e.g., purpura and severe bleeding problems.
  • platelet adhesion and aggregation play an important role in acute coronary diseases
  • TANGO 269 nucleic acids, proteins and modulators thereof can be used to modulate coronary diseases (e.g., cardiovascular diseases including unstable angina, acute myocardial infarction, coronary artery disease, coronary revascularization, ventricular thromboembolism, atherosclerosis, coronary artery disease, plaque formation).
  • coronary diseases e.g., cardiovascular diseases including unstable angina, acute myocardial infarction, coronary artery disease, coronary revascularization, ventricular thromboembolism, atherosclerosis, coronary artery disease, plaque formation.
  • coronary diseases e.g., cardiovascular diseases including unstable angina, acute myocardial infarction, coronary artery disease, coronary revascularization, ventricular thromboembolism, atherosclerosis, coronary artery disease, plaque formation.
  • TANGO 269 expression is greatly reduced in megakaryocytes obtained from gata-1 knockout mice compared to normal mice
  • TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat disease and/or disorders associated with gata-1 dysfunction, e.g., bone marrow, blood, and hematopoietic associated diseases and disorders discussed herein.
  • a cDNA encoding human TANGO 298 was identified by analyzing the sequences of clones present in a normal human bone marrow cDNA library. This analysis led to the identification of a clone, jyhMal 18f02, encoding full-length human TANGO 298.
  • the human TANGO 298 cDNA of this clone is 1078 nucleotides long ( Figure 10; SEQ ID NO:25).
  • the signal peptide prediction program SIGNALP Nielsen et al. (1997) Protein
  • human TANGO 298 includes a 31 amino acid signal peptide (amino acid 1 to about amino acid 31 of SEQ ID NO:25)(SEQ ID NO:29) preceding the mature TANGO 298 protein (corresponding to about amino acid 32 to amino acid 283 of SEQ ID NO:25)(SEQ ID NO:28).
  • the TANGO 298 protein molecular weight is 30.3 kDa prior to the cleavage of the signal peptide, 27.2 kDa after cleavage of the signal peptide.
  • N-glycosylation site having the sequence NGSA is found from amino acids 129 to 132 of SEQ ID NO:27.
  • a second N-glycosylation site having the sequence NSSW is found from amino acids 189 to 192.
  • a cAMP and cGMP dependent protein kinase phosphorylation site having the sequence RRSS is found from amino acids 263 to 266.
  • a protein kinase C phosphorylation site having the sequence SVR is found from amino acids 50 to 52.
  • a second protein kinase C phosphorylation site having the sequence SHR is found from amino acids 77 to 79.
  • a third protein kinase C phosphorylation site having the sequence SWK is found from amino acids 191 to 193.
  • a fourth protein kinase C phosphorylation site having the sequence SHR is found from amino acids 208 to 210.
  • a fifth protein kinase C phosphorylation site having the sequence TTR is found from amino acids 275 to 277.
  • a casein kinase II phosphorylation site having the sequence SHRD is found from amino acids 77 to 80.
  • a second casein kinase II phosphorylation site having the sequence STAE is found from amino acids 94 to 97.
  • a third casein kinase II phosphorylation site having the sequence THPD is found from amino acids 110 to 113.
  • a fourth casein kinase II phosphorylation site having the sequence SDFE is found from amino acids 166 to 169.
  • N-myristoylation site having the sequence GSWGAQ is found from amino acids 28 to 33.
  • a second N-myristoylation site having the sequence GQHHCG is found from amino acids 55 to 60.
  • a third N-myristoylation site having the sequence GLMEAK is found from amino acids 174 to 179.
  • a fourth N-myristoylation site having the sequence GLWCGD is found from amino acids 236 to 241.
  • An amidation site having the sequence PGRR is found from amino acids 144 to 147.
  • a serine protease, trypsin family, histidine active site having the sequence VSAAHC is found from amino acids 70 to 75.
  • Chromosomal mapping was performed by computerized comparison of TANGO 298 cDNA sequences against a chromosomal mapping database in order to identify the approximate location of the gene encoding human TANGO 298 protein.
  • Homology to EST HUMGS00642 (accession number D19687) showed that the TANGO 298 gene maps to chromosome 19, between markers D19S886 and D19S216.
  • the region between D19S886 and D19S216 lies 19pl3.3, the area which is the location for cyclic hematopoiesis (also called cyclic neutropenia).
  • a serine protease gene cluster maps to this region as well, further suggesting that this is the area to which TANGO 298 maps.
  • TANGO 298 The homology between TANGO 298 and EST HUMGS00642 (accession number D19687) appears in Figure 17.
  • the nucleotide sequences of HUMGS00642 and human TANGO 298 are about 93% identical over a 278 base pair overlap, from base pairs 801-1078 of human TANGO 298. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
  • TANGO 298 includes a trypsin domain (about amino acids 34 to 258 of SEQ ID NO:26; SEQ ID NO:30).
  • Figures 12A-12B show an alignment of the human TANGO 298 full length nucleic acid sequence (SEQ ID NO:24) with the human adipsin full length nucleic acid sequence (SEQ ID NO:32).
  • Figures 13A-13B show an alignment of the human TANGO 298 nucleotide coding region (SEQ ID NO:25) with the human adipsin nucleotide coding region (SEQ ID NO:33).
  • Figure 14 shows an alignment of the human TANGO 298 protein sequence (SEQ ID NO:26) with the human adipsin protein sequence (SEQ ID NO:34).
  • the human TANGO 298 signal sequence (SEQ ID NO:28) is represented by amino acids 1 to 31 (and encoded by nucleotides 31 to 123 of SEQ ID NO:26 (SEQ ID NO:29)).
  • the human TANGO 298 trypsin domain sequence (SEQ ID NO:30) is represented by amino acids 34 to 258 (and encoded by nucleotides 130 to 804 of SEQ ID NO:26 (SEQ ID NO:31)
  • the human adipsin trypsin domain sequence is represented by amino acids 1 to 223 (and encoded by nucleotides 55 to 723 of SEQ ID NO:32).
  • Figures 12A-12B and Figures 13A-13B show that there is a 51.8% identity between the human TANGO 298 full length nucleic acid molecule and the human adipsin full length nucleic acid molecule, and an overall 50.8% identity between the open reading frame of the human TANGO 298 nucleic acid molecule and the open reading frame of the human adipsin nucleic acid molecule, respectively.
  • the amino acid alignment in Figure 14 shows a 34.0% overall amino acid sequence identity between human TANGO 298 and human adipsin.
  • Clone EpT298, which encodes human TANGO 298, was deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110-2209) on April 20, 1999 and assigned Accession Number 207216. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. ⁇ 112.
  • Figure 11 depicts a hydropathy plot of human TANGO 298. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) and potential N- glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace.
  • the dashed vertical line separates the signal sequence (amino acids 1 to 31 of SEQ ID NO:27; SEQ ID NO:29) on the left from the mature protein (amino acids 32 to 283 of SEQ ID NO:27; SEQ ID NO:28) on the right. Thicker gray horizontal bars below the dashed horizontal line indicate extracellular (“out”), transmembrane ("TM”), and intracellular (“in”) regions of the molecule.
  • TaqMan analysis (described below in the examples) of human TANGO 298 expression in human tissues and cell lines showed moderate levels of expression in fetal liver, K562 (an erythroleukemia), and CD34 " cells from mobilized bone marrow (MBM CD34 " ), and extremely high levels of expression in CD34 + cells from mobilized peripheral blood (mPB CD34 + ), adult resting bone marrow (ABM CD34 + ), G-CSF mobilized bone marrow (mBM CD34 + ), and G-CSF mobilized peripheral blood leukocytes (mPB leukocytes). Low levels of expression were seen in CD34 " cells from mobilized bone marrow (MBM CD34 ), resting CD19 + B cells, resting peripheral blood mononuclear cells (PBMC), and granulocytes.
  • MBM CD34 + mobilized peripheral blood leukocytes
  • TaqMan analysis of human TANGO 298 expression also reveals highest expression in the erythroblast samples, the megakaryocyte samples, the neutrophil samples, and in cells populations positive for CD34 + marker (including cells from mobilized bone marrow, adult resting bone marrow, umbilical cord blood, and fetal liver).
  • the samples from each group manifest higher levels of TANGO 298 expression in the earlier stages of development, and expression tapers off after a certain point in time. For example, in the erythroblast cell samples tested, levels of TANGO 298 expression increase in proportion to the time the cells were cultured, from 24 hours to 48 hours, then decrease with culture times of 6 days, 10 days, and 12 days.
  • TANGO 298 Due to TANGO 298 's homology to adipsin (also known as complement factor D), as evidenced by similar domains and expression patterns seen between the two molecules, TANGO 298 nucleic acids, proteins, and modulators thereof can be used to treat disorders in which adipsin plays a role, some of which are described in the following references: White et al. (1992) JBiol Chem. 13:9210-9213; Spiegelman et al. (1989) J Biol Chem. 264:1811-1815; Yamauchi et al. (1994) JJmmunol. 152:3645-3653; Meri et al. (1998) Vox Sang.
  • TANGO 298 Like complement factor D's role in the alternative complement pathway, TANGO 298 also has utility in the same pathway.
  • disorders known as complement regulator deficiencies include, e.g., hereditary angioedema (an allergic disorder) and proxysmal nocturnal hemoglobinuria (the presence of hemoglobin in the urine), which TANGO 298 nucleic acids, proteins, and modulators thereof can be used to treat.
  • TANGO 298 nucleic acids, proteins, and modulators thereof play a role in the modulation, e.g., treatment and regulation, of obesity, as adipsin is synthesized in adipose tissue and secreted into the bloodstream, is highly expressed in fat, and is also observed in many common models of obesity. Adipsin levels are increased during appetite suppression, and are lowered in obese mice, including those genetically obese due to the db/db and ob/ob genes, and those in a hyperglycemic, hyperinsulinemic state. For these reasons, TANGO 298 nucleic acids, proteins, and modulators thereof can play also a role in regulation of systemic energy balance and in the treatment of diabetes.
  • TANGO 298 nucleic acids, proteins, and modulators thereof can be used to treat immune related diseases and disorders.
  • Such disorders include, e.g., autoimmune disorders (e.g., arthritis, graft rejection (e.g., allograft rejection), and T cell autoimmune disorders (e.g., AIDS)) and inflammatory disorders (e.g., bacterial infection, psoriasis, septicemia, cerebral malaria, inflammatory bowel disease, multiple sclerosis, arthritis (e.g., rheumatoid arthritis, osteoarthritis), and allergic inflammatory disorders (e.g., asthma, psoriasis)).
  • autoimmune disorders e.g., arthritis, graft rejection (e.g., allograft rejection)
  • T cell autoimmune disorders e.g., AIDS
  • inflammatory disorders e.g., bacterial infection, psoriasis, septicemia, cerebral malaria, inflammatory bowel disease, multiple sclerosis, arthritis (e.g., rheumatoid arthritis, osteoarthritis), and allergic inflammatory disorders (e.
  • adipsin is a serine protease and as both adipsin and TANGO 298 share a trypsin domain commonly seen in serine proteases, TANGO 298 nucleic acids, proteins, and modulators thereof can be used to treat disorders involving abnormal serine protease function.
  • serine protease inhibitors are abundant in plaques found in Alzheimer's patients, and may be responsible for preventing some types of metalloproteinase from breaking down the beta-amyloid proteins that make up these plaques.
  • modulation of the serine protease activity may modulate formation of Alzheimer's plaques, and consequently, TANGO 298 nucleic acids, proteins, and modulators thereof can be used to treat Alzheimer's disease.
  • Adipsin has also been seen in high levels in a patient with Fanconi anemia (FA), and thus TANGO 298 nucleic acids, proteins, and modulators thereof can be used to treat such patients (persons with FA experience bone marrow failure, suffer severe life-threatening aplastic anemia, and possess blood systems that cannot successfully combat infection, fatigue or spontaneous hemorrhage or bleeding).
  • TANGO 298 is found in an bone marrow library, is upregulated in megakaryocytes, and is expressed in cells such as neutrophils, erythroids, megakaryocytes, cord blood cells, and fetal liver
  • TANGO 298 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, morphology, migration, differentiation, and/or function of hematopoietic cells, e.g., hematopoietic stem cells and blood cells, and can play a role in a variety of hematological disorders.
  • Hematological disorders include, but are not limited to, disorders associated with abnormal differentiation or hematopoiesis, morphology, migration, proliferation, or function of blood cells derived, for example, from myeloid multipotential cells in bone marrow, such as megakaryocytes (and ultimately platelets), monocytes, erythroids, and granulocytes (e.g., neutrophils, eosinophils, and basophils), and from lymphoid multipotential cells, such as T and B lymphocytes.
  • myeloid multipotential cells in bone marrow such as megakaryocytes (and ultimately platelets), monocytes, erythroids, and granulocytes (e.g., neutrophils, eosinophils, and basophils)
  • lymphoid multipotential cells such as T and B lymphocytes.
  • TANGO 298 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, morphology, migration, differentiation, and/or function of megakaryocytes and/or platelets, and can play a role in a variety of platelet associated disorders.
  • Platelet associated disorders include, but are not limited to, bleeding disorders, e.g., hemophilia (e.g., hemophilia A), thrombocytopenia (e.g., thrombocytopenia due to a reduced number of megakaryocytes in the bone marrow, for example, as a result of chemotherapy or radiation therapy); invasive disorders, such as leukemia, idiopathic or drug- or toxin-induced aplasia of the marrow, or rare hereditary amegakaryocytic thrombocytopenias; ineffective thrombopoiesis, for example, as a result of megaloblastic anemia, alcohol toxicity, vitamin B 12 or folate deficiency, myelodysplastic disorders, or rare hereditary disorders (e.g., Wiskott-Aldrich syndrome and May-hegglin anomaly); a reduction in platelet distribution, for example, as a result of cirrhosis, a splenic invasive disease
  • thrombocytopenia secondary to intravascular clotting and thrombin induced damage to platelets as a result of, for example, obstetric complications, metastatic tumors, severe gram-negative bacteremia, thrombotic thrombocytopenic purpura, or severe illness. Also included is dilutional thrombocytopenia, for example, due to massive hemorrhage.
  • Platelet associated disorders also include, but are not limited to, essential thrombocytosis and thrombocytosis associated with, for example, splenectomy, acute or chronic inflammatory diseases, anemia (e.g., hemolytic anemia), carcinoma, Hodgkin's disease, lymphoproliferative disorders, and malignant lymphomas.
  • anemia e.g., hemolytic anemia
  • carcinoma e.g., Hodgkin's disease
  • lymphoproliferative disorders e.g., lymphoproliferative disorders
  • malignant lymphomas e.g., malignant lymphomas.
  • TANGO 298 nucleic acids, proteins and modulators thereof can be used to modulate coronary diseases (e.g. , cardiovascular diseases including unstable angina, acute myocardial infarction, coronary artery disease, coronary revascularization, ventricular thromboembolism, atherosclerosis, coronary artery disease, plaque formation).
  • coronary diseases e.g. , cardiovascular diseases including unstable angina, acute myocardial infarction, coronary artery disease, coronary revascularization, ventricular thromboembolism, atherosclerosis, coronary artery disease, plaque formation.
  • TANGO 298 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, mo ⁇ hology, migration, differentiation, and/or function of erythrocytes, and can play a role in a variety of erythrocyte associated disorders.
  • Erythrocyte associated disorders include anemias such as, for example, hemolytic anemias due to hereditary cell membrane abnormalities, such as hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis; hemolytic anemias due to acquired cell membrane defects, such as paroxysmal nocturnal hemoglobinuria and spur cell anemia; hemolytic anemias caused by antibody reactions, for example to the RBC antigens, or antigens of the ABO system, Lewis system, Ii system, Rh system, Kidd system, Duffy system, and Kell system; methemoglobinemia; a failure of erythropoiesis, for example, as a result of aplastic anemia, pure red cell aplasia, myelodysplastic syndromes, sideroblastic anemias, and congenital dyserythropoietic anemia; secondary anemia in nonhematolic disorders, for example, as a result of chemotherapy, alcohol
  • erythrocyte associated disorders include polycythemias such as, for example, polycythemia vera, secondary polycythemia, and relative polycythemia.
  • TANGO 298 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, mo ⁇ hology, migration, differentiation, and/or function of neutrophils, and can play a role in a variety of neutrophil associated disorders.
  • Neutrophil associated disorders include neutropenias that result from or accompany a number of conditions, including, but not limited to, chemotherapy; neutropenias (e.g., chronic idopathic neutropenia and cyclic neutropenia and its accompanying symptoms, including fever, malaise, mucosal ulcers, and life-threatening infections); Felty's syndrome; acute infectious disease; lymphoma or aleukemic lymphocytic leukemia; myelodysplastic syndrome; and rheumatic diseases such as systemic lupus erythematosus, rheumatoid arthritis, and polymyositis. Also included is neutrophilia, for example, accompanying chronic myelogenous leukemia.
  • neutropenias e.g., chronic idopathic neutropenia and cyclic neutropenia and its accompanying symptoms, including fever, malaise, mucosal ulcers, and life-threatening infections
  • Felty's syndrome acute infectious disease
  • TANGO 298 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, mo ⁇ hology, migration, differentiation, and/or function of monocytes and/or macrophages, and can play a role in a variety of monocyte and/or macrophage associated disorders.
  • Monocyte associated disorders include disorders associated with abnormal monocyte and/or macrophage function, such as impaired phagocytosis, chemotaxis, or secretion of cytokines, growth factors and acute-phase reactants, resulting from certain diseases, e.g., lysosomal storage diseases (e.g., Gaucher's disease); impaired monocyte cytokine production, for example, found in some patients with disseminated nontuberculous mycobacterial infection who are not infected with HIV; leukocyte adhesion deficiency (LAD), hyperimmunoglobulin E- recurrent infection (HIE) or Job's syndrome, Chediak-Higashi syndrome (CHS), and chronic granulomatous diseases (CGD), certain autoimmune diseases, such as systemic lupus erythematosus and other autoimmune diseases characterized by tissue deposition of immune complexes, as seen in Sj ⁇ gren's syndrome, mixed cryoglobulinemia, dermatitis he ⁇ eti
  • Monocyte associated disorders also include monocytoses such as, for example, monocytoses associated with certain infections such as tuberculosis, brucellosis, subacute bacterial endocarditis, Rocky Mountain spotted fever, malaria, and visceral leishmaniasis (kala azar), in malignancies, leukemias (e.g., acute myeloid leukemia), myeloproliferative syndromes, hemolytic anemias, chronic idiopathic neutropenias, and granulomatous diseases such as sarcoidosis, regional enteritis, and some collagen vascular diseases.
  • monocytoses such as, for example, monocytoses associated with certain infections such as tuberculosis, brucellosis, subacute bacterial endocarditis, Rocky Mountain spotted fever, malaria, and visceral leishmaniasis (kala azar), in malignancies, leukemias (e.g., acute myeloid leukemia), myeloproliferative syndromes,
  • monocyte associated disorders include monocytopenias such as, for example, monocytopenias that can occur with acute infections, with stress, following administration of glucocorticoids, aplastic anemia, hairy cell leukemia, and acute myelogenous leukemia and as a direct result of administration of myelotoxic and immunosuppressive drugs.
  • TANGO 298 is a protease highly expressed in a rare but very mobile population
  • TANGO 298 nucleic acids, proteins and modulators thereof can be used to treat, e.g., inhibit, tumor cell travel, e.g., in metastasis in tumors.
  • TANGO 298 is highly expressed in CD34 + bone marrow cells and upregulated in Mobilized Peripheral Blood (MPB)
  • MPB Mobilized Peripheral Blood
  • TANGO 298 nucleic acids, proteins and modulators thereof can modulate stem cell engraftment, homing, and or mobilization, e.g., in bone marrow transplantation, stem cell mobilization to harvest grafts, and/or stem cell recovery post chemotherapy.
  • Tables 1 and 2 below provide a summary of the sequence information for TANGO 269 and TANGO 298.
  • nucleic acid molecules that encode a polypeptide of the invention or a biologically active portion thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • an “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • an “isolated” nucleic acid molecule is free of sequences (preferably protein encoding sequences) which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the term "isolated" when referring to a nucleic acid molecule does not include an isolated chromosome.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, 2, 11, 12, 24, or 25, or a complement thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequences of SEQ ID NO:l, 2, 11, 12, 24, or 25 as a hybridization probe, nucleic acid molecules of the invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • a nucleic acid molecule of the invention can be amplified using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to all or a portion of a nucleic acid molecule of the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence of SEQ ID NO: 1, 2, 11, 12, 24, or 25, or a portion thereof.
  • a nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence thereby forming a stable duplex.
  • a nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence encoding a full length polypeptide of the invention for example, a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a polypeptide of the invention.
  • the nucleotide sequence determined from the cloning one gene allows for the generation of probes and primers designed for use in identifying and/or cloning homologues in other cell types, e.g., from other tissues, as well as homologues from other mammals.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides of the sense or anti-sense sequence of SEQ ID NO: 1, 2, 11, 12, 24, or 25 or of a naturally occurring mutant of SEQ ID NO:l, 2, 11, 12, 24, or 25.
  • the TANGO 269 nucleic acid molecule described herein does not include or contain, is free of, and/or is not adjacent to or flanked by an EcoRI restriction endonuclease site (GAATTC) and/or a Pad restriction endonuclease site (TTAATTAA).
  • GATTC EcoRI restriction endonuclease site
  • TTAATTAA Pad restriction endonuclease site
  • the TANGO 269 nucleic acid molecule described herein does not include or contain, is free of, and/or is not adjacent to, 10, 15, 25, 50, 60, 70, 80, 90, 100, or 150 bases which flank the nucleotide insert of the vector designated as IMAGE clone 212698.
  • TANGO 269 is in a vector containing at least one regulatory sequence which allows for transcription of the TANGO 269 nucleic acid molecule, e.g., a promoter.
  • TANGO 269 nucleic acid molecule is in a form suitable for expression of the TANGO 269 nucleic acid molecule, e.g., is associated with (e.g., is adjacent to) nucleotide sequences which allow for expression of the TANGO 269 nucleic acid molecule.
  • the TANGO 269 nucleic acid molecule described herein does not include or contain, is free of, and/or is not adjacent to 25, 30, 40, 50, 75, or 100 bases of the modified version of the pT7T3D vector sold by Pharmacia, New Jersey (modification described at http://www.ncbi.nlm.nih.gov/irx/cgi-bin/birx_doc?dbest+33286 or http://www.ncbi.nlm.nih.gov/irx/cgi-bin/birx_doc?dbest+33175, the contents of which are inco ⁇ orated herein by reference).
  • Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts or genomic sequences encoding the same protein molecule encoded by a selected nucleic acid molecule.
  • the probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which mis-express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted.
  • a nucleic acid fragment encoding a "biologically active portion" of a polypeptide of the invention can be prepared by isolating a portion of any of SEQ ID NO:2, 12, or 25, expressing the encoded portion of the polypeptide protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the polypeptide.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of SEQ ID NO:l, 2, 11, 12, 24, or 25, due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequence of SEQ ID NO:2, 12, or 25.
  • DNA sequence polymo ⁇ hisms that lead to changes in the amino acid sequence may exist within a population (e.g., the human population). Such genetic polymo ⁇ hisms may exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes which occur alternatively at a given genetic locus.
  • TANGO 269 has been mapped to chromosome 12, between markers D12S98 and D12S358, and therefore TANGO 269 family members include nucleotide sequences which include polymo ⁇ hisms (i.e., nucleotide sequences which vary from the nucleotide sequence of SEQ ID NO:2) but which map to chromosome 12, between markers D12S09 and D12S358.
  • nucleotide sequences are allelic variants of the TANGO 269 molecules described herein. l o
  • the phrase "allelic variant" refers to a nucleotide sequence which occurs at a given locus or to a polypeptide encoded by the nucleotide sequence.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention. Such natural allelic variations can typically result in 1- 5% variance in the nucleotide sequence of a given gene. Alternative alleles can be identified by
  • nucleic acid molecules encoding proteins of the invention from other species
  • nucleic acid molecules corresponding to natural allelic variants and homologues of a cDNA of the invention can be isolated based on their identity to the human nucleic acid molecule disclosed herein using the
  • a cDNA encoding a soluble form of a membrane-bound protein of the invention isolated based on its hybridization to a nucleic acid molecule encoding all or part of the membrane-bound form.
  • a cDNA encoding a membrane-bound form can be isolated based on its hybridization to a nucleic acid molecule encoding all or part of the soluble form.
  • an isolated nucleic acid molecule of the invention is at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1290) nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence, preferably the coding sequence, of SEQ ID NO: 1, 2, 11, 12, 24, or 25, or a complement thereof.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, preferably 75%) identical to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65° C.
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1, 2, 11, 12, 24, or 25, or a complement thereof, corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally- occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • allelic variants of a nucleic acid molecule of the invention sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation thereby leading to changes in the amino acid sequence of the encoded protein, without altering the biological activity of the protein. For example, one can make nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues.
  • a "non-essential” amino acid residue is a residue that can be altered from the wild-type sequence without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are not conserved or only semi-conserved among homologues of various species may be non-essential for activity and thus would be likely targets for alteration.
  • amino acid residues that are conserved among the homologues of various species e.g., murine and human
  • amino acid residues that are conserved among the homologues of various species may be essential for activity and thus would not be likely targets for alteration.
  • nucleic acid molecules encoding a polypeptide of the invention that contain changes in amino acid residues that are not essential for activity. Such polypeptides differ in amino acid sequence from SEQ ID NO:3, 13, or 26, yet retain biological activity.
  • the isolated nucleic acid molecule includes a nucleotide sequence encoding a protein that includes an amino acid sequence that is at least about 45% identical, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:3, 13, or 26.
  • An isolated nucleic acid molecule encoding a variant protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 1, 2, 11, 12, 24, or 25 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
  • a mutant polypeptide that is a variant of a polypeptide of the invention can be assayed for: (1) the ability to form proteimprotein interactions with proteins in a signaling pathway of the polypeptide of the invention; (2) the ability to bind a ligand of the polypeptide of the invention; or (3) the ability to bind to an intracellular target protein of the polypeptide of the invention.
  • the mutant polypeptide can be assayed for the ability to modulate cellular proliferation, cellular migration or chemotaxis, or cellular differentiation.
  • the present invention encompasses antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid encoding a polypeptide of the invention, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame).
  • An antisense nucleic acid molecule can be antisense to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding a polypeptide of the invention.
  • the non-coding regions (“5* and 3' untranslated regions") are the 5' and 3' sequences which flank the coding region and are not translated into amino acids.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides or more in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxy
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a selected polypeptide of the invention to thereby inhibit expression, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • An antisense nucleic acid molecule of the invention can be an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131- 6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)
  • a ribozyme having specificity for a nucleic acid molecule encoding a polypeptide of the invention can be designed based upon the nucleotide sequence of a cDNA disclosed herein.
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742.
  • an mRNA encoding a polypeptide of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261 :1411-1418.
  • the invention also encompasses nucleic acid molecules which form triple helical structures.
  • expression of a polypeptide of the invention can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g., the promoter and/or enhancer) to form triple helical structures that prevent transcription of the gene in target cells. See generally Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992) Ann. N. Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14(12):807-15.
  • the nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996)
  • PNAs refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675.
  • PNAs can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
  • PNAs can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup (1996), supra; or as probes or primers for DNA sequence and hybridization (Hyrup (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675).
  • PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras can be generated which may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, e.g., RNAse H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996), supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W0 89/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl.
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio/Techniques 6:958-976) or intercalating agents (see, e.g., Zon (1988) Pharm. Res. 5:539-549).
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • One aspect of the invention pertains to isolated proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise antibodies directed against a polypeptide of the invention.
  • the native polypeptide can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • polypeptides of the invention are produced by recombinant DNA techniques.
  • a polypeptide of the invention can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein").
  • the protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.
  • Biologically active portions of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the protein (e.g., the amino acid sequence shown in any of SEQ ID NO:3, 4, 13, 14,
  • biologically active portions comprise a domain or motif with at least one activity of the corresponding protein.
  • a biologically active portion of a protein of the invention can be a polypeptide which is, for example, 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length.
  • other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the invention.
  • Preferred polypeptides have the amino acid sequence of SEQ ID NO:3, 4, 13, 14, 26, or
  • Other useful proteins are substantially identical (e.g., at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99%) to any of SEQ ID NO:3, 4, 13, 14, 26, or 27, and retain the functional activity of the protein of the corresponding naturally-occurring protein yet differ in amino acid sequence due to natural allelic variation or mutagenesis.
  • the sequences are aligned for optimal comparison pu ⁇ oses (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
  • Such an algorithm is inco ⁇ orated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
  • PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • Gapped BLAST, and PSI-Blast programs the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
  • a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is inco ⁇ orated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 70:3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 55:2444-8.
  • FASTA parameters see http://bioweb.pasteur.fr/docs/man/man/fasta. I.html#sect2, the contents of which are inco ⁇ orated herein by reference.
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
  • a "chimeric protein” or “fusion protein” comprises all or part (preferably biologically active) of a polypeptide of the invention operably linked to a heterologous polypeptide (i.e., a polypeptide other than the polypeptide of the invention).
  • a heterologous polypeptide i.e., a polypeptide other than the polypeptide of the invention.
  • the term "operably linked” is intended to indicate that the polypeptide of the invention and the heterologous polypeptide are fused in- frame to each other.
  • the heterologous polypeptide can be fused to the N-terminus or C-terminus of the polypeptide of the invention.
  • One useful fusion protein is a GST fusion protein in which the polypeptide of the invention is fused to the C-terminus of GST sequences. Such fusion proteins can facilitate the purification of a recombinant polypeptide of the invention.
  • the fusion protein contains a heterologous signal sequence at its N-terminus.
  • the native signal sequence of a polypeptide of the invention can be removed and replaced with a signal sequence from another protein.
  • the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992).
  • Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California).
  • useful prokaryotic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).
  • the fusion protein is an immunoglobulin fusion protein in which all or part of a polypeptide of the invention is fused to sequences derived from a member of the immunoglobulin protein family.
  • the immunoglobulin fusion proteins of the invention can be inco ⁇ orated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand (soluble or membrane-bound) and a protein on the surface of a cell (receptor), to thereby suppress signal transduction in vivo.
  • the immunoglobulin fusion protein can be used to affect the bioavailability of a cognate ligand of a polypeptide of the invention.
  • Inhibition of ligand/receptor interaction may be useful therapeutically, both for treating proliferative and differentiative disorders and for modulating (e.g. promoting or inhibiting) cell survival.
  • the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies directed against a polypeptide of the invention in a subject, to purify ligands and in screening assays to identify molecules which inhibit the interaction of receptors with ligands.
  • Chimeric and fusion proteins of the invention can be produced by standard recombinant DNA techniques.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel et al., supra).
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide of the invention.
  • a signal sequence of a polypeptide of the invention (SEQ ID NO:5, 15, and 28) can be used to facilitate secretion and isolation of the secreted protein or other proteins of interest. Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway.
  • a nucleic acid sequence encoding a signal sequence of the invention can be operably linked in an expression vector to a protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate.
  • the signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved.
  • the protein can then be readily purified from the extracellular medium by art recognized methods.
  • the signal sequence can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.
  • the signal sequences of the present invention can be used to identify regulatory sequences, e.g., promoters, enhancers, repressors. Since signal sequences are the most amino-terminal sequences of a peptide, it is expected that the nucleic acids which flank the signal sequence on its amino-terminal side will be regulatory sequences which affect transcription. Thus, a nucleotide sequence which encodes all or a portion of a signal sequence can be used as a probe to identify and isolate signal sequences and their flanking regions, and these flanking regions can be studied to identify regulatory elements therein.
  • regulatory sequences e.g., promoters, enhancers, repressors.
  • the present invention also pertains to variants of the polypeptides of the invention.
  • variants have an altered amino acid sequence which can function as either agonists (mimetics) or as antagonists.
  • Variants can be generated by mutagenesis, e.g., discrete point mutation or truncation.
  • An agonist can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the protein.
  • An antagonist of a protein can inhibit one or more of the activities of the naturally occurring form of the protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest.
  • specific biological effects can be elicited by treatment with a variant of limited function.
  • Variants of a protein of the invention which function as either agonists (mimetics) or as antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the protein of the invention for agonist or antagonist activity.
  • a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).
  • methods which can be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acid Res. 11 :477).
  • libraries of fragments of the coding sequence of a polypeptide of the invention can be used to generate a variegated population of polypeptides for screening and subsequent selection of variants.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N- terminal and internal fragments of various sizes of the protein of interest.
  • REM Recursive ensemble mutagenesis
  • An isolated polypeptide of the invention, or a fragment thereof, can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length polypeptide or protein can be used or, alternatively, the invention provides antigenic peptide fragments for use as immunogens.
  • the antigenic peptide of a protein of the invention comprises at least 8 (preferably 10, 15, 20, or 30) amino acid residues of the amino acid sequence of SEQ ID NO:3, 13, or 26, and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.
  • Preferred epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, e.g., hydrophilic regions.
  • Figures 2, 7, and 11 are hydropathy plots of the proteins of the invention. These plots or similar analyses can be used to identify hydrophilic regions.
  • An immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal).
  • a suitable subject e.g., rabbit, goat, mouse or other mammal.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed or chemically synthesized polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention.
  • a molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies.
  • the term "monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope.
  • Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide of the invention as an immunogen.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well- known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody- producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques.
  • standard techniques such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques.
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.
  • a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide of interest.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene Sur ⁇ APTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No.
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Patent No. 4,816,567; European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
  • Patent 5,225,539 Jones et al. (1986) Nature 321 :552-525; Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al. (1988) J. Immunol. 141 :4053-4060.
  • Fully human antibodies are particularly desirable for therapeutic treatment of human patients.
  • Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
  • Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA and IgE antibodies.
  • Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g., a murine antibody
  • a completely human antibody recognizing the same epitope is used to guide the selection of a completely human antibody recognizing the same epitope.
  • An antibody directed against a polypeptide of the invention can be used to isolate the polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, such an antibody can be used to detect the protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide.
  • the antibodies can also be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta- galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and
  • suitable radioactive material include l25 1, 131 1, 35 S or 3 H.
  • an antibody may be conjugated to or administered with a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine
  • antimetabolites e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine
  • alkylating agents e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine
  • CCNU cyclothosphamide
  • busulfan dibromomannitol
  • streptozotocin mitomycin C
  • anthracyclines e.g., daunorubicin (formerly daunomycin) and doxorubicin
  • antibiotics e.g. , dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)
  • anti-mitotic agents e.g., vincristine and vinblastine.
  • the conjugates of the invention can be used for modifying a given biological response.
  • the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta- interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
  • vectors preferably expression vectors, containing a nucleic acid encoding a polypeptide of the invention (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors, expression vectors are capable of directing the expression of genes to which they are operably linked.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors).
  • the invention is intended to include such other forms of expression vectors, such as viral vectors
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell.
  • the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
  • the recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells (using baculovirus expression vectors), yeast cells or mammalian cells). Suitable host cells are discussed further in Goeddel, supra.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three pu ⁇ oses: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • GST glutathione S-transferase
  • maltose E binding protein or protein A, respectively, to the target recombinant protein.
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 1 Id (Studier et al., Gene Expression
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or
  • HMS174(DE3) from a resident ⁇ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerivisae include pYepSecl (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Co ⁇ oration, San Diego, CA), and pPicZ (Invitrogen Co ⁇ , San Diego, CA).
  • the expression vector is a baculovirus expression vector.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187- 195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • viral regulatory elements For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al., supra.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBOJ.
  • promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the beta-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to the mRNA encoding a polypeptide of the invention.
  • Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • host cell and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell (e.g., insect cells, yeast or mammalian cells).
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have inco ⁇ orated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce a polypeptide of the invention.
  • the invention further provides methods for producing a polypeptide of the invention using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced.
  • the method further comprises isolating the polypeptide from the medium or the host cell.
  • the host cells of the invention can also be used to produce nonhuman transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a sequence encoding a polypeptide of the invention has been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous sequences encoding a polypeptide of the invention have been introduced into their genome or homologous recombinant animals in which endogenous encoding a polypeptide of the invention sequences have been altered.
  • Such animals are useful for studying the function and/or activity of the polypeptide and for identifying and/or evaluating modulators of polypeptide activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • an "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing nucleic acid encoding a polypeptide of the invention (or a homologue thereof) into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue- specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the polypeptide of the invention to particular cells.
  • transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of mRNA encoding the transgene in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying the transgene can further be bred to other transgenic animals carrying other transgenes.
  • a vector which contains at least a portion of a gene encoding a polypeptide of the invention into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the gene.
  • the vector is designed such that, upon homologous recombination, the endogenous gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous protein).
  • the altered portion of the gene is flanked at its 5' and 3' ends by additional nucleic acid of the gene to allow for homologous recombination to occur between the exogenous gene carried by the vector and an endogenous gene in an embryonic stem cell.
  • the additional flanking nucleic acid sequences are of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5' and 3' ends
  • flanking DNA both at the 5' and 3' ends
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced gene has homologously recombined with the endogenous gene are selected (see, e.g., Li et al. (1992) Cell 69:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see, e.g., Bradley in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed. (IRL, Oxford, 1987) pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene.
  • cre/loxP recombinase system of bacteriophage PI is the cre/loxP recombinase system of bacteriophage PI.
  • a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. ( 1991 ) Science 251 : 1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 385:810-813 and PCT Publication NOS. WO 97/07668 and WO 97/07669. IV. Pharmaceutical Compositions
  • compositions suitable for administration can be inco ⁇ orated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be inco ⁇ orated into the compositions.
  • the invention includes methods for preparing pharmaceutical compositions for modulating the expression or activity of a polypeptide or nucleic acid of the invention. Such methods comprise formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of the invention. Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of the invention and one or more additional active compounds.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules,
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF; Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by including in the composition an agent which delays abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by inco ⁇ orating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or saccharin
  • the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Co ⁇ oration and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • anti-TANGO 269 or anti-TANGO 298 antibodies are administered parenterally, preferably intravenously or intramuscularly daily, monthly, biweekly, weekly, or more frequently.
  • the preferred dosage is O.lmg/kg to 100 mg/kg of body weight, preferably 10 to 20 mg/kg of body weight. Dosages of 50 mg/kg or higher are preferred if the antibody is to be effective within the brain. Accordingly, lower dosages and less frequent administration is often possible.
  • the preferred dosage for treatment of a particular disorder can be based on results observed with other therapeutic antibodies or it can be determined by one skilled based on testing in animal models.
  • the suitable dosage of antibody in a given situation depends on the disease being treated, the severity of the disease, whether the antibody is being administered for therapeutic or preventative reasons, previous therapies administered, and the patient's clinical history. Treatment is generally continued until the desired therapeutic or preventative effect is observed. Dosage regimes of the type that can be adapted to the methods of the present invention are found in PCT Publication Number WO 94/04188. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).
  • a therapeutically effective amount of protein or polypeptide ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • the present invention encompasses agents which modulate expression or activity of TANGO 269 and/or TANGO 298.
  • An agent may, for example, be a small molecule.
  • small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with antibody, or protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Patent 5,328,470) or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) detection assays (e.g., chromosomal mapping, tissue typing, forensic biology); c) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenomics); and d) methods of treatment (e.g., therapeutic and prophylactic).
  • detection assays e.g., chromosomal mapping, tissue typing, forensic biology
  • predictive medicine e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenomics
  • methods of treatment e.g., therapeutic and prophylactic.
  • the TANGO 269 polypeptides of the invention can to used to, for example (i) mediate protein-protein interactions; (ii) modulate cellular migration, proliferation, and differentiation; (iii) modulate proteolytic enzyme activity; and/or (iv) modulate protein degradation.
  • the isolated nucleic acid molecules of the invention can be used to express proteins (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect mRNA (e.g., in a biological sample) or a genetic lesion, and to modulate activity of a polypeptide of the invention.
  • polypeptides of the invention can be used to screen drugs or compounds which modulate activity or expression of a polypeptide of the invention as well as to treat disorders characterized by insufficient or excessive production of a protein of the invention or production of a form of a protein of the invention which has decreased or aberrant activity compared to the wild type protein.
  • the antibodies of the invention can be used to detect and isolate a protein of the and modulate activity of a protein of the invention. This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to polypeptide of the invention or have a stimulatory or inhibitory effect on, for example, expression or activity of a polypeptide of the invention.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to polypeptide of the invention or have a stimulatory or inhibitory effect on, for example, expression or activity of a polypeptide of the invention.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a polypeptide of the invention or biologically active portion thereof.
  • the test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one- bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).
  • an assay is a cell-based assay in which a cell which expresses a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to the polj ⁇ eptide determined.
  • the cell for example, can be a yeast cell or a cell of mammalian origin. Determining the ability of the test compound to bind to the polypeptide can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the polypeptide or biologically active portion thereof can be determined by detecting the labeled compound in a complex.
  • test compounds can be labeled with 125 1, 35 S, l4 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay comprises contacting a cell which expresses a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or a biologically active portion thereof as compared to the known compound.
  • an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the polypeptide or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of the polypeptide or a biologically active portion thereof can be accomplished, for example, by determining the ability of the polypeptide protein to bind to or interact with a target molecule.
  • a target molecule is a molecule with which a selected polypeptide (e.g., a polypeptide of the invention) binds or interacts with in nature, for example, a molecule on the surface of a cell which expresses the selected protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.
  • a target molecule can be a polypeptide of the invention or some other polypeptide or protein.
  • a target molecule can be a component of a signal transduction pathway which facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a compound to a polypeptide of the invention) through the cell membrane and into the cell or a second intercellular protein which has catalytic activity or a protein which facilitates the association of downstream signaling molecules with a polypeptide of the invention. Determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule.
  • an extracellular signal e.g., a signal generated by binding of a compound to a polypeptide of the invention
  • the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca 2+ , diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cellular differentiation, or cell proliferation.
  • a reporter gene e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • a cellular response for example, cellular differentiation, or cell proliferation.
  • an assay of the present invention is a cell-free assay comprising contacting a polypeptide of the invention or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to the polypeptide or biologically active portion thereof. Binding of the test compound to the polypeptide can be determined either directly or indirectly as described above.
  • the assay includes contacting the polypeptide of the invention or biologically active portion thereof with a known compound which binds the polj ⁇ eptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or biologically active portion thereof as compared to the known compound.
  • an assay is a cell-free assay comprising contacting a polypeptide of the invention or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the polypeptide or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished, for example, by determining the ability of the polypeptide to bind to a target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished by determining the ability of the polypeptide of the invention to further modulate the target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as previously described.
  • the cell-free assay comprises contacting a polypeptide of the invention or biologically active portion thereof with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the polypeptide to preferentially bind to or modulate the activity of a target molecule.
  • the cell-free assays of the present invention are amenable to use of both a soluble form or the membrane-bound form of a polypeptide of the invention.
  • solubilizing agent such that the membrane-bound form of the polypeptide is maintained in solution.
  • solubilizing agents include non-ionic detergents such as n- octylglucoside, n-dodecylglucoside, n-octylmaltoside, octanoyl-N-methylglucamide, decanoyl-N- methylglucamide, Triton X-100, Triton X-l 14, Thesit, Isotridecypoly(ethylene glycol ether)n, 3- [(3-cholamidopropyl)dimethylamminio]-l -propane sulfonate (CHAPS), 3-[(3- cholamidopropyl)dimethylamminio]-2-hydroxy-l -propane sulfonate (CHAPSO),
  • binding of a test compound to the polypeptide, or interaction of the polypeptide with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or A polypeptide of the invention, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of binding or activity of the polypeptide of the invention can be determined using standard techniques.
  • polypeptide of the invention or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N-hydroxy- succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with the polypeptide of the invention or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the invention trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the polypeptide of the invention or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the polypeptide of the invention or target molecule.
  • modulators of expression of a polypeptide of the invention are identified in a method in which a cell is contacted with a candidate compound and the expression of the selected mRNA or protein (i.e., the mRNA or protein corresponding to a polypeptide or nucleic acid of the invention) in the cell is determined.
  • the level of expression of the selected mRNA or protein in the presence of the candidate compound is compared to the level of expression of the selected mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of expression of the polypeptide of the invention based on this comparison.
  • the candidate compound when expression of the selected mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of the selected mRNA or protein expression.
  • the candidate compound when expression of the selected mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of the selected mRNA or protein expression.
  • the level of the selected mRNA or protein expression in the cells can be determined by methods described herein.
  • a polypeptide of the inventions can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g. , U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and PCT Publication No.
  • binding proteins are also likely to be involved in the propagation of signals by the polypeptide of the inventions as, for example, upstream or downstream elements of a signaling pathway involving the polypeptide of the invention.
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.
  • sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. Accordingly, nucleic acid molecules described herein or fragments thereof, can be used to map the location of the corresponding genes on a chromosome. The mapping of the sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
  • genes can be mapped to chromosomes by preparing PCR primers (preferably 15- 25 bp in length) from the sequence of a gene of the invention.
  • Computer analysis of the sequence of a gene of the invention can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process.
  • These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the gene sequences will yield an amplified fragment.
  • D'Eustachio et al. see D'Eustachio et al. ((1983) Science 220:919-924).
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the nucleic acid sequences of the invention to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a gene to its chromosome include in situ hybridization (described in Fan et al. (1990) Proc. Natl. Acad. Sci. USA 87:6223-27), pre-screening with labeled flow-sorted chromosomes (CITE), and preselection by hybridization to chromosome specific cDNA libraries.
  • Fluorescence in situ hybridization of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
  • FISH Fluorescence in situ hybridization
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping pu ⁇ oses. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymo ⁇ hisms.
  • the nucleic acid sequences of the present invention can also be used to identify individuals from minute biological samples.
  • the United States military for example, is considering the use of restriction fragment length polymo ⁇ hism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymo ⁇ hism
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the nucleic acid sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the nucleic acid sequences of the invention uniquely represent portions of the human genome.
  • allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
  • Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification pu ⁇ oses. Because greater numbers of polymo ⁇ hisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals.
  • the noncoding sequences of SEQ ID NO:l or 12 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:2, 12, or 25 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • a panel of reagents from the nucleic acid sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • positive identification of the individual, living or dead can be made from extremely small tissue samples.
  • DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a pe ⁇ etrator of a crime.
  • PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
  • sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual).
  • an "identification marker” i.e. another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
  • Sequences targeted to noncoding regions are particularly appropriate for this use as greater numbers of polymo ⁇ hisms occur in the noncoding regions, making it easier to differentiate individuals using this technique.
  • polynucleotide reagents include the nucleic acid sequences of the invention or portions thereof, e.g., fragments derived from noncoding regions having a length of at least 20 or 30 bases.
  • the nucleic acid sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such probes can be used to identify tissue by species and/or by organ type.
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trails are used for prognostic (predictive) pu ⁇ oses to thereby treat an individual prophylactically.
  • diagnostic assays for determining expression of a polypeptide or nucleic acid of the invention and/or activity of a polypeptide of the invention, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant expression or activity of a polypeptide of the invention.
  • a biological sample e.g., blood, serum, cells, tissue
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with aberrant expression or activity of a polypeptide of the invention. For example, mutations in a gene of the invention can be assayed in a biological sample. Such assays can be used for prognostic or predictive pu ⁇ ose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with aberrant expression or activity of a polypeptide of the invention.
  • Another aspect of the invention provides methods for expression of a nucleic acid or polypeptide of the invention or activity of a polypeptide of the invention in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics").
  • Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent).
  • Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs or other compounds) on the expression or activity of a polypeptide of the invention in clinical trials.
  • agents e.g., drugs or other compounds
  • An exemplary method for detecting the presence or absence of a polypeptide or nucleic acid of the invention in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of the invention such that the presence of a polypeptide or nucleic acid of the invention is detected in the biological sample.
  • a preferred agent for detecting mRNA or genomic DNA encoding a polypeptide of the invention is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA encoding a polypeptide of the invention.
  • the nucleic acid probe can be, for example, a full-length cDNA, such as the nucleic acid of SEQ ID NO: 1, 2, 11, 12, 24, or 25, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a polypeptide of the invention.
  • a full-length cDNA such as the nucleic acid of SEQ ID NO: 1, 2, 11, 12, 24, or 25, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a polypeptide of the invention.
  • Other suitable probes for use in the diagnostic assays of the invention are described herein.
  • a preferred agent for detecting a polypeptide of the invention is an antibody capable of binding to a polypeptide of the invention, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab') 2 ) can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of a polypeptide of the invention include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of a polypeptide of the invention include introducing into a subject a labeled antibody directed against the polypeptide.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting a polypeptide of the invention or mRNA or genomic DNA encoding a polypeptide of the invention, such that the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide is detected in the biological sample, and comparing the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide in the control sample with the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide in the test sample.
  • kits for detecting the presence of a polj ⁇ eptide or nucleic acid of the invention in a biological sample can be used to determine if a subject is suffering from or is at increased risk of developing a disorder associated with aberrant expression of a polypeptide of the invention (e.g., a proliferative disorder, e.g., psoriasis or cancer).
  • a disorder associated with aberrant expression of a polypeptide of the invention e.g., a proliferative disorder, e.g., psoriasis or cancer.
  • the kit can comprise a labeled compound or agent capable of detecting the polypeptide or mRNA encoding the polypeptide in a biological sample and means for determining the amount of the polj ⁇ eptide or mRNA in the sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide).
  • Kits can also include instructions for observing that the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of the polypeptide if the amount of the polypeptide or mRNA encoding the polypeptide is above or below a normal level.
  • the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a polj ⁇ eptide of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.
  • a first antibody e.g., attached to a solid support
  • a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.
  • the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule encoding a polypeptide of the invention.
  • the kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent.
  • the kit can also comprise components necessary for detecting the detectable agent (e.g., an enzj ⁇ ne or a substrate).
  • the kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained.
  • Each component of the kit is usually enclosed within an individual container and all of the various containers are within a single package along with instructions for observing whether the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of the polypeptide.
  • the methods described herein can furthermore be utilized as diagnostic or prognostic assays to identify subjects having or at risk of developing a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention.
  • the assays described herein such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with aberrant expression or activity of a polypeptide of the invention, e.g., a neurological disorder, e.g., Alzheimer's disease, a bone disorder, or a cardiovascular disorder.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing such a disease or disorder.
  • test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • such methods can be used to determine whether a subject can be effectively treated with a specific agent or class of agents (e.g., agents of a type which decrease activity of the
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant expression or activity of a polypeptide of the invention in which a test sample is obtained and the polypeptide or nucleic acid encoding the polj ⁇ eptide is detected (e.g., wherein the presence of the polypeptide or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant expression or activity of the polypeptide).
  • the methods of the invention can also be used to detect genetic lesions or mutations in a gene of the invention, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized aberrant expression or activity of a polypeptide of the invention.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion or mutation characterized by at least one of an alteration affecting the integrity of a gene encoding the polypeptide of the invention, or the mis-expression of the gene encoding the polypeptide of the invention.
  • such genetic lesions or mutations can be detected by ascertaining the existence of at least one of: 1) a deletion of one or more nucleotides from the gene; 2) an addition of one or more nucleotides to the gene; 3) a substitution of one or more nucleotides of the gene; 4) a chromosomal rearrangement of the gene; 5) an alteration in the level of a messenger RNA transcript of the gene; 6) an aberrant modification of the gene, such as of the methylation pattern of the genomic DNA; 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; 8) a non-wild type level of a the protein encoded by the gene; 9) an allelic loss of the gene; and 10) an inappropriate post-translational modification of the protein encoded by the gene.
  • assay techniques known in the art which can be used for detecting lesions in a gene.
  • detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91 : 360-364), the latter of which can be particularly useful for detecting point mutations in a gene (see, e.g., Abravaya et al.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to the selected gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • nucleic acid e.g., genomic, mRNA or both
  • Alternative amplification methods include: self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art.
  • mutations in a selected gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes can be used to score for the presence of specific mutations by development or loss of a ribozjrme cleavage site.
  • genetic mutations can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759).
  • genetic mutations can be identified in two- dimensional arrays containing light-generated DNA probes as described in Cronin et al., supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes.
  • This step allows the identification of point mutations.
  • This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
  • Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the selected gene and detect mutations by comparing the sequence of the sample nucleic acids with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Bio/Techniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT Publication No.
  • WO 94/16101 Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. ( ⁇ 99?>) Appl. Biochem. Biotechnol. 38: 147-159).
  • Other methods for detecting mutations in a selected gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242).
  • the technique of "mismatch cleavage” entails providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • RNA/DNA duplexes can be treated with RNase to digest mismatched regions, and DNA/DNA hybrids can be treated with SI nuclease to digest mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295.
  • the control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
  • a probe based on a selected sequence is hybridized to a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in genes.
  • single strand conformation potymo ⁇ hism may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766; see also Cotton (1993) Mutat. Res. 285:125-144; Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, and the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a 'GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chem. 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324: 163); Saiki et al. (1989) Proc. Natl.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent or reduce polymerase extension (Prossner (1993) Tibtech 11 :238).
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a gene encoding a polypeptide of the invention.
  • any cell type or tissue, e.g., hepatocytes, in which the polypeptide of the invention is expressed may be utilized in the prognostic assays described herein.
  • Agents, or modulators which have a stimulatory or inhibitory effect on activity or expression of a polypeptide of the invention as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders associated with aberrant activity of the polypeptide.
  • the pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
  • the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of a polypeptide of the invention, expression of a nucleic acid of the invention, or mutation content of a gene of the invention in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Linder (1997) Clin. Chem. 43(2):254-266.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body are referred to as “altered drug action.” Genetic conditions transmitted as single factors altering the way the body acts on drugs are referred to as "altered drug metabolism”. These pharmacogenetic conditions can occur either as rare defects or as polymo ⁇ hisms.
  • G6PD glucose-6-phosphate dehydrogenase deficiency
  • oxidant drugs anti- malarials, sulfonamides, analgesics, nitrofurans
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • drug metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • the gene coding for CYP2D6 is highly polymo ⁇ hic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, a PM will show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite mo ⁇ hine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • the activity of a polypeptide of the invention, expression of a nucleic acid encoding the polypeptide, or mutation content of a gene encoding the polypeptide in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • pharmacogenetic studies can be used to apply genotyping of polymo ⁇ hic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a modulator of activity or expression of the polypeptide, such as a modulator identified by one of the exemplary screening assays described herein.
  • a polypeptide of the invention e.g., the ability to modulate aberrant cell proliferation chemotaxis, and/or differentiation
  • agents e.g., drugs, compounds
  • the effectiveness of an agent, as determined by a screening assay as described herein, to increase gene expression, protein levels or protein activity can be monitored in clinical trials of subjects exhibiting decreased gene expression, protein levels, or protein activity.
  • the effectiveness of an agent, as determined by a screening assay, to decrease gene expression, protein levels or protein activity can be monitored in clinical trials of subjects exhibiting increased gene expression, protein levels, or protein activity.
  • expression or activity of a polypeptide of the invention and preferably, that of other polypeptide that have been implicated in for example, a cellular proliferation disorder can be used as a marker of the immune responsiveness of a particular cell.
  • genes including those of the invention, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates activity or expression of a polypeptide of the invention (e.g., as identified in a screening assay described herein) can be identified.
  • an agent e.g., compound, drug or small molecule
  • a polypeptide of the invention e.g., as identified in a screening assay described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of a gene of the invention and other genes implicated in the disorder.
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of a gene of the invention or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of the polypeptide or nucleic acid of the invention in the preadministration sample; (iii) obtaining one or more post- administration samples from the subject; (iv) detecting the level the of the polypeptide or nucleic acid of the invention in the post-administration samples; (v) comparing the level of the polypeptide or nucleic acid of the invention in the pre-administration sample with the level of the polypeptide or nucleic acid of the invention in the post-administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.
  • an agent e.g., an agonist, antagonist,
  • increased administration of the agent may be desirable to increase the expression or activity of the polypeptide to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of the polypeptide to lower levels than detected, i.e., to decrease the effectiveness of the agent.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant expression or activity of a polypeptide of the invention.
  • disorders characterized by aberrant expression or activity of the polj ⁇ eptides of the invention include atherosclerosis and obesity-related disorders.
  • polypeptides of the invention can be used to bind Ox- LDL, to modulate the alternative complement pathway, to treat immune related disorders (e.g., autoimmune disorders (e.g., arthritis and T cell autoimmune disorders (e.g., AIDS)) and inflammatory disorders (e.g., bacterial infection, and arthritis (e.g., rheumatoid arthritis, osteoarthritis)), and obesity related disorders (e.g., hypertension and energy balance regulation), as well as other uses described herein.
  • autoimmune disorders e.g., arthritis and T cell autoimmune disorders (e.g., AIDS)
  • inflammatory disorders e.g., bacterial infection, and arthritis (e.g., rheumatoid arthritis, osteoarthritis)
  • obesity related disorders e.g., hypertension and energy balance regulation
  • the TANGO 269 and TANGO 298 polypeptides of the invention can be used to modulate hepatocyte and bone marrow cell proliferation and differentiation (e.g.
  • the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant expression or activity of a polypeptide of the invention, by administering to the subject an agent which modulates expression or at least one activity of the polypeptide.
  • Subjects at risk for a disease which is caused or contributed to by aberrant expression or activity of a polypeptide of the invention can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • an agonist or antagonist agent can be used for treating the subject.
  • an antagonist of a TANGO 269 protein can be used to treat a blood related disorder, e.g., acute myeloid leukemia, hemophilia, leukemia, anemia (e.g., sickle cell anemia), and thalassemia, associated with aberrant TANGO 269 expression or activity
  • a blood related disorder e.g., acute myeloid leukemia, hemophilia, leukemia, anemia (e.g., sickle cell anemia), and thalassemia
  • an antagonist of a TANGO 298 protein may be used to treat an obesity related disorder, e.g., hyperglycemic shock or poor regulation of energy balance, associated with aberrant TANGO 269 expression or activity.
  • the appropriate agent can be determined based on screening assays described herein.
  • the modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of the polypeptide.
  • An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of the polypeptide, a peptide, a peptidomimetic, or other small molecule.
  • the agent stimulates one or more of the biological activities of the polypeptide. Examples of such stimulatory agents include the active polypeptide of the invention and a nucleic acid molecule encoding the polypeptide of the invention that has been introduced into the cell.
  • the agent inhibits one or more of the biological activities of the polypeptide of the invention.
  • inhibitory agents include antisense nucleic acid molecules and antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a polj ⁇ eptide of the invention.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) expression or activity.
  • an agent e.g., an agent identified by a screening assay described herein
  • the method involves administering a polypeptide of the invention or a nucleic acid molecule of the invention as therapy to compensate for reduced or aberrant expression or activity of the polypeptide.
  • Stimulation of activity is desirable in situations in which activity or expression is abnormally low or downregulated and/or in which increased activity is likely to have a beneficial effect, e.g., in treatment of immune disorders.
  • inhibition of activity is desirable in situations in which activity or expression is abnormally high or upregulated and/or in which decreased activity is likely to have a beneficial effect, e.g., in treatment of a obesity related disorder.
  • Human TANGO 298 expression was measured by TaqMan ® quantitative PCR (Perkin Elmer Applied Biosystems) in cDNA prepared from the following normal human tissues or cell lines: lymph node, spleen, kidney, brain, lung, skeletal muscle, fetal liver, tonsil, colon, heart, and liver from two adult donors; fibrotic liver samples prepared from seven different donors; transformed human cell lines K562, an erythroleukemia; Hep3B hepatocellular liver carcinoma cells cultured in reduced oxygen tension (Hep3B hj ⁇ oxia); CD34 + cells from mobilized peripheral blood (mPB CD34 + ), adult resting bone marrow (ABM CD34 + ), and G-CSF mobilized bone marrow (mBM CD34 + ); CD34 " cells purified from mPB leukocytes (mPB CD34 ); G-CSF mobilized peripheral blood leukocytes (mPB leukocytes); CD34 " cells from mobilized bone marrow (MBM CD34 ); Thl and Th2 cells
  • Probes were designed by PrimerExpress software (PE Biosystems) based on the sequence of the human TANGO 298 gene.
  • the primers and probes for expression analysis of human TANGO 298 were as follows: human TANGO 298 Forward Primer: 5'-AGCAGGTGTTTGGCATCGAT-3' (SEQ ID NO:35) human TANGO 298 Reverse Primer: 5'-GTCGTTGGCGTGGGTCAT-3' (SEQ ID NO:36) human TANGO 298 TaqMan Probe: FAM-TCACCACGCACCCCGACTACCAC-TAMRA
  • Each human TANGO 298 gene probe was labeled using FAM (6-carboxyfluorescein), and the ⁇ 2-microglobulin reference probe was labeled with a different fluorescent dye, VIC.
  • FAM 6-carboxyfluorescein
  • VIC a different fluorescent dye
  • Forward and reverse primers and the probes for both ⁇ 2 -microglobulin and target gene were added to the TaqMan ® Universal PCR Master Mix (PE Applied Biosystems). Although the final concentration of primer and probe could vary, each was internally consistent within a given experiment.
  • a typical experiment contained 200nM of forward and reverse primers plus lOOnM probe for ⁇ -2 microglobulin and 600 nM forward and reverse primers plus 200 nM probe for the target gene.
  • TaqMan matrix experiments were carried out on an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems).
  • the thermal cycler conditions were as follows: hold for 2 min at 50°C and 10 min at 95°C, followed by two-step PCR for 40 cycles of 95°C for 15 sec followed by 60°C for 1 min.
  • the following method was used to quantitatively calculate human TANGO 298 gene expression in the various tissues relative to ⁇ -2 microglobulin expression in the same tissue.
  • the threshold cycle (Ct) value is defined as the cycle at which a statistically significant increase in fluorescence is detected. A lower Ct value is indicative of a higher mRNA concentration.
  • Figure 15 shows expression of human TANGO 298 in various tissues and cell lines as described above, relative to expression in tonsil cells.
  • the results indicate significant expression in CD34 + cells from mobilized peripheral blood (mPB CD34 + ), adult resting bone marrow (ABM CD34 + ), G-CSF mobilized bone marrow (mBM CD34 + ), and G-CSF mobilized peripheral blood leukocytes (mPB leukocytes); and moderate expression in fetal liver, cell line K526 (an ei throleukemia), and CD34 " cells from mobilized bone marrow (MBM CD34 ).
  • TANGO 298 expression was measured by TaqMan ® quantitative PCR (Perkin Elmer Applied Biosystems) in cDNA prepared from the following normal human tissues or cell lines: lung; brain; colon; heart; spleen; kidney; fetal liver; skeletal muscle; mononuclear cells from mobilized bone marrow (mBM MNC); mononuclear cells from mobilized bone marrow (mBM MNC); CD34 + cells from mobilized bone marrow (CD34 is a progenitor cell marker) (mBM CD34 + ); CD34 + cells from mobilized peripheral blood (mPB CD34 + ); CD34 + cells from mobilized peripheral blood (mPB CD34 + ); CD34 + cells from mobilized peripheral blood (mPB CD34 + ); CD34 + cells from mobilized peripheral blood (mPB CD34 + ); CD34 + cells from adult resting bone marrow (ABM CD34 + ); CD34 + cells from adult resting bone marrow (ABM CD34 + ); CD34 + cells from umbilical cord
  • Probes were designed by PrimerExpress software (PE Biosystems) based on the sequence of the human TANGO 298 gene.
  • the primers and probes for expression analysis of human TANGO 298 were as follows:
  • human TANGO 298 Forward Primer 5*-AGCAGGTGTTTGGCATCGAT-3' (SEQ ID NO:35)
  • human TANGO 298 Reverse Primer 5'-GTCGTTGGCGTGGGTCAT-3' (SEQ ID NO:36)
  • human TANGO 298 TaqMan Probe FAM-TCACCACGCACCCCGACTACCAC-TAMRA
  • Figures 16A-16B show expression of human TANGO 298 in various tissues and cell lines as described above, relative to expression in CDl 57CD1 lb + cells from mobilized bone marrow (mBMCF157CDl lb + ).
  • the figures are from the same experiment, but are shown on two pages for ease of display.
  • expression is revealed to be highest in the erythroblast samples, the megakaryocyte samples, the neutrophil samples, and in cells populations positive for CD34 + marker (including cells from mobilized bone marrow, adult resting bone marrow, umbilical cord blood, and fetal liver).
  • the samples from each group manifest higher levels of TANGO 298 expression in the earlier stages of development, and expression tapers off after a certain point in time.
  • levels of TANGO 298 expression increase in proportion to the time the cells were cultured, from 24 hours to 48 hours, then decrease with culture times of 6 days, 10 days, and 12 days.

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Abstract

The invention provides isolated nucleic acid molecules, designated TANGO 269 nucleic acid molecules, which encode type II transmembrane proteins with homology to the human LOX-1 (lectin-like oxidized low-density lipoprotein receptor) protein. The invention also provides isolated nucleic acid molecules, designated TANGO 298 nucleic acid molecules, which encode wholly secreted proteins with homology to the human adipsin proteins. The invention also provides antisense nucleic acid molecules, expression vectors containing the nucleic acid molecules of the invention, host cells into which the expression vectors have been introduced, and non-human transgenic animals in which a nucleic acid molecule of the invention has been introduced or disrupted. The invention still further provides isolated polypeptides, fusion polypeptides, antigenic peptides and antibodies. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

Description

SECRETED PROTEINS AND USES THEREOF
Background of the Invention Many secreted proteins, for example, cytokines and cytokine receptors, play a vital role in the regulation of cell growth, cell differentiation, and a variety of specific cellular responses. A number of medically useful proteins, including erythropoietin, granulocyte-macrophage colony stimulating factor, human growth hormone, and various interleukins, are secreted proteins. Thus, an important goal in the design and development of new therapies is the identification and characterization of secreted and transmembrane proteins and the genes which encode them. Many secreted proteins are receptors which bind a ligand and transduce an intracellular signal, leading to a variety of cellular responses. The identification and characterization of such a receptor enables one to identify both the ligands which bind to the receptor and the intracellular molecules and signal transduction pathways associated with the receptor, permitting one to identify or design modulators of receptor activity, e.g., receptor agonists or antagonists and modulators of signal transduction.
Summary of the Invention The present invention is based, at least in part, on the discovery of cDNA molecules which encode the TANGO 269 and 298 proteins, both of which are wholly secreted or transmembrane proteins.
The TANGO 269 proteins share homology to lectin-like oxidized low-density lipoprotein receptor (LOX-1), a cell surface receptor and membrane protein that belongs structurally to the C-type lectin family, and is expressed in vivo in vascular endothelium and vascular-rich organs. The TANGO 298 proteins share homology to adipsin, a serine protease homolog that is synthesized and secreted by adipose cells and which has been proven to be the same as complement factor D. Complement factor D is known to play an important role in obesity and energy regulation pathways.
The TANGO 269 and TANGO 298 proteins, fragments, derivatives, and variants thereof are collectively referred to as "polypeptides of the invention" or "proteins of the invention." Nucleic acid molecules encoding the polypeptides or proteins of the invention are collectively referred to as "nucleic acids of the invention."
The nucleic acids and polypeptides of the present invention are useful as modulating agents in regulating a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding a polypeptide of the invention or a biologically active portion thereof. The present invention also provides nucleic acid molecules which are suitable for use as primers or hybridization probes for the detection of nucleic acids encoding a polypeptide of the invention.
The invention features nucleic acid molecules which are at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:l 1, the nucleotide sequence of the cDNA insert of a clone deposited with ATCC as Accession Number 207218, or a complement thereof. The invention features nucleic acid molecules which are at least 37% (or 40%, 45%, 50%,
55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequence of SEQ ID NO:24, the nucleotide sequence of the cDNA insert of a clone deposited with ATCC as Accession Number 207216, or a complement thereof.
The invention also features nucleic acid molecules which are at least 25% (or 30%, 35%, 40%, 45%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%) identical to the nucleotide sequence of SEQ ID NO: 1 , 11 , or 24, the nucleotide sequence of the cDNA insert of a clone deposited with ATCC as Accession Number 207218 or 207216, or a complement thereof, wherein such nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or functional feature of a polypeptide of the invention. The invention features nucleic acid molecules which include a fragment of at least 500
(525, 550, 575, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, or 1100) nucleotides of the nucleotide sequence of SEQ ID NO:l, the nucleotide sequence of the cDNA of ATCC Accession Number 207218, or a complement thereof.
The invention features nucleic acid molecules which include a fragment of at least 400 (425, 475, 500, 525, 550, 575, 600, 650, 700, 750, 800, 850, 900, or 930) nucleotides of the nucleotide sequence of SEQ ID NO: 11 or a complement thereof.
The invention features nucleic acid molecules which include a fragment of at least 400 (425, 475, 500, 525, 550, 575, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, or 1070) nucleotides of the nucleotide sequence of SEQ ID NO:24, the nucleotide sequence of the cDNA of ATCC Accession Number 207216, or a complement thereof. The invention also features nucleic acid molecules comprising at least 50, 75, 100, 125, 150, 75, 200, 225, 250, 275, 300, or 325 nucleotides of the nucleotide sequence from nucleotide 475 to 800 of SEQ ID NO:24, or a complement thereof. The invention also features nucleic acid molecules which include a fragment of at least 50
(75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850, 900) nucleotides of the nucleotide sequence of SEQ ID NO:l, 11, or 24, or a complement thereof, wherein such nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or functional feature of a polypeptide of the invention.
The invention also features nucleic acid molecules which include a nucleotide sequence encoding a protein having an amino acid sequence that is at least 25% (or 35%, 45%, 55%, 65%, 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NO:3 or 13, the amino acid sequence encoded by the cDNA of ATCC Accession Number 207218, or a complement thereof.
The invention also features nucleic acid molecules which include a nucleotide sequence encoding a protein having an amino acid sequence that is at least 40% (or 45%, 55%, 65%, 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NO:26, the amino acid sequence encoded by the cDNA of ATCC Accession Number 207216, or a complement thereof. In preferred embodiments, the nucleic acid molecules have the nucleotide sequence of
SEQ ID NO:l, 11, 24 or the nucleotide sequence of the cDNA of ATCC Accession Number 207218, 207216.
Also within the invention are nucleic acid molecules which encode a fragment (e.g., a biologically active fragment) of a polypeptide having the amino acid sequence of SEQ ID NO: 3 or 13, or a fragment including at least 15 (25, 30, 50, 75, 100, 125, 150, 175, 200, or 225) contiguous amino acids of SEQ ID NO:3 or 13, or the amino acid sequence encoded by the cDNA of ATCC Accession Number 207218.
Also within the invention are nucleic acid molecules which encode a fragment of a polypeptide having the amino acid sequence of SEQ ID NO:26, or a fragment including at least 15 (25, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, or 280) contiguous amino acids of SEQ ID NO:26, or the amino acid sequence encoded by the cDNA of ATCC Accession Number 207216. The invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 13, or 26, or the amino acid sequence encoded by the cDNA of ATCC Accession Number 207218 or 207216, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule consisting of a nucleic acid sequence encoding SEQ ID NO:3, 13, or 26, the amino acid sequence encoded by the cDNA of ATCC Accession Number 207218 or 207216, or a complement thereof under stringent conditions. Also within the invention are isolated polypeptides or proteins having an amino acid sequence that is at least about 25%, preferably 35%, 45%, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 3 or 13, or the amino acid sequence encoded by the cDNA of ATCC Accession Number 207218.
Also within the invention are isolated polypeptides or proteins having an amino acid sequence that is at least about 40%, preferably 45%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:26 or the amino acid sequence encoded by the cDNA of ATCC Accession Number 207216.
Also within the invention are isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 90%, preferably 92%, 94%, 96%, or 98% identical to the nucleic acid sequence encoding SEQ ID NO:3, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 or 2, a complement thereof, or the non-coding strand of the cDNA of ATCC Accession Number 207218. Also within the invention are isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to the nucleic acid sequence encoding SEQ ID NO: 13, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l 1 or 12, a complement thereof, or the non-coding strand of the cDNA of ATCC Accession Number 207216.
Also within the invention are isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 30%, preferably 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% identical to the nucleic acid sequence encoding SEQ ID NO:26, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:24 or 25, a complement thereof, or the non-coding strand of the cDNA of ATCC Accession Number 207216. Also within the invention are polypeptides which are naturally occurring allelic variants of a polypeptide that includes the amino acid sequence of SEQ ID NO:3, 13, or 26, or the amino acid sequence encoded by the cDNA of ATCC Accession Number 207218 or 207216, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule having the sequence of SEQ ID NO:l, 2, 11, 12, 24, or 25, or a complement thereof under stringent conditions.
The invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, the cDNA of ATCC Accession Number 207218, or a complement thereof. In other embodiments, the nucleic acid molecules are at least 500 (525, 550, 575, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, or 1080) nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, the cDNA of ATCC Accession Number 207218, or a complement thereof.
The invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l 1 or a complement thereof. In other embodiments, the nucleic acid molecules are at least 400 (425, 475, 500, 525, 550, 575, 600, 650, 700, 750, 800, 850, 900, or 930) nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 11 or a complement thereof.
The invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:24, the cDNA of ATCC Accession Number 207216, or a complement thereof. In other embodiments, the nucleic acid molecules are at least 400 (425, 475, 500, 525, 550, 575, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, or 1070) nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:24, the cDNA of ATCC Accession Number 207216, or a complement thereof.
The invention also features nucleic acid molecules at least 15, preferably 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or more nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, 11, or 24, the cDNA of ATCC Accession Number 207216 or 207218, or a complement thereof, wherein such nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or functional feature of a polypeptide of the invention.
In one embodiment, the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a nucleic acid of the invention. Another aspect of the invention provides vectors, e.g., recombinant expression vectors, comprising a nucleic acid molecule of the invention. In another embodiment, the invention provides host cells containing such a vector or a nucleic acid molecule of the invention. The invention also provides methods for producing a polypeptide of the invention by culturing, in a suitable medium, a host cell of the invention containing a recombinant expression vector such that a polypeptide is produced.
Another aspect of this invention features isolated or recombinant proteins and polypeptides of the invention. Preferred proteins and polypeptides possess at least one biological activity possessed by the corresponding naturally-occurring human polypeptide. An activity, a biological activity, or a functional activity of a polypeptide or nucleic acid of the invention refers to an activity exerted by a protein, polypeptide or nucleic acid molecule of the invention on a responsive cell as determined in vivo, or in vitro, according to standard techniques. Such activities can be a direct activity, such as an association with or an enzymatic activity on a second protein, or an indirect activity, such as a cellular signaling activity mediated by interaction of the protein with a second protein. For TANGO 269, biological activities can include, e.g., (1) the ability to form, e.g., stabilize, promote, inhibit, or disrupt, protein-protein interactions (e.g., homophilic and/or heterophilic) with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; (3) the ability to interact with a TANGO 269 receptor; and (4) the ability to modulate the function, migration, proliferation (e.g., suppress cell growth), and/or differentiation of cells, e.g., cells in tissues in which it is expressed (see description of expression data below) and, in particular, hepatocytes.
Other TANGO 269 biological activities can include, e.g., (1) the ability to bind proteins, e.g., Hpoproteins, e.g., low-density Hpoproteins, e.g., oxidatively modified low-density Hpoproteins; (2) the ability to modulate internalization of proteins, e.g., Hpoproteins, e.g., low- density Hpoproteins, e.g., oxidatively modified low-density Hpoproteins; (3) the ability to modulate degradation, e.g., proteolytic degradation, of proteins, e.g., Hpoproteins, e.g., low- density Hpoproteins, e.g., oxidatively modified low-density Hpoproteins; (4) the ability to modulate, e.g., increase, uptake of proteins, e.g., Hpoproteins, e.g., low-density Hpoproteins, e.g., oxidatively modified low-density Hpoproteins, by cells, e.g., macrophages and muscle cells, e.g., smooth muscle cells; (5) the ability to modulate the function of a cell expressing LOX-1; (6) the ability to modulate the binding of a protein, e.g., Ox-LDL (oxidized low-density lipoprotein), to a cell which expresses LOX-1; and (7) the ability to modulate the binding of a protein, e.g., Ox- LDL, to LOX-1. Other TANGO 269 biological activities can include, e.g., (1) the ability to modulate, e.g., prevent, lipid deposition, e.g., in arteries; (2) the ability to modulate, e.g., induce or prevent, changes in cells, e.g., transformation of cells (e.g., macrophages and smooth muscle cells) into foam cells and functional alteration of cells (e.g., endothelial cells, e.g., intimal neovascular endothelial cells); (3) the ability to bind and phagocytose cells, e.g., aged and apoptotic cells; (4) the ability to modulate, e.g., prevent, intimal thickening, e.g., by preventing deposit and buildup of substances (e.g., lipids) on blood vessel walls; and (5) the ability to remove debris, e.g., aged and apoptotic (dead) cells, from blood vessel walls.
Still other TANGO 269 biological activities can include, e.g., (1) the ability to modulate homeostasis, e.g., vascular homeostasis, e.g., by modulating, e.g., preventing the impairment of, nitric oxide production; (2) the ability to modulate, e.g., inhibit, the expression of molecules, e.g., adhesion molecules (e.g., leukocyte adhesion molecules) and growth factors (e.g., smooth-muscle growth factors); (3) the ability to alter, e.g., increase, expression in response to stimuli, e.g., TNF- α, shear stress, and pathophysiological stimuli relevant to disorders (e.g., atherosclerosis and inflammation).
Still other TANGO 269 biological activities can include, e.g., (1) the ability to form, e.g., stabilize, promote, facilitate, inhibit, or disrupt, cell-extracellular matrix interactions, e.g., adhesion between cells and extracellular matrix; (2) the ability to form, e.g., stabilize, promote, facilitate, inhibit, or disrupt, cell to cell and cell to blood product interaction, e.g., between leukocytes and platelets or leukocytes and vascular endothelial cells; and (3) the ability to recognize large molecules, e.g., carbohydrates.
In addition, TANGO 269 biological activities can also include, e.g., the ability to perform one or more of the functions of LOX-1 described, for example, in the following: Sawamura et al. (1997) Nature 386:73-77; Kataoka et al. (1999) Circulation 99:3110-3117; and Kita (1999) Circulation Research 84 : 1113 - 1115 , the contents of all of which are incorporated herein by reference.
For TANGO 298, biological activities can include, e.g., (1) the ability to form, e.g., stabilize, promote, facilitate, inhibit, or disrupt, protein-protein interactions (e.g., homophilic and/or heterophilic) with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; and (3) the ability to interact with a TANGO 298 receptor.
Other TANGO 298, biological activities can include, e.g., (1) the ability to alter e.g., enzymatically cleave (e.g., by hydrolysis), proteins, e.g., alternative complement factor B; (2) the ability to act as a proteolytic enzyme cleaving either itself or other substrates; (3) the ability to modulate, e.g., activate, the alternative complement cascade; (4) the ability to alter, e.g., lyse (e.g., via activation of the alternative complement cascade), cells, e.g., red blood cells; (5) the ability to modulate systemic energy balance, e.g., via the alternative complement cascade; and (6) the ability to modulate host defense, e.g., by suppressing infection or invasion by foreign agents, e.g., bacteria, viruses, parasites, neoplastic cells, e.g., via the alternative complement cascade. Still other TANGO 298 biological activities can include, e.g., (1) the ability to modulate, e.g., inhibit or activate, the immune response, e.g., by modulating inflammation; (2) the ability to modulate, e.g., inhibit, cellular (e.g., immune cells, e.g., neutrophils, monocytes, lymphocytes) and tissue destruction; and (3) the ability to modulate adipocyte proliferation, differentiation, and/or function.
Other TANGO 298 biological activities can include, e.g., (1) the ability to modulate cyclic hematopoiesis, e.g., the ability to modulate the cyclic change in numbers of blood cells (e.g., neutrophils, monocytes, platelets, lymphocytes, and erythrocytes) in circulation. In addition, the various biological activities of TANGO 298 described herein can be modulated by serpins, a gene family that encompasses a wide variety of protein molecules, such as protease inhibitors. Thus, other TANGO 298 modulators, e.g., antisense molecules and antibodies, can also be used to modulate, e.g., inhibit, these TANGO 298 biological activities.
In addition, TANGO 298 biological activities can include, e.g., the ability to perform one or more of the functions of adipsin described, for example, in the following: White et al. (1992) J Biol Chem. 13:9210-9213; Spiegelman et al. (1989) J Biol Chem. 264:1811-1815; Yamauchi et al. (1994) J mmunol. 152:3645-3653; Meri et al. (1998) Vox Sang. 74 Suppl 2:291-302; and PCT Publication Number WO 90/06365, the contents of all of which are incorporated herein by reference.
In one embodiment, a polypeptide of the invention has an amino acid sequence sufficiently identical to an identified domain of a polypeptide of the invention. As used herein, the term "sufficiently identical" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity. For example, amino acid or nucleotide sequences which contain a common structural domain having about 60% identity, preferably 65% identity, more preferably 75%, 85%, 95%, 98% or more identity are defined herein as sufficiently identical.
In one embodiment, a TANGO 269 protein includes at least one of the following domains: a signal sequence, an extracellular link domain, and a C-type lectin domain. In another embodiment, a TANGO 269 protein includes two or more of the following domains: a signal sequence, an extracellular link domain, and a C-type lectin domain, and is a type II transmembrane protein.
In one embodiment, a TANGO 298 protein includes at least one of the following domains: a signal sequence and a trypsin domain.
In another embodiment, a nucleic acid molecule of the present invention encodes a TANGO 269 protein which includes at least one of the following domains: a signal sequence, an extracellular link domain, and a C-type lectin domain. In another embodiment, a nucleic acid molecule of the present invention encodes a TANGO 269 protein which includes two or more of the following domains: a signal sequence, an extracellular link domain, and a C-type lectin domain.
In another embodiment, a nucleic acid molecule of the present invention encodes a TANGO 298 protein which includes at least one of the following domains: a signal sequence and a trypsin domain. The polypeptides of the present invention, or biologically active portions thereof, can be operably linked to a heterologous amino acid sequence to form fusion proteins. The invention further features antibodies, such as monoclonal or polyclonal antibodies or fragments thereof, that specifically bind to a polypeptide of the invention. The antibodies of the invention can be conjugated antibodies comprising, for example, therapeutic or diagnostic agents. For example, the antibodies can be conjugated to a therapeutic moiety such as a chemotherapeutic cytotoxin, e.g., a cytostatic or cytocidal agent (e.g., paclitaxol, cytochalasin B or diphtheria toxin), a thrombotic or anti-angiogenic agent or a radioactive or fluorescent label.
In addition, the polypeptides of the invention or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
In another aspect, the present invention provides methods for detecting the presence of the activity or expression of a polypeptide of the invention in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of activity such that the presence of activity is detected in the biological sample. In another aspect, the invention provides methods for modulating activity of a polypeptide of the invention comprising contacting a cell with an agent that modulates (inhibits or stimulates) the activity or expression of a polypeptide of the invention such that activity or expression in the cell is modulated. In one embodiment, the agent is an antibody that specifically binds to a polypeptide of the invention.
In another embodiment, the agent modulates expression of a polypeptide of the invention by modulating transcription, splicing, or translation of an mRNA encoding a polypeptide of the invention. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an mRNA encoding a polypeptide of the invention.
The present invention also provides methods to treat a subject having a disorder characterized by aberrant activity of a polypeptide of the invention or aberrant expression of a nucleic acid of the invention by administering an agent which is a modulator of the activity of a polypeptide of the invention or a modulator of the expression of a nucleic acid of the invention to the subject. In one embodiment, the modulator is a protein of the invention. In another embodiment, the modulator is a nucleic acid of the invention. In other embodiments, the modulator is a peptide, peptidomimetic, or other small molecule.
The present invention also provides diagnostic assays for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of: (i) aberrant modification or mutation of a gene encoding a polypeptide of the invention, (ii) mis-regulation of a gene encoding a polypeptide of the invention, and (iii) aberrant post-translational modification of the invention wherein a wild-type form of the gene encodes a protein having the activity of the polypeptide of the invention.
In another aspect, the invention provides a method for identifying a compound that binds to or modulates the activity of a polypeptide of the invention. In general, such methods entail measuring a biological activity of the polypeptide in the presence and absence of a test compound and identifying those compounds which alter the activity of the polypeptide.
The invention also features methods for identifying a compound which modulates the expression of a polypeptide or nucleic acid of the invention by measuring the expression of the polypeptide or nucleic acid in the presence and absence of the compound.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
Brief Description of the Drawings
Figure 1 depicts the cDNA sequence of human TANGO 269 (SEQ ID NO: 1) and the predicted amino acid sequence of TANGO 269 (SEQ ID NO:3). The open reading frame of SEQ ID NO:l extends from nucleotide 66 to nucleotide 749 of SEQ ID NO:l (SEQ ID NO:2).
Figure 2 depicts a hydropathy plot of human TANGO 269. Relatively hydrophobic regions of the protein are above the dashed horizontal line, and relatively hydrophilic regions of the protein are below the dashed horizontal line. The cysteine residues (cys) and potential N- glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1 to 47 of SEQ ID NO:3; SEQ ID NO:5) on the left from the mature protein (amino acids 48 to 228 of SEQ ID NO:3; SEQ ID NO:4) on the right. Thicker gray horizontal bars below the dashed horizontal line indicate extracellular ("out"), transmembrane ("TM"), and intracellular ("in") regions of the molecule.
Figures 3A-3B depict a local alignment of the nucleotide sequence of human LOX-1 (SEQ ID NO:21; GenBank Accession Number ABO 10710) and the nucleotide sequence of human TANGO 269 (SEQ ID NO:l). The nucleotide sequences of human LOX-1 and human TANGO 269 are about 46% identical over a 1084 base pair overlap. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
Figures 4A-4B depict an alignment of the nucleotide sequence of the open reading frames of human LOX-1 (nucleotides 62 to 880 of SEQ ID NO:22 (GenBank Accession Number AB010710)) and human TANGO 269 (SEQ ID NO:2). The nucleotide sequences of the open reading frames of human LOX-1 and human TANGO 269 (SEQ ID NO:l) are 44.9 % identical. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
Figure 5 depicts an alignment of the amino acid sequence of human LOX-1 (SEQ ID NO:23 (GenBank Accession Number AB010710)) and the amino acid sequence of human TANGO 269 (SEQ ID NO:3). The amino acid sequences of human LOX-1 and human TANGO 269 are 23.8% identical. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4. Figure 6 depicts the cDNA sequence of murine TANGO 269 (SEQ ID NO: 11 ) and the predicted amino acid sequence of murine TANGO 269 (SEQ ID NO: 13). The open reading frame of SEQ ID NO: l 1 extends from nucleotide 106 to nucleotide 792 of SEQ ID NO: l 1 (SEQ ID NO: 12).
Figure 7 depicts a hydropathy plot of murine TANGO 269. Relatively hydrophobic regions of the protein are above the dashed horizontal line, and relatively hydrophilic regions of the protein are below the dashed horizontal line. The cysteine residues (cys) and potential N- glycosylation sites (Ngty) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1 to 46 of SEQ ID NO: 13; SEQ ID NO: 15) on the left from the mature protein (amino acids 47 to 229 of SEQ ID NO: 13; SEQ ID NO: 14) on the right. Thicker gray horizontal bars below the dashed horizontal line indicate extracellular ("out"), transmembrane ("TM"), and intracellular ("in") regions of the molecule.
Figures 8A-8B depict an alignment of the open reading frames of the nucleotide sequence of murine TANGO 269 (SEQ ID NO: 11) and the nucleotide sequence of human TANGO 269 (SEQ ID NO: 1). The open reading frames of murine TANGO 269 and human TANGO 269 are 74.1% identical. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4. There is also about a 57% identity between the human and murine TANGO 269 full length nucleic acid sequences. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
Figure 9 depicts an alignment of the amino acid sequence of murine TANGO 269 (SEQ ID NO:13) and the amino acid sequence of human TANGO 269 (SEQ ID NO:3). The amino acid sequences of murine TANGO 269 and human TANGO 269 are 63.0 % identical. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
Figure 10 depicts the cDNA sequence of human TANGO 298 (SEQ ID NO:25) and the predicted amino acid sequence of TANGO 298 (SEQ ID NO:27). The open reading frame of SEQ ID NO:25 extends from nucleotide 31 to nucleotide 879 of SEQ ID NO:25 (SEQ ID NO:26).
Figure 11 depicts a hydropathy plot of human TANGO 298. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) and potential N- glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1 to 31 of SEQ ID NO:27; SEQ ID NO:29) on the left from the mature protein (amino acids 32 to 283 of SEQ ID NO:27; SEQ ID NO:28) on the right. Thicker gray horizontal bars below the dashed horizontal line indicate extracellular ("out"), transmembrane ("TM"), and intracellular ("in") regions of the molecule. Figures 12A-12B depict an alignment of the nucleotide sequence of human adipsin (SEQ
ID NO:32; GenBank Accession Number NM 001928) and the nucleotide sequence of human TANGO 298 (SEQ ID NO:25). The nucleotide sequences of human adipsin and human TANGO 298 are 51.8% identical. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4. Figures 13A-13B depict an alignment of the nucleotide sequence of the open reading frames of human adipsin (nucleotides 55 to 738 of SEQ ID NO:32 (SEQ ID NO:33); GenBank Accession Number NM_001928) and human TANGO 298 (SEQ ID NO:26). The nucleotide sequences of the open reading frames of human adipsin and human TANGO 298 (SEQ ID NO:26) are 50.8% identical. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
Figure 14 depicts an alignment of the amino acid sequence of human adipsin (SEQ ID NO: 34; GenBank Accession Number NM_001919) and the amino acid sequence of human TANGO 298 (SEQ ID NO:27). The amino acid sequences of human adipsin and human TANGO 298 are 34.0% identical. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
Figure 15 shows a TaqMan analysis of human TANGO 298 expression in various tissues and cell types relative to expression in tonsil.
Figures 16A-16B show a TaqMan analysis of human TANGO 298 expression in various tissues and cell types relative to expression in CDl 5 CDl lb+ cells from mobilized bone marrow (mBMCF157CDl lb+).
Figure 77 shows an alignment between TANGO 298 and HUMGS00642, an EST (accession number D19687) which maps to chromosome 19, between markers D19S886 and D19S216. In the region between D19S886 and D19S216 lies 19pl3.3, the area which is the location for cyclic hematopoiesis (also called cyclic neutropenia).
Detailed Description of the Invention The TANGO 269 proteins and nucleic acid molecules comprise a family of molecules having certain conserved structural and functional features. Similarly, the TANGO 298 proteins and nucleic acid molecules comprise a family of molecules having conserved functional and structural features. As used herein, the term "family" is intended to mean two or more proteins or nucleic acid molecules having a common structural domain and having sufficient amino acid or nucleotide sequence identity as defined herein. Family members can be from either the same or different species. For example, a family can comprises two or more proteins of human origin, or can comprise one or more proteins of human origin and one or more of non-human origin. Members of the same family may also have common structural domains.
For example, TANGO 269 proteins and TANGO 298 proteins of the invention have signal sequences. As used herein, a "signal sequence" includes a peptide of at least about 15 or 20 amino acid residues in length which occurs at the N-terminus of secretory and membrane- bound proteins and which contains at least about 70% hydrophobic amino acid residues such as alanine, leucine, isoleucine, phenylalanine, proline, tyrosine, tryptophan, or valine. In a preferred embodiment, a signal sequence contains at least about 15 to 25 amino acid residues, preferably about 18 to 22 amino acid residues, and has at least about 60-80%, more preferably 65-75%, and more preferably at least about 70% hydrophobic residues. A signal sequence serves to direct a protein containing such a sequence to a lipid bilayer. Thus, in one embodiment, a human TANGO 269 protein contains a signal sequence of about amino acids 1 to 44, 1 to 45, 1 to 46, 1 to 47, 1 to 48, or 1 to 49 of SEQ ID NO:3 (SEQ ID NO:5). In such embodiments of the invention, the domains and mature protein resulting from cleavage of such signal peptides are also included herein. For example, the cleavage of a signal sequence consisting of amino acids 1 to 47 results in a mature human TANGO 269 protein corresponding to amino acids 48 to 228.
In another embodiment, a murine TANGO 269 protein contains a signal sequence of about amino acids 1 to 44, 1 to 45, 1 to 46, 1 to 47, or 1 to 48 of SEQ ID NO: 13 (SEQ ID NO: 15). In such embodiments of the invention, the domains and mature protein resulting from cleavage of such signal peptides are also included herein. For example, the cleavage of a signal sequence consisting of amino acids 1 to 46 results in a mature murine TANGO 269 protein corresponding to amino acids 47 to 229.
In another embodiment, a TANGO 298 protein contains a signal sequence of about amino acids 1 to 28, 1 to 29, 1 to 30, 1 to 31, 1 to 32, or 1 to 33 of SEQ ID NO:27 (SEQ ID NO:29). In such embodiments of the invention, the domains and mature protein resulting from cleavage of such signal peptides are also included herein. For example, the cleavage of a signal sequence consisting of amino acids 1 to 31 results in a mature TANGO 298 protein corresponding to amino acids 32 to 283.
TANGO 269 family members can also include a C-type lectin domain. C-type lectin domains are found in a variety of families, including type-II membrane proteins, large proteoglycans, proteins collectively known as collectins, and proteins collectively known as selectins, which are cell adhesion molecules implicated in the interaction between leukocytes and platelets or leukocytes and vascular endothelium. As used herein, the term "C-type lectin domain" refers to a calcium-dependent carbohydrate-recognition domain that includes about 60 to 150 amino acid residues, preferably about 70 to 140 amino acid residues, and more preferably about 80 to 130 amino acid residues, and contains about 2 to 10, preferably 3 to 8, still more preferably 4, cysteine residues. Typically, the four cysteines are conserved, and involved in two disulfide bonds. In addition, a C-type lectin domain includes at least the following consensus sequence: C-[LIVMFATG]-Xaa(nl)-[WLF]-Xaa-[DNSR]-Xaa(n2)-C-Xaa(n3)-[FYWLIVSTA]- [LIVSTAM]-C, wherein C is a cysteine residue, brackets ([]) indicate one of the group of amino acids contained therein, L is a leucine residue, I is an isoleucine residue, V is a valine residue, M is a methionine residue, F is a phenylalanine residue, A is an alanine residue, T is a threonine residue, G is a glycine residue, Xaa is any amino acid, nl is about 1 to 20 amino acid residues, more preferably about 1 to 15 amino acid residues, and more preferably 2 to 12 amino acid residues in length, W is a tryptophan residue, D is an aspartic acid residue, N is an asparagine residue, S is a serine residue, R is an arginine residue, n2 is about 1 to 15 amino acid residues, more preferably about 1 to 10 amino acid residues, and more preferably about 2 to 7 amino acid residues in length, n3 is about 2 to 10 amino acid residues, more preferably about 3 to 8 amino acid residues, and more preferably about 5 to 6 amino acid residues in length, and Y is a tyrosine residue.
In one embodiment, a TANGO 269 family member includes an C-type lectin domain having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 127 to 215 of SEQ ID NO:3 or 128 to 216 of SEQ ID NO: 13, which are the C-type lectin domains of human and murine TANGO 269, respectively (the C-type lectin domains are also represented as SEQ ID NO:8 and 19). In another embodiment, a TANGO 269 family member includes a C-type lectin domain having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 127 to 215 of SEQ ID NO:3 or 128 to 216 of SEQ ID NO:13 (SEQ ID NO:8 and 19), includes 4 conserved cysteine residues, and a C-type lectin domain consensus sequence as described herein. In yet another embodiment, a TANGO 269 family member includes a C-type lectin domain having an amino acid sequence that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 127 to 215 of SEQ ID NO :3 or 128 to 216 of SEQ ID NO: 13 (SEQ ID NO:8 and 19), includes 4 conserved cysteine residues, a C-type lectin domain consensus sequence as described herein, and has at least one TANGO 269 biological activity as described herein. In one embodiment, a TANGO 269 family member has the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 13 wherein the 4 conserved cysteine residues are located at amino acid positions 130, 195, 208, and 216 of the human protein (SEQ ID NO:3), and amino acid positions 129, 194, 207, and 215 of the mouse protein (SEQ ID NO: 13), and the C-type lectin domain consensus sequence is located at amino acid residues 127 to 215 of the human protein and amino acids 128 to 216 of the mouse protein.
A TANGO 269 family member can also include an extracellular link domain. As used herein, the term "extracellular link domain" refers to a protein domain that includes about 10-40 amino acid residues, preferably about 15-35 amino acid residues, more preferably about 18-30 amino acid residues, and most preferably about 20-28 amino acid residues. Typically, an extracellular link domain includes the following consensus sequence: R-Xaa(2)-L-T-Xaa-E-E- Xaa(4)-C-Xaa(4)-A, wherein R is an arginine residue, Xaa is any amino acid, L is a leucine residue, T is a threonine residue, E is a glutamic acid residue, C is a cysteine residue, and A is an alanine residue. In one embodiment, a TANGO 269 family member includes an extracellular link domain having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 117 to 136 of SEQ ID NO:3 or 110 to 137 of SEQ ID NO: 13, which are the extracellular link domains of human and murine TANGO 269, respectively (the extracellular link domains are also represented as SEQ ID NO:7 and 17). In another embodiment, a TANGO 269 family member includes an extracellular link domain having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 117 to 136 of SEQ ID NO:3 or 110 to 137 of SEQ ID NO:13 (SEQ ID NO:7 and 17), includes one conserved cysteine residue and two conserved glutamic acid residues, and an extracellular link domain consensus sequence as described herein. In yet another embodiment, a TANGO 269 family member includes an extracellular link domain having an amino acid sequence that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 117 to 136 of SEQ ID NO:3 or 110 to 137 of SEQ ID NO: 13 (SEQ ID NO:7 and 17), includes one conserved cysteine residue and two conserved glutamic acid residues, an extracellular link domain consensus sequence as described herein, and has at least one TANGO 269 biological activity as described herein. In one embodiment, a TANGO 269 family member has the amino acid sequence of SEQ
ID NO:3 or SEQ ID NO: 13 wherein the conserved cysteine residue is located at amino acid position 129 of the human protein (SEQ ID NO:3), and amino acid position 130 of the mouse protein (SEQ ID NO: 13), the conserved glutamic acid residues are located at amino acid positions 123 and 124 of the human protein (SEQ ID NO:3), and amino acid positions 124 and 125 of the mouse protein (SEQ ID NO: 13), and the extracellular link domain consensus sequence is located at amino acid residues 117 to 136 of the human protein and amino acids 110 to 137 of the mouse protein.
A TANGO 298 family member can include a trypsin domain. Trypsin domains are typically found in serine proteases, and can be found in, among other proteins, blood coagulation factors VII, XI, and X, thrombin, plasminogen, tryptases, mast cell proteases, and members of the complement system, which is known for regulation of energy balance and suppression of infectious agents. As used herein, the term "trypsin domain" refers to a protein domain that can be found in serine proteases and other proteins with catalytic activity, and includes about 100-400 amino acid residues, preferably about 150-350 amino acid residues, more preferably about 200- 300 amino acid residues, and most preferably about 225-260 amino acid residues. Typically, a trypsin domain has about 2 to 15, preferably about 5 to 10, more preferably about 8 conserved cysteine residues, about 2 to 25, preferably about 5 to 15, more preferably about 13 conserved glycine residues, and about 2 to 15, preferably about 5 to 12, more preferably about 9 conserved valine residues. A trypsin domain also typically has at least one of the following two consensus sequences: [LV]-[ST]-A-A-H-C, wherein L is a leucine residue, V is a valine residue, S is a serine residue, T is a threonine residue, A is an alanine residue, H is a histidine residue, and C is a cysteine residue; and [DG]-[AF]-C-Xaa-[GA]-D-S-G-G-P-L-V-C, wherein D is a aspartic acid residue, G is a glycine residue, F is a phenylalanine residue, Xaa is any amino acid, and P is a proline residue. In the first consensus sequence, the histidine residue serves as the active site, to which an aspartic acid residue binds in order to confer upon the domain its catalytic activity.
In one embodiment, a TANGO 298 family member includes a trypsin domain having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 34 to 258 of SEQ ID NO:27, which is the trypsin domain of human TANGO 298 (the trypsin domain is also represented as SEQ ID NO:31). In another embodiment, a TANGO 298 family member includes a trypsin domain having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 34 to 258 of SEQ ID NO:27 (SEQ ID NO:31), includes 8 conserved cysteine residues, 13 conserved glycine residues, and 9 valine residues, and a trypsin domain consensus sequence as described herein. In yet another embodiment, a TANGO 298 family member includes a trypsin domain having an amino acid sequence that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 34 to 258 of SEQ ID NO:27 (SEQ ID NO:31), includes 8 conserved cysteine residues, 13 conserved glycine residues, and 9 valine residues, a trypsin domain consensus sequence as described herein, and has at least one TANGO 298 biological activity as described herein. In one embodiment, a TANGO 298 family member has the amino acid sequence of SEQ
ID NO:27 wherein the 8 conserved cysteine residues are located at amino acid positions 59, 75, 157, 188, 202, 214, 224, and 239, the 13 conserved glycine residues are located at amino acid positions 36, 37, 55, 60, 61, 89, 154, 161, 163, 194, 219, 220, 230, the 9 valine residues are located at amino acid positions 69, 86, 138, 159, 180, 223, 232, 247, and 251, and the active site histidine residue is located at amino acid position 74 of the protein (SEQ ID NO:3), and the trypsin domain consensus sequence is located at amino acid residues 34 to 258 of the protein. Various features of TANGO 269 and TANGO 298 are summarized below. Human TANGO 269
A cDNA encoding human TANGO 269 was identified by analyzing the sequences of clones present in a human adrenal gland cDNA library.
This analysis led to the identification of a clone, jthAa35cl2, encoding full-length human TANGO 269. The human TANGO 269 cDNA of this clone is 1084 nucleotides long (Figure 1 ; SEQ ID NO: 1). The open reading frame of this cDNA, nucleotides 72 to 755 of SEQ ID NO: 1 (SEQ ID NO:2), encodes a 228 amino acid type II transmembrane protein (Figure 1; SEQ ID NO:3).
The signal peptide prediction program SIGNALP (Nielsen et al. (1997) Protein Engineering 10:1-6) predicted that human TANGO 269 includes a 47 amino acid signal peptide (amino acid 1 to about amino acid 47 of SEQ ID NO:3)(SEQ ID NO:5) preceding the mature TANGO 269 protein (corresponding to about amino acid 48 to amino acid 228 of SEQ ID NO:3)(SEQ ID NO:4). The TANGO 269 protein molecular weight is 26.6 kDa prior to the cleavage of the signal peptide, 21.4 kDa after cleavage of the signal peptide. An N-glycosylation site having the sequence NRTG is found from amino acids 68 to 71 of SEQ ID NO:3. A second N-glycosylation site having the sequence NLTW is found from amino acids 119 to 122. A third N-glycosylation site having the sequence NATL is found from amino acids 133 to 136. A protein kinase C phosphorylation site having the sequence TRK is found from amino acids 14 to 16. A second protein kinase C phosphorylation site having the sequence SHK is found from amino acids 95 to 97. A casein kinase II phosphorylation site having the sequence SPCD is found from amino acids 99 to 102. A second casein kinase II phosphorylation site having the sequence TWEE is found from amino acids 121 to 124. A third casein kinase II phosphorylation site having the sequence TFCE is found from amino acids 205 to 208. A fourth casein kinase II phosphorylation site having the sequence TKVD is found from amino acids 222 to 225. A tyrosine kinase phosphorylation site having the sequence
RYYGDSCY is found from amino acids 106 to 113. An N-myristoylation site having the sequence GMVVGL is found from amino acids 43 to 48. A second N-myristoylation site having the sequence GSVISE is found from amino acids 174 to 179. A third N-myristoylation site having the sequence GNMNCA is found from amino acids 190 to 195. Chromosomal mapping was performed by computerized comparison of human TANGO 269 cDNA sequences against a chromosomal mapping database in order to identify the approximate location of the gene encoding human TANGO 269 protein. This analysis showed that the gene was located on chromosome 12 between markers D12S98 and D12S358. LOX-1 is also mapped to chromosome 12, as seen in Aoyama et al. (1999) Biochem J. 339: 177-184, the contents of which are incorporated herein by reference.
Human TANGO 269 includes an extracellular link domain (about amino acids 117 to 136 of SEQ ID NO:3; SEQ ID NO:7) and a C-type lectin domain (about amino acids 127 to 215 of SEQ ID NO:3; SEQ ID NO:8). Figures 3A-3B show a local alignment of the human TANGO 269 full length nucleic acid sequence (SEQ ID NO: 1) with the human LOX-1 full length nucleic acid sequence (SEQ ID NO:21). Figures 4A-4B show an alignment of the human TANGO 269 nucleotide coding region (SEQ ID NO:2) with the human LOX-1 nucleotide coding region (SEQ ID NO:22). Figure 5 shows an alignment of the human TANGO 269 protein sequence (SEQ ID NO:3) with the human LOX-1 protein sequence (SEQ ID NO:23). As shown in Figure 5, the human TANGO 269 signal sequence (SEQ ID NO:5) is represented by amino acids 1-47 (and encoded by nucleotides 66 to 206 of SEQ ID NO:l (SEQ ID NO:6)), and the human LOX-1 signal sequence is represented by amino acids 1 to 61 (and encoded by nucleotides 62-244 of SEQ ID NO:21). The human TANGO 269 extracellular link domain sequence (SEQ ID NO: 7) is represented by amino acids 117 to 136 (and encoded by nucleotides 414-473 of SEQ ID NO:l (SEQ ID NO:9)). The human TANGO 269 C-type lectin domain-type domain sequence (SEQ ID NO: 8) is represented by amino acids 127 to 215 (and encoded by nucleotides 444-710 of SEQ ID NO:l (SEQ ID NO: 10)), and the human LOX-1 C-type lectin domain-type domain sequence is represented by amino acids 170 to 264 (and encoded by nucleotides 563 to 857 of SEQ ID NO:21). Figures 3A-3B and Figures 4A-4B show that there is about a 46% identity over 1084 base pairs between the human TANGO 269 nucleic acid molecule and the human LOX-1 nucleic acid molecule, and an overall 44.9% identity between the open reading frame of the human TANGO 269 nucleic acid molecule and the open reading frame of the human LOX-1 nucleic acid molecule, respectively. The amino acid alignment in Figure 5 shows a 23.8% overall amino acid sequence identity between human TANGO 269 and human LOX-1.
Human TANGO 269 is homologous to murine TANGO 269. Figures 8A-8B depict an alignment of the open reading frame of murine TANGO 269 (SEQ ID NO: 12) with the open reading frame of human TANGO 269 (SEQ ID NO:2). Figure 9 depicts an alignment of the amino acid sequence of murine TANGO 269 (SEQ ID NO: 13) with the amino acid sequence of human TANGO 269 (SEQ ID NO:3). As shown in Figure 9, the human TANGO 269 signal sequence (SEQ ID NO:5) is represented by amino acids 1 to 47 (and encoded by nucleotides 66 to 206 of SEQ ID NO: 1 (SEQ ID NO:6)). The murine TANGO 269 signal sequence (SEQ ID NO: 15) is represented by amino acids 1 to 46 (and encoded by nucleotides 106 to 243 of SEQ ID NO: 11 (SEQ ID NO: 16)) The human TANGO 269 extracellular link domain sequence (SEQ ID NO:7) is represented by amino acids 117 to 136 (and encoded by nucleotides 414 to 473 of SEQ ID NO:l (SEQ ID NO:9)), and the murine TANGO 269 extracellular link domain sequence (SEQ ID NO: 19) is represented by amino acids 110 to 137 (and encoded by nucleotides 433 to 516 of SEQ ID NO: 11 (SEQ ID NO: 19)). The human TANGO 269 C-type lectin domain-type domain sequence (SEQ ID NO:8) is represented by amino acids 127 to 215 (and encoded by nucleotides 444 to 713 of SEQ ID NO:l (SEQ ID NO: 10)), and the murine TANGO 269 C-type lectin domain-type domain sequence is represented by amino acids 128 to 216 (and encoded by nucleotides 487 to 753 of SEQ ID NO: 11 (SEQ ID NO:20)).
Figures 8A-8B and 9 show that there is an 74.1% identity between the human and murine TANGO 269 open reading frames, and a 63.0% identity between the TANGO 269 amino acid sequence and the murine TANGO 269 amino acid sequence. There is also about a 57% identity between the full length human TANGO 269 nucleic acid molecule and the full length murine TANGO 269 molecule.
Clone EpT269, which encodes human TANGO 269, was deposited with the American Type Culture Collection ( 10801 University Boulevard, Manassas, VA 20110-2209) on April 20, 1999 and assigned Accession Number 207218. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112. Figure 2 depicts a hydropathy plot of human TANGO 269. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1 to 47 of SEQ ID NO:3; SEQ ID NO:5) on the left from the mature protein (amino acids 48 to 228 of SEQ ID NO:3; SEQ ID NO:4) on the right. Thicker gray horizontal bars below the dashed horizontal line indicate extracellular ("out"), transmembrane ("TM"), and intracellular ("in") regions of the molecule. This hydropathy plot indicates that TANGO 269 is a type II transmembrane protein. Northern analysis of human TANGO 269 expression in human tissues showed that an approximately 1.0 kB transcript is highly expressed in human fetal liver and is weakly expressed in bone marrow and peripheral blood leukocytes. There was no expression seen in spleen, lymph node, and thymus. A second Northern analysis of different tissues showed an approximately 1.0 kB transcript highly expressed in adult liver and no expression in heart, brain, placenta, lung, skeletal muscle, kidney, and pancreas.
Murine TANGO 269
A cDNA encoding murine TANGO 269 was identified by analyzing the sequences of clones present in a mouse megakaryocyte cDNA library. This analysis led to the identification of a clone, jtmea040e07, encoding full-length murine TANGO 269. The murine TANGO 269 cDNA of this clone is 932 nucleotides long (Figure 6; SEQ ID NO:l 1). The open reading frame of this cDNA, nucleotides 106 to 792 of SEQ ID NO:l 1 (SEQ ID NO: 12), encodes a 229 amino acid type II transmembrane protein (Figure 6; SEQ ID NO: 13). The signal peptide prediction program SIGNALP (Nielsen et al. (1997) Protein
Engineering 10:1-6) predicted that murine TANGO 269 includes a 46 amino acid signal peptide (amino acid 1 to about amino acid 46 of SEQ ID NO:13)(SEQ ID NO: 15) preceding the mature murine TANGO 269 protein (corresponding to about amino acid 47 to amino acid 229 of SEQ ID NO: 13)(SEQ ID NO: 14). The TANGO 269 protein molecular weight is 26.1 kDa prior to the cleavage of the signal peptide, 21.2 kDa after cleavage of the signal peptide.
An N-glycosylation site having the sequence NLSA is found from amino acids 67 to 70 of SEQ ID NO: 13. A second N-glycosylation site having the sequence NLTW is found from amino acids 120 to 123. A third N-glycosylation site having the sequence NATL is found from amino acids 134 to 137. A fourth N-glycosylation site having the sequence NLSG is found from amino acids 184 to 187. A protein kinase C phosphorylation site having the sequence SVR is found from amino acids 155 to 157. A second protein kinase C phosphorylation site having the sequence SKK is found from amino acids 166 to 168. A third protein kinase C phosphorylation site having the sequence SCK is found from amino acids 207 to 209. A casein kinase II phosphorylation site having the sequence SSAE is found from amino acids 20 to 23. A second casein kinase II phosphorylation site having the sequence SCQE is found from amino acids 79 to 82. A third casein kinase II phosphorylation site having the sequence STFE is found from amino acids 93 to 96. A fourth casein kinase II phosphorylation site having the sequence TWEE is found from amino acids 122 to 125. A fifth casein kinase II phosphorylation site having the sequence SLTD is found from amino acids 144 to 147. A sixth casein kinase II phosphorylation site having the sequence SKKD is found from amino acids 166 to 169. A seventh casein kinase II phosphorylation site having the sequence SCKE is found from amino acids 207 to 210. An eighth casein kinase II phosphorylation site having the sequence TRVD is found from amino acids 223 to 226. A tyrosine kinase phosphorylation site having the sequence RYHGDSCY is found from amino acids 107 to 114. An N-myristoylation site having the sequence GLWGL is found from amino acids 42 to 47. A second N-myristoylation site having the sequence GINLSG is found from amino acids 182 to 187.
Murine TANGO 269 includes an extracellular link domain (about amino acids 110 to 137 of SEQ ID NO:13; SEQ ID NO: 17) and a C-type lectin domain (about amino acids 128 to 216 of SEQ ID NO:13; SEQ ID NO:18).
Figure 7 depicts a hydropathy plot of murine TANGO 269. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1 to 46 of SEQ ID NO:13; SEQ ID NO:15) on the left from the mature protein (amino acids 47 to 229 of SEQ ID NO:13; SEQ ID NO:14) on the right. Thicker gray horizontal bars below the dashed horizontal line indicate extracellular ("out"), transmembrane ("TM"), and intracellular ("in") regions of the molecule. This hydropathy plot indicates that TANGO 269 is a type II transmembrane protein.
Northern analysis of murine TANGO 269 expression in murine tissues showed that an approximately 1.0 kB transcript is highly expressed in liver, moderately expressed in spleen, and weakly expressed in heart. There was no expression seen in brain, lung, skeletal muscle, kidney, and testis. A murine in situ expression analysis of TANGO 269 revealed expression only in the developing fetal liver, with a multifocal signal pattern suggestive of a scattered cell population. No signal was detected in adult liver or spleen. The signal pattern is suggestive of megakaryocyte expression.
Mice in which a transcription factor responsible for proper development of erythrocytes and megakaryocytes (and which is involved in other hematopoietic cell lineages) is knocked out of the gata-1 gene were produced. Gata-1 is a transcription factor involved in the development of hematopoietic cell lineages: gata-1 expression is required for proper development of erythrocytes and megakaryocytes. Although deletion of the gata-1 gene is lethal at the embryonic stage due to a failure to form red blood cells, deletion of only the element of the gata-1 gene responsible for megakaryocyte-specific expression (a lOkb region of genomic DNA containing a megakaryocyte specific DNase I hypersensitive) is not lethal and results in a reduction in gata-1 expression in the megakaryocyte without affecting gata-1 expression in red blood cells. The megakaryocytes of mice with this element of the gata-1 gene knocked out fail to develop mature platelets, and the mice experience abnormal bleeding due to their profound thrombocytopenia. TANGO 269 was found to be present in the wild type animals but its expression was greatly reduced when the megakaryocyte-specific element was knocked out.
TANGO 269 has been found to be upregulated in megakaryocytes and downregulated in immature megakaryocytes. Uses of TANGO 269 Nucleic acids, Polypeptides, and Modulators Thereof
As human TANGO 269 was originally found in an adrenal gland library, TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat adrenal disorders, such as hypoadrenalism (e.g., primary chronic or acute adrenocortical insufficiency, and secondary adrenocortical insufficiency), hyperadrenalism (Cushing's syndrome, primary hyperaldosteronism, adrenal virilism, and adrenal hyperplasia), or neoplasia (e.g., adrenal adenoma and cortical carcinoma).
As human and murine TANGO 269 are highly expressed in liver tissue, both in TaqMan experiments and in Northern blots, TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat hepatic (liver) disorders, such as jaundice, hepatic failure, hereditary hyperbiliruinemias (e.g., Gilbert's syndrome, Crigler-Naijar syndromes and Dubin- Johnson and Rotor's syndromes), hepatic circulatory disorders (e.g., hepatic vein thrombosis and portal vein obstruction and thrombosis), hepatitis (e.g., chronic active hepatitis, acute viral hepatitis, and toxic and drug-induced hepatitis), cirrhosis (e.g., alcoholic cirrhosis, biliary cirrhosis, and hemochromatosis), and malignant tumors (e.g., primary carcinoma, hepatoblastoma, and angiosarcoma).
As human TANGO 269 is expressed in bone marrow and peripheral blood leukocytes, and as murine TANGO 269 was originally found in a megakaryocyte library, TANGO 269 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, differentiation, and/or function of cells that appear in the bloodstream and in the bone marrow, e.g., stem cells (e.g., hematopoietic stem cells), and blood cells, e.g., erythrocytes, platelets, and leukocytes. Thus TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat bone marrow, blood, and hematopoietic associated diseases and disorders, e.g., acute myeloid leukemia, hemophilia, leukemia, anemia (e.g., sickle cell anemia), and thalassemia. As murine TANGO 269 is moderately expressed in spleen, TANGO 269 nucleic acids, proteins, and modulators thereof can be used to can be used to modulate the proliferation, differentiation, and/or function of cells that form the spleen, e.g., cells of the splenic connective tissue, e.g., splenic smooth muscle cells and/or endothelial cells of the splenic blood vessels. TANGO 269 nucleic acids, proteins, and modulators thereof can also be used to modulate the proliferation, differentiation, and/or function of cells that are processed, e.g., regenerated or phagocytized within the spleen, e.g., erythrocytes and/or B and T lymphocytes and macrophages. Thus TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat spleen, e.g., the fetal spleen, associated diseases and disorders. Examples of splenic diseases and disorders include e.g., splenic lymphoma and/or splenomegaly, phagocytotic disorders, e.g., those inhibiting macrophage engulfment of bacteria and viruses in the bloodstream, and/or diseases and disorders associated with cells processed in the spleen, e.g., erythrocytes (e.g., leukemia and hemophilia).
As murine TANGO 269 is expressed in heart, TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat cardiovascular disorders, such as ischemic heart disease (e.g., angina pectoris, myocardial infarction, and chronic ischemic heart disease), hypertensive heart disease, pulmonary heart disease, valvular heart disease (e.g., rheumatic fever and rheumatic heart disease, endocarditis, mitral valve prolapse, and aortic valve stenosis), congenital heart disease (e.g., valvular and vascular obstructive lesions, atrial or ventricular septal defect, and patent ductus arteriosus), and myocardial disease (e.g., myocarditis, congestive cardiomyopathy, and hypertrophic cariomyopathy).
Due to TANGO 269 's homology to LOX-1, as evidenced by the presence of similar domains, mapping coordinates, and expression patterns seen between the two molecules, TANGO 269 nucleic acids, proteins, and modulators thereof can play a role in treating disorders in which LOX-1 plays a role, some of which are described in the following references: Sawamura et al. (1997) Nature 386:73-77; Kataoka et al. (1999) Circulation 99:3110-3117; and Kita (1999) Circulation Research 84:1113-1115, the contents of all of which are incorporated herein by reference.
Therefore, TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat atherosclerosis, e.g., by binding Ox-LDL (oxidatively modified low density lipoprotein) and its lipid constituents, thus preventing lipid deposition and intimal thickening in the arteries, and thus induction of endothelial expression of leukocyte adhesion molecules and smooth-muscle growth factors (both which are implicated in atherogenesis). TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat immune related diseases and disorders, as LOX-1 is implicated in inflammation, and as LOX-1 has highest homology with the NKR-P1 family of proteins, which are involved in target-cell recognition and natural killer cell activation. Such immune disorders include, e.g., autoimmune disorders (e.g., arthritis, graft rejection (e.g., allograft rejection), and T cell autoimmune disorders (e.g., AIDS)) and inflammatory disorders (e.g., bacterial infection, psoriasis, septicemia, cerebral malaria, inflammatory bowel disease, multiple sclerosis, arthritis (e.g., rheumatoid arthritis, osteoarthritis), and allergic inflammatory disorders (e.g., asthma, psoriasis)).
TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat TNF- related disorders, as LOX-1 expression is induced by tumor necrosis factor-α. Such disorders include, e.g., acute myocarditis, myocardial infarction, congestive heart failure, T cell disorders (e.g., dermatitis, fibrosis)), differentiative and apoptotic disorders, and disorders related to angiogenesis (e.g., tumor formation and/or metastasis). As LOX-1 expression is upregulated in hypertensive rats, and as LOX-1 levels are downregulated in patients treated with ACE (angiotensin converting enzyme) inhibitors, TANGO 269 can also can be used to treat hypertension and congestive heart failure.
As both TANGO 269 and LOX-1 have C-type lectin domains, and are similar in that respect to the selectins, which are implicated in cell-cell recognition (including endothelial- leukocyte adhesion), TANGO 269 nucleic acids, proteins, and modulators thereof, and LOX-1, can be used to treat cell adhesion and cell migration/motility related disorders. Such disorders include, e.g., disorders associated with adhesion and migration of cells, e.g., platelet aggregation disorders (e.g., Glanzmann's thromboasthemia, which is a bleeding disorders characterized by failure of platelet aggregation in response to cell stimuli), inflammatory disorders (e.g., leukocyte adhesion deficiency, which is a disorder associated with impaired migration of neutrophils to sites of extravascular inflammation), disorders associated with abnormal tissue migration during embryo development, and tumor metastasis.
As murine TANGO 269 was originally found in an megakaryocyte library, TANGO 269 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, morphology, migration, differentiation, and/or function of megakaryocytes and platelets, including during development, e.g., embryogenesis. TANGO 269 nucleic acids, proteins, and modulators thereof can also be used to modulate leukocyte-platelet and platelet-endothelium interactions in inflammation and/or thrombosis. Further, TANGO 269 nucleic acids, proteins, and modulators thereof can be used to modulate platelet aggregation and degranulation.
TANGO 269 nucleic acids, proteins, and modulators thereof can also be used to modulate disorders associated with abnormal or aberrant megakaryocyte and/or platelet proliferation, differentiation, morphology, migration, aggregation, degranulation and/or function. Examples of these disorders include, but are not limited to, bleeding disorders (e.g., bleeding tendency and/or prolonged bleeding time) such as thrombocytopenia (e.g., idiopathic thrombocytopenic purpura (ITP) or immune thrombocytopenia or thrombocytopenia induced by chemotherapy or radiation therapy). TANGO 269 nucleic acids, proteins, and modulators thereof can also be used to modulate thrombotic or hemorrhagic disorders and diseases exhibiting quantitative or qualitative platelet dysfunction.
Further, TANGO 269 nucleic acids, proteins, and modulators thereof can be used to modulate symptoms associated with platelet disorders and/or diseases (e.g., bleeding disorders, e.g., purpura and severe bleeding problems). As platelet adhesion and aggregation play an important role in acute coronary diseases,
TANGO 269 nucleic acids, proteins and modulators thereof can be used to modulate coronary diseases (e.g., cardiovascular diseases including unstable angina, acute myocardial infarction, coronary artery disease, coronary revascularization, ventricular thromboembolism, atherosclerosis, coronary artery disease, plaque formation). As murine TANGO 269 expression is greatly reduced in megakaryocytes obtained from gata-1 knockout mice compared to normal mice, TANGO 269 nucleic acids, proteins, and modulators thereof can be either a direct or indirect target of gata-1 and can have profound biological implications. TANGO 269 nucleic acids, proteins, and modulators thereof can be used to treat disease and/or disorders associated with gata-1 dysfunction, e.g., bone marrow, blood, and hematopoietic associated diseases and disorders discussed herein. Human TANGO 298
A cDNA encoding human TANGO 298 was identified by analyzing the sequences of clones present in a normal human bone marrow cDNA library. This analysis led to the identification of a clone, jyhMal 18f02, encoding full-length human TANGO 298. The human TANGO 298 cDNA of this clone is 1078 nucleotides long (Figure 10; SEQ ID NO:25). The open reading frame of this cDNA, nucleotides 31 to 879 of SEQ ID NO:25 (SEQ ID NO:26), encodes a 283 amino acid secreted protein (Figure 10; SEQ ID NO:27). The signal peptide prediction program SIGNALP (Nielsen et al. (1997) Protein
Engineering 10:1-6) predicted that human TANGO 298 includes a 31 amino acid signal peptide (amino acid 1 to about amino acid 31 of SEQ ID NO:25)(SEQ ID NO:29) preceding the mature TANGO 298 protein (corresponding to about amino acid 32 to amino acid 283 of SEQ ID NO:25)(SEQ ID NO:28). The TANGO 298 protein molecular weight is 30.3 kDa prior to the cleavage of the signal peptide, 27.2 kDa after cleavage of the signal peptide.
An N-glycosylation site having the sequence NGSA is found from amino acids 129 to 132 of SEQ ID NO:27. A second N-glycosylation site having the sequence NSSW is found from amino acids 189 to 192. A cAMP and cGMP dependent protein kinase phosphorylation site having the sequence RRSS is found from amino acids 263 to 266. A protein kinase C phosphorylation site having the sequence SVR is found from amino acids 50 to 52. A second protein kinase C phosphorylation site having the sequence SHR is found from amino acids 77 to 79. A third protein kinase C phosphorylation site having the sequence SWK is found from amino acids 191 to 193. A fourth protein kinase C phosphorylation site having the sequence SHR is found from amino acids 208 to 210. A fifth protein kinase C phosphorylation site having the sequence TTR is found from amino acids 275 to 277. A casein kinase II phosphorylation site having the sequence SHRD is found from amino acids 77 to 80. A second casein kinase II phosphorylation site having the sequence STAE is found from amino acids 94 to 97. A third casein kinase II phosphorylation site having the sequence THPD is found from amino acids 110 to 113. A fourth casein kinase II phosphorylation site having the sequence SDFE is found from amino acids 166 to 169. An N-myristoylation site having the sequence GSWGAQ is found from amino acids 28 to 33. A second N-myristoylation site having the sequence GQHHCG is found from amino acids 55 to 60. A third N-myristoylation site having the sequence GLMEAK is found from amino acids 174 to 179. A fourth N-myristoylation site having the sequence GLWCGD is found from amino acids 236 to 241. An amidation site having the sequence PGRR is found from amino acids 144 to 147. A serine protease, trypsin family, histidine active site having the sequence VSAAHC is found from amino acids 70 to 75.
Chromosomal mapping was performed by computerized comparison of TANGO 298 cDNA sequences against a chromosomal mapping database in order to identify the approximate location of the gene encoding human TANGO 298 protein. Homology to EST HUMGS00642 (accession number D19687) showed that the TANGO 298 gene maps to chromosome 19, between markers D19S886 and D19S216. In the region between D19S886 and D19S216 lies 19pl3.3, the area which is the location for cyclic hematopoiesis (also called cyclic neutropenia). A serine protease gene cluster maps to this region as well, further suggesting that this is the area to which TANGO 298 maps.
The homology between TANGO 298 and EST HUMGS00642 (accession number D19687) appears in Figure 17. The nucleotide sequences of HUMGS00642 and human TANGO 298 are about 93% identical over a 278 base pair overlap, from base pairs 801-1078 of human TANGO 298. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.
TANGO 298 includes a trypsin domain (about amino acids 34 to 258 of SEQ ID NO:26; SEQ ID NO:30).
Figures 12A-12B show an alignment of the human TANGO 298 full length nucleic acid sequence (SEQ ID NO:24) with the human adipsin full length nucleic acid sequence (SEQ ID NO:32). Figures 13A-13B show an alignment of the human TANGO 298 nucleotide coding region (SEQ ID NO:25) with the human adipsin nucleotide coding region (SEQ ID NO:33). Figure 14 shows an alignment of the human TANGO 298 protein sequence (SEQ ID NO:26) with the human adipsin protein sequence (SEQ ID NO:34). As shown in Figure 14, the human TANGO 298 signal sequence (SEQ ID NO:28) is represented by amino acids 1 to 31 (and encoded by nucleotides 31 to 123 of SEQ ID NO:26 (SEQ ID NO:29)). The human TANGO 298 trypsin domain sequence (SEQ ID NO:30) is represented by amino acids 34 to 258 (and encoded by nucleotides 130 to 804 of SEQ ID NO:26 (SEQ ID NO:31)), and the human adipsin trypsin domain sequence is represented by amino acids 1 to 223 (and encoded by nucleotides 55 to 723 of SEQ ID NO:32).
Figures 12A-12B and Figures 13A-13B show that there is a 51.8% identity between the human TANGO 298 full length nucleic acid molecule and the human adipsin full length nucleic acid molecule, and an overall 50.8% identity between the open reading frame of the human TANGO 298 nucleic acid molecule and the open reading frame of the human adipsin nucleic acid molecule, respectively. The amino acid alignment in Figure 14 shows a 34.0% overall amino acid sequence identity between human TANGO 298 and human adipsin.
Clone EpT298, which encodes human TANGO 298, was deposited with the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110-2209) on April 20, 1999 and assigned Accession Number 207216. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
Figure 11 depicts a hydropathy plot of human TANGO 298. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) and potential N- glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1 to 31 of SEQ ID NO:27; SEQ ID NO:29) on the left from the mature protein (amino acids 32 to 283 of SEQ ID NO:27; SEQ ID NO:28) on the right. Thicker gray horizontal bars below the dashed horizontal line indicate extracellular ("out"), transmembrane ("TM"), and intracellular ("in") regions of the molecule.
TaqMan analysis (described below in the examples) of human TANGO 298 expression in human tissues and cell lines showed moderate levels of expression in fetal liver, K562 (an erythroleukemia), and CD34" cells from mobilized bone marrow (MBM CD34"), and extremely high levels of expression in CD34+ cells from mobilized peripheral blood (mPB CD34+), adult resting bone marrow (ABM CD34+), G-CSF mobilized bone marrow (mBM CD34+), and G-CSF mobilized peripheral blood leukocytes (mPB leukocytes). Low levels of expression were seen in CD34" cells from mobilized bone marrow (MBM CD34 ), resting CD19+ B cells, resting peripheral blood mononuclear cells (PBMC), and granulocytes.
TaqMan analysis of human TANGO 298 expression also reveals highest expression in the erythroblast samples, the megakaryocyte samples, the neutrophil samples, and in cells populations positive for CD34+ marker (including cells from mobilized bone marrow, adult resting bone marrow, umbilical cord blood, and fetal liver). In particular, the samples from each group manifest higher levels of TANGO 298 expression in the earlier stages of development, and expression tapers off after a certain point in time. For example, in the erythroblast cell samples tested, levels of TANGO 298 expression increase in proportion to the time the cells were cultured, from 24 hours to 48 hours, then decrease with culture times of 6 days, 10 days, and 12 days.
Uses of TANGO 298 Nucleic acids, Polypeptides, and Modulators Thereof
Due to TANGO 298 's homology to adipsin (also known as complement factor D), as evidenced by similar domains and expression patterns seen between the two molecules, TANGO 298 nucleic acids, proteins, and modulators thereof can be used to treat disorders in which adipsin plays a role, some of which are described in the following references: White et al. (1992) JBiol Chem. 13:9210-9213; Spiegelman et al. (1989) J Biol Chem. 264:1811-1815; Yamauchi et al. (1994) JJmmunol. 152:3645-3653; Meri et al. (1998) Vox Sang. 74 Suppl 2:291-302; and PCT Publication Number WO 90/06365, the contents of all of which are incorporated herein by reference. Like complement factor D's role in the alternative complement pathway, TANGO 298 also has utility in the same pathway. For example, disturbances of the complement regulation system lead to disorders known as complement regulator deficiencies, which include, e.g., hereditary angioedema (an allergic disorder) and proxysmal nocturnal hemoglobinuria (the presence of hemoglobin in the urine), which TANGO 298 nucleic acids, proteins, and modulators thereof can be used to treat. TANGO 298 nucleic acids, proteins, and modulators thereof play a role in the modulation, e.g., treatment and regulation, of obesity, as adipsin is synthesized in adipose tissue and secreted into the bloodstream, is highly expressed in fat, and is also observed in many common models of obesity. Adipsin levels are increased during appetite suppression, and are lowered in obese mice, including those genetically obese due to the db/db and ob/ob genes, and those in a hyperglycemic, hyperinsulinemic state. For these reasons, TANGO 298 nucleic acids, proteins, and modulators thereof can play also a role in regulation of systemic energy balance and in the treatment of diabetes.
As adipsin is also highly expressed in monocytes and macrophages, and as the alternative complement pathway is know for suppression of infectious agents and fomenting inflammation, TANGO 298 nucleic acids, proteins, and modulators thereof can be used to treat immune related diseases and disorders. Such disorders include, e.g., autoimmune disorders (e.g., arthritis, graft rejection (e.g., allograft rejection), and T cell autoimmune disorders (e.g., AIDS)) and inflammatory disorders (e.g., bacterial infection, psoriasis, septicemia, cerebral malaria, inflammatory bowel disease, multiple sclerosis, arthritis (e.g., rheumatoid arthritis, osteoarthritis), and allergic inflammatory disorders (e.g., asthma, psoriasis)). TANGO 298 nucleic acids, proteins, and modulators thereof, can also be used to rid the body of invading or infecting agents, e.g., bacteria, viruses, parasites, neoplastic cells.
As adipsin is a serine protease and as both adipsin and TANGO 298 share a trypsin domain commonly seen in serine proteases, TANGO 298 nucleic acids, proteins, and modulators thereof can be used to treat disorders involving abnormal serine protease function. For example, it is known that serine protease inhibitors are abundant in plaques found in Alzheimer's patients, and may be responsible for preventing some types of metalloproteinase from breaking down the beta-amyloid proteins that make up these plaques. Thus, modulation of the serine protease activity may modulate formation of Alzheimer's plaques, and consequently, TANGO 298 nucleic acids, proteins, and modulators thereof can be used to treat Alzheimer's disease. Adipsin has also been seen in high levels in a patient with Fanconi anemia (FA), and thus TANGO 298 nucleic acids, proteins, and modulators thereof can be used to treat such patients (persons with FA experience bone marrow failure, suffer severe life-threatening aplastic anemia, and possess blood systems that cannot successfully combat infection, fatigue or spontaneous hemorrhage or bleeding).
As TANGO 298 is found in an bone marrow library, is upregulated in megakaryocytes, and is expressed in cells such as neutrophils, erythroids, megakaryocytes, cord blood cells, and fetal liver, TANGO 298 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, morphology, migration, differentiation, and/or function of hematopoietic cells, e.g., hematopoietic stem cells and blood cells, and can play a role in a variety of hematological disorders.
Hematological disorders include, but are not limited to, disorders associated with abnormal differentiation or hematopoiesis, morphology, migration, proliferation, or function of blood cells derived, for example, from myeloid multipotential cells in bone marrow, such as megakaryocytes (and ultimately platelets), monocytes, erythroids, and granulocytes (e.g., neutrophils, eosinophils, and basophils), and from lymphoid multipotential cells, such as T and B lymphocytes. Due to its expression in megakaryocytes, which are platelet precursor cells, TANGO 298 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, morphology, migration, differentiation, and/or function of megakaryocytes and/or platelets, and can play a role in a variety of platelet associated disorders. Platelet associated disorders include, but are not limited to, bleeding disorders, e.g., hemophilia (e.g., hemophilia A), thrombocytopenia (e.g., thrombocytopenia due to a reduced number of megakaryocytes in the bone marrow, for example, as a result of chemotherapy or radiation therapy); invasive disorders, such as leukemia, idiopathic or drug- or toxin-induced aplasia of the marrow, or rare hereditary amegakaryocytic thrombocytopenias; ineffective thrombopoiesis, for example, as a result of megaloblastic anemia, alcohol toxicity, vitamin B 12 or folate deficiency, myelodysplastic disorders, or rare hereditary disorders (e.g., Wiskott-Aldrich syndrome and May-hegglin anomaly); a reduction in platelet distribution, for example, as a result of cirrhosis, a splenic invasive disease (e.g., Gaucher's disease), or myelofibrosis with extramedullary myeloid metaplasia; increased platelet destruction, for example, as a result of removal of IgG-coated platelets by the mononuclear phagocytic system (e.g., idiopathic thrombocytopenic purpura (ITP), secondary immune thrombocytopenia (e.g., systemic lupus erythematosus, lymphoma, or chronic lymphocytic leukemia), drug-related immune thrombocytopenias (e.g., as with quinidine, aspirin, and heparin), post-transfusion purpura, and neonatal thrombocytopenia as a result of maternal platelet autoantibodies or maternal platelet alloantibodies). Also included are thrombocytopenia secondary to intravascular clotting and thrombin induced damage to platelets as a result of, for example, obstetric complications, metastatic tumors, severe gram-negative bacteremia, thrombotic thrombocytopenic purpura, or severe illness. Also included is dilutional thrombocytopenia, for example, due to massive hemorrhage.
Platelet associated disorders also include, but are not limited to, essential thrombocytosis and thrombocytosis associated with, for example, splenectomy, acute or chronic inflammatory diseases, anemia (e.g., hemolytic anemia), carcinoma, Hodgkin's disease, lymphoproliferative disorders, and malignant lymphomas.
In addition, as platelet adhesion and aggregation play an important role in acute coronary diseases, TANGO 298 nucleic acids, proteins and modulators thereof can be used to modulate coronary diseases (e.g. , cardiovascular diseases including unstable angina, acute myocardial infarction, coronary artery disease, coronary revascularization, ventricular thromboembolism, atherosclerosis, coronary artery disease, plaque formation).
Due to its expression in erythroids, which are erythrocyte precursor cells, TANGO 298 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, moφhology, migration, differentiation, and/or function of erythrocytes, and can play a role in a variety of erythrocyte associated disorders.
Erythrocyte associated disorders include anemias such as, for example, hemolytic anemias due to hereditary cell membrane abnormalities, such as hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis; hemolytic anemias due to acquired cell membrane defects, such as paroxysmal nocturnal hemoglobinuria and spur cell anemia; hemolytic anemias caused by antibody reactions, for example to the RBC antigens, or antigens of the ABO system, Lewis system, Ii system, Rh system, Kidd system, Duffy system, and Kell system; methemoglobinemia; a failure of erythropoiesis, for example, as a result of aplastic anemia, pure red cell aplasia, myelodysplastic syndromes, sideroblastic anemias, and congenital dyserythropoietic anemia; secondary anemia in nonhematolic disorders, for example, as a result of chemotherapy, alcoholism, or liver disease; anemia of chronic disease, such as chronic renal failure; and endocrine deficiency diseases.
Other erythrocyte associated disorders include polycythemias such as, for example, polycythemia vera, secondary polycythemia, and relative polycythemia.
Due to TANGO 298's expression in neutrophils, and the fact that it exhibits strong homology to a cosmid that maps to the region of 19pl3.3 (site of cyclic neutropenia gene), TANGO 298 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, moφhology, migration, differentiation, and/or function of neutrophils, and can play a role in a variety of neutrophil associated disorders.
Neutrophil associated disorders include neutropenias that result from or accompany a number of conditions, including, but not limited to, chemotherapy; neutropenias (e.g., chronic idopathic neutropenia and cyclic neutropenia and its accompanying symptoms, including fever, malaise, mucosal ulcers, and life-threatening infections); Felty's syndrome; acute infectious disease; lymphoma or aleukemic lymphocytic leukemia; myelodysplastic syndrome; and rheumatic diseases such as systemic lupus erythematosus, rheumatoid arthritis, and polymyositis. Also included is neutrophilia, for example, accompanying chronic myelogenous leukemia.
Due to its expression in hematopoeitic stem cells, and the fact that it is homologous to adipsin, which is highly expressed in monocytes and macrophages, TANGO 298 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, moφhology, migration, differentiation, and/or function of monocytes and/or macrophages, and can play a role in a variety of monocyte and/or macrophage associated disorders.
Monocyte associated disorders include disorders associated with abnormal monocyte and/or macrophage function, such as impaired phagocytosis, chemotaxis, or secretion of cytokines, growth factors and acute-phase reactants, resulting from certain diseases, e.g., lysosomal storage diseases (e.g., Gaucher's disease); impaired monocyte cytokine production, for example, found in some patients with disseminated nontuberculous mycobacterial infection who are not infected with HIV; leukocyte adhesion deficiency (LAD), hyperimmunoglobulin E- recurrent infection (HIE) or Job's syndrome, Chediak-Higashi syndrome (CHS), and chronic granulomatous diseases (CGD), certain autoimmune diseases, such as systemic lupus erythematosus and other autoimmune diseases characterized by tissue deposition of immune complexes, as seen in Sjδgren's syndrome, mixed cryoglobulinemia, dermatitis heφetiformis, and chronic progressive multiple sclerosis. Also included are disorders or infections that impair mononuclear phagocyte function, for example, influenza virus infection and AIDS.
Monocyte associated disorders also include monocytoses such as, for example, monocytoses associated with certain infections such as tuberculosis, brucellosis, subacute bacterial endocarditis, Rocky Mountain spotted fever, malaria, and visceral leishmaniasis (kala azar), in malignancies, leukemias (e.g., acute myeloid leukemia), myeloproliferative syndromes, hemolytic anemias, chronic idiopathic neutropenias, and granulomatous diseases such as sarcoidosis, regional enteritis, and some collagen vascular diseases.
Other monocyte associated disorders include monocytopenias such as, for example, monocytopenias that can occur with acute infections, with stress, following administration of glucocorticoids, aplastic anemia, hairy cell leukemia, and acute myelogenous leukemia and as a direct result of administration of myelotoxic and immunosuppressive drugs.
As TANGO 298 is a protease highly expressed in a rare but very mobile population, TANGO 298 nucleic acids, proteins and modulators thereof can be used to treat, e.g., inhibit, tumor cell travel, e.g., in metastasis in tumors. Further, as TANGO 298 is highly expressed in CD34+ bone marrow cells and upregulated in Mobilized Peripheral Blood (MPB), TANGO 298 nucleic acids, proteins and modulators thereof can modulate stem cell engraftment, homing, and or mobilization, e.g., in bone marrow transplantation, stem cell mobilization to harvest grafts, and/or stem cell recovery post chemotherapy.
Tables 1 and 2 below provide a summary of the sequence information for TANGO 269 and TANGO 298.
TABLE 1 : Summary of TANGO 269 and TANGO 298 Sequence Information
Figure imgf000041_0001
TABLE 2: Summary of Domains of TANGO 269 and TANGO 298 Proteins
Figure imgf000041_0002
Various aspects of the invention are described in further detail in the following subsections: I. Isolated Nucleic Acid Molecules
One aspect of the invention pertains to isolated nucleic acid molecules that encode a polypeptide of the invention or a biologically active portion thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Preferably, an "isolated" nucleic acid molecule is free of sequences (preferably protein encoding sequences) which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. As used herein, the term "isolated" when referring to a nucleic acid molecule does not include an isolated chromosome.
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, 2, 11, 12, 24, or 25, or a complement thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequences of SEQ ID NO:l, 2, 11, 12, 24, or 25 as a hybridization probe, nucleic acid molecules of the invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
A nucleic acid molecule of the invention can be amplified using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to all or a portion of a nucleic acid molecule of the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence of SEQ ID NO: 1, 2, 11, 12, 24, or 25, or a portion thereof. A nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence thereby forming a stable duplex. Moreover, a nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence encoding a full length polypeptide of the invention for example, a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a polypeptide of the invention. The nucleotide sequence determined from the cloning one gene allows for the generation of probes and primers designed for use in identifying and/or cloning homologues in other cell types, e.g., from other tissues, as well as homologues from other mammals. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides of the sense or anti-sense sequence of SEQ ID NO: 1, 2, 11, 12, 24, or 25 or of a naturally occurring mutant of SEQ ID NO:l, 2, 11, 12, 24, or 25.
In one embodiment, the TANGO 269 nucleic acid molecule described herein does not include or contain, is free of, and/or is not adjacent to or flanked by an EcoRI restriction endonuclease site (GAATTC) and/or a Pad restriction endonuclease site (TTAATTAA). In another embodiment, the TANGO 269 nucleic acid molecule described herein does not include or contain, is free of, and/or is not adjacent to, 10, 15, 25, 50, 60, 70, 80, 90, 100, or 150 bases which flank the nucleotide insert of the vector designated as IMAGE clone 212698. In another embodiment, TANGO 269 is in a vector containing at least one regulatory sequence which allows for transcription of the TANGO 269 nucleic acid molecule, e.g., a promoter. In yet another embodiment, TANGO 269 nucleic acid molecule is in a form suitable for expression of the TANGO 269 nucleic acid molecule, e.g., is associated with (e.g., is adjacent to) nucleotide sequences which allow for expression of the TANGO 269 nucleic acid molecule. In still another embodiment, the TANGO 269 nucleic acid molecule described herein does not include or contain, is free of, and/or is not adjacent to 25, 30, 40, 50, 75, or 100 bases of the modified version of the pT7T3D vector sold by Pharmacia, New Jersey (modification described at http://www.ncbi.nlm.nih.gov/irx/cgi-bin/birx_doc?dbest+33286 or http://www.ncbi.nlm.nih.gov/irx/cgi-bin/birx_doc?dbest+33175, the contents of which are incoφorated herein by reference).
Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts or genomic sequences encoding the same protein molecule encoded by a selected nucleic acid molecule. The probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which mis-express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted.
A nucleic acid fragment encoding a "biologically active portion" of a polypeptide of the invention can be prepared by isolating a portion of any of SEQ ID NO:2, 12, or 25, expressing the encoded portion of the polypeptide protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the polypeptide.
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of SEQ ID NO:l, 2, 11, 12, 24, or 25, due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequence of SEQ ID NO:2, 12, or 25. In addition to the nucleotide sequences of SEQ ID NO:2 and 13, it will be appreciated by those skilled in the art that DNA sequence polymoφhisms that lead to changes in the amino acid sequence may exist within a population (e.g., the human population). Such genetic polymoφhisms may exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes which occur alternatively at a given genetic locus. For 5 example, TANGO 269 has been mapped to chromosome 12, between markers D12S98 and D12S358, and therefore TANGO 269 family members include nucleotide sequences which include polymoφhisms (i.e., nucleotide sequences which vary from the nucleotide sequence of SEQ ID NO:2) but which map to chromosome 12, between markers D12S09 and D12S358. Such nucleotide sequences are allelic variants of the TANGO 269 molecules described herein. l o As used herein, the phrase "allelic variant" refers to a nucleotide sequence which occurs at a given locus or to a polypeptide encoded by the nucleotide sequence. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention. Such natural allelic variations can typically result in 1- 5% variance in the nucleotide sequence of a given gene. Alternative alleles can be identified by
15 sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid polymoφhisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention.
20 Moreover, nucleic acid molecules encoding proteins of the invention from other species
(homologues), which have a nucleotide sequence which differs from that of the human protein described herein are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of a cDNA of the invention can be isolated based on their identity to the human nucleic acid molecule disclosed herein using the
25 human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. For example, a cDNA encoding a soluble form of a membrane-bound protein of the invention isolated based on its hybridization to a nucleic acid molecule encoding all or part of the membrane-bound form. Likewise, a cDNA encoding a membrane-bound form can be isolated based on its hybridization to a nucleic acid molecule encoding all or part of the soluble form.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1290) nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence, preferably the coding sequence, of SEQ ID NO: 1, 2, 11, 12, 24, or 25, or a complement thereof.
As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, preferably 75%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1, 2, 11, 12, 24, or 25, or a complement thereof, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally- occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In addition to naturally-occurring allelic variants of a nucleic acid molecule of the invention sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation thereby leading to changes in the amino acid sequence of the encoded protein, without altering the biological activity of the protein. For example, one can make nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are not conserved or only semi-conserved among homologues of various species may be non-essential for activity and thus would be likely targets for alteration. Alternatively, amino acid residues that are conserved among the homologues of various species (e.g., murine and human) may be essential for activity and thus would not be likely targets for alteration.
Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding a polypeptide of the invention that contain changes in amino acid residues that are not essential for activity. Such polypeptides differ in amino acid sequence from SEQ ID NO:3, 13, or 26, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule includes a nucleotide sequence encoding a protein that includes an amino acid sequence that is at least about 45% identical, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:3, 13, or 26.
An isolated nucleic acid molecule encoding a variant protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 1, 2, 11, 12, 24, or 25 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
In a preferred embodiment, a mutant polypeptide that is a variant of a polypeptide of the invention can be assayed for: (1) the ability to form proteimprotein interactions with proteins in a signaling pathway of the polypeptide of the invention; (2) the ability to bind a ligand of the polypeptide of the invention; or (3) the ability to bind to an intracellular target protein of the polypeptide of the invention. In yet another preferred embodiment, the mutant polypeptide can be assayed for the ability to modulate cellular proliferation, cellular migration or chemotaxis, or cellular differentiation. The present invention encompasses antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid encoding a polypeptide of the invention, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can be antisense to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding a polypeptide of the invention. The non-coding regions ("5* and 3' untranslated regions") are the 5' and 3' sequences which flank the coding region and are not translated into amino acids. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides or more in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4- thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5- methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a selected polypeptide of the invention to thereby inhibit expression, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred. An antisense nucleic acid molecule of the invention can be an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131- 6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
The invention also encompasses ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. A ribozyme having specificity for a nucleic acid molecule encoding a polypeptide of the invention can be designed based upon the nucleotide sequence of a cDNA disclosed herein. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742. Alternatively, an mRNA encoding a polypeptide of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261 :1411-1418. The invention also encompasses nucleic acid molecules which form triple helical structures. For example, expression of a polypeptide of the invention can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g., the promoter and/or enhancer) to form triple helical structures that prevent transcription of the gene in target cells. See generally Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992) Ann. N. Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14(12):807-15.
In various embodiments, the nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996)
Bioorganic & Medicinal Chemistry 4(1): 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675.
PNAs can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup (1996), supra; or as probes or primers for DNA sequence and hybridization (Hyrup (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675).
In another embodiment, PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNAse H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996), supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs. Compounds such as 5'-(4- methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite can be used as a link between the PNA and the 5' end of DNA (Mag et al. (1989) Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser et al. (1975) Bioorganic Med. Chem. Lett. 5:1119-11124).
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W0 89/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio/Techniques 6:958-976) or intercalating agents (see, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
II. Isolated Proteins and Antibodies
One aspect of the invention pertains to isolated proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise antibodies directed against a polypeptide of the invention. In one embodiment, the native polypeptide can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, polypeptides of the invention are produced by recombinant DNA techniques. Alternative to recombinant expression, a polypeptide of the invention can be synthesized chemically using standard peptide synthesis techniques.
An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein"). When the protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.
Biologically active portions of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the protein (e.g., the amino acid sequence shown in any of SEQ ID NO:3, 4, 13, 14,
26, or 27), which include fewer amino acids than the full length protein, and exhibit at least one activity of the corresponding full-length protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the corresponding protein. A biologically active portion of a protein of the invention can be a polypeptide which is, for example, 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the invention.
Preferred polypeptides have the amino acid sequence of SEQ ID NO:3, 4, 13, 14, 26, or
27. Other useful proteins are substantially identical (e.g., at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99%) to any of SEQ ID NO:3, 4, 13, 14, 26, or 27, and retain the functional activity of the protein of the corresponding naturally-occurring protein yet differ in amino acid sequence due to natural allelic variation or mutagenesis.
To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison puφoses (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions (e.g., overlapping positions) x 100). In one embodiment, the two sequences are the same length.
The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incoφorated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison puφoses, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST,
Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incoφorated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 70:3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 55:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. If ktup=2, similar regions in the two sequences being compared are found by looking at pairs of aligned residues; if ktup=l, single aligned amino acids are examined, ktup can be set to 2 or 1 for protein sequences, or from 1 to 6 for DNA sequences. The default if ktup is not specified is 2 for proteins and 6 for DNA. For a further description of FASTA parameters, see http://bioweb.pasteur.fr/docs/man/man/fasta. I.html#sect2, the contents of which are incoφorated herein by reference.
The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
The invention also provides chimeric or fusion proteins. As used herein, a "chimeric protein" or "fusion protein" comprises all or part (preferably biologically active) of a polypeptide of the invention operably linked to a heterologous polypeptide (i.e., a polypeptide other than the polypeptide of the invention). Within the fusion protein, the term "operably linked" is intended to indicate that the polypeptide of the invention and the heterologous polypeptide are fused in- frame to each other. The heterologous polypeptide can be fused to the N-terminus or C-terminus of the polypeptide of the invention.
One useful fusion protein is a GST fusion protein in which the polypeptide of the invention is fused to the C-terminus of GST sequences. Such fusion proteins can facilitate the purification of a recombinant polypeptide of the invention.
In another embodiment, the fusion protein contains a heterologous signal sequence at its N-terminus. For example, the native signal sequence of a polypeptide of the invention can be removed and replaced with a signal sequence from another protein. For example, the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992). Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California). In yet another example, useful prokaryotic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).
In yet another embodiment, the fusion protein is an immunoglobulin fusion protein in which all or part of a polypeptide of the invention is fused to sequences derived from a member of the immunoglobulin protein family. The immunoglobulin fusion proteins of the invention can be incoφorated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand (soluble or membrane-bound) and a protein on the surface of a cell (receptor), to thereby suppress signal transduction in vivo. The immunoglobulin fusion protein can be used to affect the bioavailability of a cognate ligand of a polypeptide of the invention. Inhibition of ligand/receptor interaction may be useful therapeutically, both for treating proliferative and differentiative disorders and for modulating (e.g. promoting or inhibiting) cell survival. Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies directed against a polypeptide of the invention in a subject, to purify ligands and in screening assays to identify molecules which inhibit the interaction of receptors with ligands.
Chimeric and fusion proteins of the invention can be produced by standard recombinant DNA techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel et al., supra). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide of the invention. A signal sequence of a polypeptide of the invention (SEQ ID NO:5, 15, and 28) can be used to facilitate secretion and isolation of the secreted protein or other proteins of interest. Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway. Thus, the invention pertains to the described polypeptides having a signal sequence, as well as to the signal sequence itself and to the polypeptide in the absence of the signal sequence (i.e., the cleavage products). In one embodiment, a nucleic acid sequence encoding a signal sequence of the invention can be operably linked in an expression vector to a protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate. The signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by art recognized methods. Alternatively, the signal sequence can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.
In another embodiment, the signal sequences of the present invention can be used to identify regulatory sequences, e.g., promoters, enhancers, repressors. Since signal sequences are the most amino-terminal sequences of a peptide, it is expected that the nucleic acids which flank the signal sequence on its amino-terminal side will be regulatory sequences which affect transcription. Thus, a nucleotide sequence which encodes all or a portion of a signal sequence can be used as a probe to identify and isolate signal sequences and their flanking regions, and these flanking regions can be studied to identify regulatory elements therein.
The present invention also pertains to variants of the polypeptides of the invention. Such variants have an altered amino acid sequence which can function as either agonists (mimetics) or as antagonists. Variants can be generated by mutagenesis, e.g., discrete point mutation or truncation. An agonist can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the protein. An antagonist of a protein can inhibit one or more of the activities of the naturally occurring form of the protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein can have fewer side effects in a subject relative to treatment with the naturally occurring form of the protein. Variants of a protein of the invention which function as either agonists (mimetics) or as antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the protein of the invention for agonist or antagonist activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display). There are a variety of methods which can be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acid Res. 11 :477).
In addition, libraries of fragments of the coding sequence of a polypeptide of the invention can be used to generate a variegated population of polypeptides for screening and subsequent selection of variants. For example, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N- terminal and internal fragments of various sizes of the protein of interest.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 59:7811- 7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331). An isolated polypeptide of the invention, or a fragment thereof, can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. The full-length polypeptide or protein can be used or, alternatively, the invention provides antigenic peptide fragments for use as immunogens. The antigenic peptide of a protein of the invention comprises at least 8 (preferably 10, 15, 20, or 30) amino acid residues of the amino acid sequence of SEQ ID NO:3, 13, or 26, and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.
Preferred epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, e.g., hydrophilic regions. Figures 2, 7, and 11 are hydropathy plots of the proteins of the invention. These plots or similar analyses can be used to identify hydrophilic regions.
An immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal). An appropriate immunogenic preparation can contain, for example, recombinantly expressed or chemically synthesized polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
Accordingly, another aspect of the invention pertains to antibodies directed against a polypeptide of the invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention. A molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope.
Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide of the invention as an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well- known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody- producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology (1994) CoHgan et al. (eds.) John Wiley & Sons, Inc., New York, NY). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide of interest. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurβAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246: 1275-1281; Griffiths et al. (1993) EMBOJ. 12:725-734.
Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Patent No. 4,816,567; European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancerlnst. 80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Patent 5,225,539; Jones et al. (1986) Nature 321 :552-525; Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al. (1988) J. Immunol. 141 :4053-4060.
Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Patent 5,625,126; U.S. Patent 5,633,425; U.S. Patent 5,569,825; U.S. Patent 5,661,016; and U.S. Patent 5,545,806. In addition, companies such as Abgenix, Inc. (Freemont, CA), can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above. Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al. (1994) Bio/technology 12:899-903).
An antibody directed against a polypeptide of the invention (e.g., monoclonal antibody) can be used to isolate the polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, such an antibody can be used to detect the protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide. The antibodies can also be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta- galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include l251, 1311, 35S or 3H. Further, an antibody (or fragment thereof) may be conjugated to or administered with a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine
(CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g. , dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
The conjugates of the invention can be used for modifying a given biological response. The drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta- interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thoφe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thoφe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62: 119-58 (1982).
Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
III. Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide of the invention (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors, expression vectors, are capable of directing the expression of genes to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, the invention is intended to include such other forms of expression vectors, such as viral vectors
(e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell. This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
The recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells (using baculovirus expression vectors), yeast cells or mammalian cells). Suitable host cells are discussed further in Goeddel, supra. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non- fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three puφoses: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 1 Id (Studier et al., Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or
HMS174(DE3) from a resident λ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerivisae include pYepSecl (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Coφoration, San Diego, CA), and pPicZ (Invitrogen Coφ, San Diego, CA).
Alternatively, the expression vector is a baculovirus expression vector. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39). In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187- 195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al., supra.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBOJ. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the beta-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to the mRNA encoding a polypeptide of the invention. Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al. (Reviews - Trends in Genetics, Vol. 1(1) 1986).
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell (e.g., insect cells, yeast or mammalian cells).
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incoφorated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce a polypeptide of the invention. Accordingly, the invention further provides methods for producing a polypeptide of the invention using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell. The host cells of the invention can also be used to produce nonhuman transgenic animals.
For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a sequence encoding a polypeptide of the invention has been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous sequences encoding a polypeptide of the invention have been introduced into their genome or homologous recombinant animals in which endogenous encoding a polypeptide of the invention sequences have been altered. Such animals are useful for studying the function and/or activity of the polypeptide and for identifying and/or evaluating modulators of polypeptide activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, an "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
A transgenic animal of the invention can be created by introducing nucleic acid encoding a polypeptide of the invention (or a homologue thereof) into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue- specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the polypeptide of the invention to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, U.S. Patent No. 4,873,191 and in Hogan, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of mRNA encoding the transgene in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying the transgene can further be bred to other transgenic animals carrying other transgenes.
To create an homologous recombinant animal, a vector is prepared which contains at least a portion of a gene encoding a polypeptide of the invention into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the gene. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous protein). In the homologous recombination vector, the altered portion of the gene is flanked at its 5' and 3' ends by additional nucleic acid of the gene to allow for homologous recombination to occur between the exogenous gene carried by the vector and an endogenous gene in an embryonic stem cell. The additional flanking nucleic acid sequences are of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see, e.g., Thomas and Capecchi (1987) Cell 51 :503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced gene has homologously recombined with the endogenous gene are selected (see, e.g., Li et al. (1992) Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see, e.g., Bradley in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley (1991) Current Opinion in Bio/Technology 2:823-829 and in PCT Publication NOS. WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169. In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. ( 1991 ) Science 251 : 1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 385:810-813 and PCT Publication NOS. WO 97/07668 and WO 97/07669. IV. Pharmaceutical Compositions
The nucleic acid molecules, polypeptides, and antibodies (also referred to herein as "active compounds") of the invention can be incoφorated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incoφorated into the compositions.
The invention includes methods for preparing pharmaceutical compositions for modulating the expression or activity of a polypeptide or nucleic acid of the invention. Such methods comprise formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of the invention. Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of the invention and one or more additional active compounds. A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF; Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absoφtion of the injectable compositions can be brought about by including in the composition an agent which delays absoφtion, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incoφorating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incoφorating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the puφose of oral therapeutic administration, the active compound can be incoφorated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Coφoration and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
In therapeutic applications, anti-TANGO 269 or anti-TANGO 298 antibodies, like other therapeutic antibodies, are administered parenterally, preferably intravenously or intramuscularly daily, monthly, biweekly, weekly, or more frequently. The preferred dosage is O.lmg/kg to 100 mg/kg of body weight, preferably 10 to 20 mg/kg of body weight. Dosages of 50 mg/kg or higher are preferred if the antibody is to be effective within the brain. Accordingly, lower dosages and less frequent administration is often possible. The preferred dosage for treatment of a particular disorder can be based on results observed with other therapeutic antibodies or it can be determined by one skilled based on testing in animal models. The suitable dosage of antibody in a given situation depends on the disease being treated, the severity of the disease, whether the antibody is being administered for therapeutic or preventative reasons, previous therapies administered, and the patient's clinical history. Treatment is generally continued until the desired therapeutic or preventative effect is observed. Dosage regimes of the type that can be adapted to the methods of the present invention are found in PCT Publication Number WO 94/04188. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).
As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
The present invention encompasses agents which modulate expression or activity of TANGO 269 and/or TANGO 298. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments. In a preferred example, a subject is treated with antibody, or protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Patent 5,328,470) or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g.,rerroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
V. Uses and Methods of the Invention
The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) detection assays (e.g., chromosomal mapping, tissue typing, forensic biology); c) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenomics); and d) methods of treatment (e.g., therapeutic and prophylactic). For example, the TANGO 269 polypeptides of the invention can to used to, for example (i) mediate protein-protein interactions; (ii) modulate cellular migration, proliferation, and differentiation; (iii) modulate proteolytic enzyme activity; and/or (iv) modulate protein degradation. The isolated nucleic acid molecules of the invention can be used to express proteins (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect mRNA (e.g., in a biological sample) or a genetic lesion, and to modulate activity of a polypeptide of the invention. In addition, the polypeptides of the invention can be used to screen drugs or compounds which modulate activity or expression of a polypeptide of the invention as well as to treat disorders characterized by insufficient or excessive production of a protein of the invention or production of a form of a protein of the invention which has decreased or aberrant activity compared to the wild type protein. In addition, the antibodies of the invention can be used to detect and isolate a protein of the and modulate activity of a protein of the invention. This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
A. Screening Assays
The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to polypeptide of the invention or have a stimulatory or inhibitory effect on, for example, expression or activity of a polypeptide of the invention.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a polypeptide of the invention or biologically active portion thereof. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one- bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91 :11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Caπell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (\994) Angew. Chem. Int. Ed. Engl. 33:2061 ; and Gallop et al. (1994) J. Med. Chem. 37:1233. Libraries of compounds may be presented in solution (e.g., Houghten (1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nαtwre 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Patent No. 5,223,409), spores (Patent NOS. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865- 1869) or phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404- 406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol. 222:301-310).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to the poljφeptide determined. The cell, for example, can be a yeast cell or a cell of mammalian origin. Determining the ability of the test compound to bind to the polypeptide can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the polypeptide or biologically active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 1251, 35S, l4C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In a preferred embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or a biologically active portion thereof as compared to the known compound. In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the polypeptide or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of the polypeptide or a biologically active portion thereof can be accomplished, for example, by determining the ability of the polypeptide protein to bind to or interact with a target molecule. Determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by one of the methods described above for determining direct binding. As used herein, a "target molecule" is a molecule with which a selected polypeptide (e.g., a polypeptide of the invention) binds or interacts with in nature, for example, a molecule on the surface of a cell which expresses the selected protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. A target molecule can be a polypeptide of the invention or some other polypeptide or protein. For example, a target molecule can be a component of a signal transduction pathway which facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a compound to a polypeptide of the invention) through the cell membrane and into the cell or a second intercellular protein which has catalytic activity or a protein which facilitates the association of downstream signaling molecules with a polypeptide of the invention. Determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cellular differentiation, or cell proliferation. In yet another embodiment, an assay of the present invention is a cell-free assay comprising contacting a polypeptide of the invention or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to the polypeptide or biologically active portion thereof. Binding of the test compound to the polypeptide can be determined either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the polypeptide of the invention or biologically active portion thereof with a known compound which binds the poljφeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or biologically active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-free assay comprising contacting a polypeptide of the invention or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the polypeptide or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished, for example, by determining the ability of the polypeptide to bind to a target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished by determining the ability of the polypeptide of the invention to further modulate the target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as previously described.
In yet another embodiment, the cell-free assay comprises contacting a polypeptide of the invention or biologically active portion thereof with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the polypeptide to preferentially bind to or modulate the activity of a target molecule. The cell-free assays of the present invention are amenable to use of both a soluble form or the membrane-bound form of a polypeptide of the invention. In the case of cell-free assays comprising the membrane-bound form of the polypeptide, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of the polypeptide is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n- octylglucoside, n-dodecylglucoside, n-octylmaltoside, octanoyl-N-methylglucamide, decanoyl-N- methylglucamide, Triton X-100, Triton X-l 14, Thesit, Isotridecypoly(ethylene glycol ether)n, 3- [(3-cholamidopropyl)dimethylamminio]-l -propane sulfonate (CHAPS), 3-[(3- cholamidopropyl)dimethylamminio]-2-hydroxy-l -propane sulfonate (CHAPSO), or N- dodecyl=N,N-dimethyl-3-ammonio-l -propane sulfonate.
In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either the polypeptide of the invention or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to the polypeptide, or interaction of the polypeptide with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or A polypeptide of the invention, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of the polypeptide of the invention can be determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the polypeptide of the invention or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N-hydroxy- succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with the polypeptide of the invention or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the invention trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the polypeptide of the invention or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the polypeptide of the invention or target molecule.
In another embodiment, modulators of expression of a polypeptide of the invention are identified in a method in which a cell is contacted with a candidate compound and the expression of the selected mRNA or protein (i.e., the mRNA or protein corresponding to a polypeptide or nucleic acid of the invention) in the cell is determined. The level of expression of the selected mRNA or protein in the presence of the candidate compound is compared to the level of expression of the selected mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of expression of the polypeptide of the invention based on this comparison. For example, when expression of the selected mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of the selected mRNA or protein expression. Alternatively, when expression of the selected mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of the selected mRNA or protein expression. The level of the selected mRNA or protein expression in the cells can be determined by methods described herein.
In yet another aspect of the invention, a polypeptide of the inventions can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g. , U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and PCT Publication No. WO 94/10300), to identify other proteins, which bind to or interact with the polypeptide of the invention and modulate activity of the polypeptide of the invention. Such binding proteins are also likely to be involved in the propagation of signals by the polypeptide of the inventions as, for example, upstream or downstream elements of a signaling pathway involving the polypeptide of the invention.
This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
B. Detection Assays
Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.
1. Chromosome Mapping
Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. Accordingly, nucleic acid molecules described herein or fragments thereof, can be used to map the location of the corresponding genes on a chromosome. The mapping of the sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
Briefly, genes can be mapped to chromosomes by preparing PCR primers (preferably 15- 25 bp in length) from the sequence of a gene of the invention. Computer analysis of the sequence of a gene of the invention can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the gene sequences will yield an amplified fragment. For a review of this technique, see D'Eustachio et al. ((1983) Science 220:919-924).
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the nucleic acid sequences of the invention to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a gene to its chromosome include in situ hybridization (described in Fan et al. (1990) Proc. Natl. Acad. Sci. USA 87:6223-27), pre-screening with labeled flow-sorted chromosomes (CITE), and preselection by hybridization to chromosome specific cDNA libraries. Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. For a review of this technique, see Verma et al., (Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York, 1988)).
Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping puφoses. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland et al. (1987) Nature 325:783-787. Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with a gene of the invention can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymoφhisms.
2. Tissue Typing
The nucleic acid sequences of the present invention can also be used to identify individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymoφhism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057). Furthermore, the sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the nucleic acid sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue. The nucleic acid sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification puφoses. Because greater numbers of polymoφhisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO:l or 12 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:2, 12, or 25 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
If a panel of reagents from the nucleic acid sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.
3. Use of Partial Gene Sequences in Forensic Biology
DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a peφetrator of a crime. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions are particularly appropriate for this use as greater numbers of polymoφhisms occur in the noncoding regions, making it easier to differentiate individuals using this technique. Examples of polynucleotide reagents include the nucleic acid sequences of the invention or portions thereof, e.g., fragments derived from noncoding regions having a length of at least 20 or 30 bases. The nucleic acid sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such probes can be used to identify tissue by species and/or by organ type.
C. Predictive Medicine
The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trails are used for prognostic (predictive) puφoses to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining expression of a polypeptide or nucleic acid of the invention and/or activity of a polypeptide of the invention, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant expression or activity of a polypeptide of the invention. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with aberrant expression or activity of a polypeptide of the invention. For example, mutations in a gene of the invention can be assayed in a biological sample. Such assays can be used for prognostic or predictive puφose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with aberrant expression or activity of a polypeptide of the invention.
Another aspect of the invention provides methods for expression of a nucleic acid or polypeptide of the invention or activity of a polypeptide of the invention in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics"). Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent).
Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs or other compounds) on the expression or activity of a polypeptide of the invention in clinical trials. These and other agents are described in further detail in the following sections.
1. Diagnostic Assays An exemplary method for detecting the presence or absence of a polypeptide or nucleic acid of the invention in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of the invention such that the presence of a polypeptide or nucleic acid of the invention is detected in the biological sample. A preferred agent for detecting mRNA or genomic DNA encoding a polypeptide of the invention is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA encoding a polypeptide of the invention. The nucleic acid probe can be, for example, a full-length cDNA, such as the nucleic acid of SEQ ID NO: 1, 2, 11, 12, 24, or 25, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a polypeptide of the invention. Other suitable probes for use in the diagnostic assays of the invention are described herein.
A preferred agent for detecting a polypeptide of the invention is an antibody capable of binding to a polypeptide of the invention, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of a polypeptide of the invention include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of a polypeptide of the invention include introducing into a subject a labeled antibody directed against the polypeptide. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting a polypeptide of the invention or mRNA or genomic DNA encoding a polypeptide of the invention, such that the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide is detected in the biological sample, and comparing the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide in the control sample with the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide in the test sample.
The invention also encompasses kits for detecting the presence of a poljφeptide or nucleic acid of the invention in a biological sample (a test sample). Such kits can be used to determine if a subject is suffering from or is at increased risk of developing a disorder associated with aberrant expression of a polypeptide of the invention (e.g., a proliferative disorder, e.g., psoriasis or cancer). For example, the kit can comprise a labeled compound or agent capable of detecting the polypeptide or mRNA encoding the polypeptide in a biological sample and means for determining the amount of the poljφeptide or mRNA in the sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide). Kits can also include instructions for observing that the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of the polypeptide if the amount of the polypeptide or mRNA encoding the polypeptide is above or below a normal level.
For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a poljφeptide of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.
For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule encoding a polypeptide of the invention. The kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit can also comprise components necessary for detecting the detectable agent (e.g., an enzjαne or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit is usually enclosed within an individual container and all of the various containers are within a single package along with instructions for observing whether the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of the polypeptide.
2. Prognostic Assays
The methods described herein can furthermore be utilized as diagnostic or prognostic assays to identify subjects having or at risk of developing a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with aberrant expression or activity of a polypeptide of the invention, e.g., a neurological disorder, e.g., Alzheimer's disease, a bone disorder, or a cardiovascular disorder. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing such a disease or disorder. Thus, the present invention provides a method in which a test sample is obtained from a subject and a poljφeptide or nucleic acid (e.g., mRNA, genomic DNA) of the invention is detected, wherein the presence of the polypeptide or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant expression or activity of the poljφeptide. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention. For example, such methods can be used to determine whether a subject can be effectively treated with a specific agent or class of agents (e.g., agents of a type which decrease activity of the polypeptide). Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant expression or activity of a polypeptide of the invention in which a test sample is obtained and the polypeptide or nucleic acid encoding the poljφeptide is detected (e.g., wherein the presence of the polypeptide or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant expression or activity of the polypeptide).
The methods of the invention can also be used to detect genetic lesions or mutations in a gene of the invention, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized aberrant expression or activity of a polypeptide of the invention. In preferred embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion or mutation characterized by at least one of an alteration affecting the integrity of a gene encoding the polypeptide of the invention, or the mis-expression of the gene encoding the polypeptide of the invention. For example, such genetic lesions or mutations can be detected by ascertaining the existence of at least one of: 1) a deletion of one or more nucleotides from the gene; 2) an addition of one or more nucleotides to the gene; 3) a substitution of one or more nucleotides of the gene; 4) a chromosomal rearrangement of the gene; 5) an alteration in the level of a messenger RNA transcript of the gene; 6) an aberrant modification of the gene, such as of the methylation pattern of the genomic DNA; 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; 8) a non-wild type level of a the protein encoded by the gene; 9) an allelic loss of the gene; and 10) an inappropriate post-translational modification of the protein encoded by the gene. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in a gene.
In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91 : 360-364), the latter of which can be particularly useful for detecting point mutations in a gene (see, e.g., Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to the selected gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art.
These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in a selected gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (.see, e.g., U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozjrme cleavage site.
In other embodiments, genetic mutations can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For example, genetic mutations can be identified in two- dimensional arrays containing light-generated DNA probes as described in Cronin et al., supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the selected gene and detect mutations by comparing the sequence of the sample nucleic acids with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Bio/Techniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (\99?>) Appl. Biochem. Biotechnol. 38: 147-159). Other methods for detecting mutations in a selected gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242). In general, the technique of "mismatch cleavage" entails providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. RNA/DNA duplexes can be treated with RNase to digest mismatched regions, and DNA/DNA hybrids can be treated with SI nuclease to digest mismatched regions.
In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662). According to an exemplary embodiment, a probe based on a selected sequence, e.g., a wild-type sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in genes. For example, single strand conformation potymoφhism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766; see also Cotton (1993) Mutat. Res. 285:125-144; Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, and the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).
In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a 'GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chem. 265:12753).
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324: 163); Saiki et al. (1989) Proc. Natl.
Acad. Sci. USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent or reduce polymerase extension (Prossner (1993) Tibtech 11 :238). In addition, it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a gene encoding a polypeptide of the invention. Furthermore, any cell type or tissue, e.g., hepatocytes, in which the polypeptide of the invention is expressed may be utilized in the prognostic assays described herein.
3. Pharmacogenomics
Agents, or modulators which have a stimulatory or inhibitory effect on activity or expression of a polypeptide of the invention as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders associated with aberrant activity of the polypeptide. In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of a polypeptide of the invention, expression of a nucleic acid of the invention, or mutation content of a gene of the invention in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Linder (1997) Clin. Chem. 43(2):254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body are referred to as "altered drug action." Genetic conditions transmitted as single factors altering the way the body acts on drugs are referred to as "altered drug metabolism". These pharmacogenetic conditions can occur either as rare defects or as polymoφhisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti- malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymoφhisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These poljmioφhisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymoφhic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, a PM will show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite moφhine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
Thus, the activity of a polypeptide of the invention, expression of a nucleic acid encoding the polypeptide, or mutation content of a gene encoding the polypeptide in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymoφhic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a modulator of activity or expression of the polypeptide, such as a modulator identified by one of the exemplary screening assays described herein.
4. Monitoring of Effects During Clinical Trials Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of a polypeptide of the invention (e.g., the ability to modulate aberrant cell proliferation chemotaxis, and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent, as determined by a screening assay as described herein, to increase gene expression, protein levels or protein activity, can be monitored in clinical trials of subjects exhibiting decreased gene expression, protein levels, or protein activity. Alternatively, the effectiveness of an agent, as determined by a screening assay, to decrease gene expression, protein levels or protein activity, can be monitored in clinical trials of subjects exhibiting increased gene expression, protein levels, or protein activity. In such clinical trials, expression or activity of a polypeptide of the invention and preferably, that of other polypeptide that have been implicated in for example, a cellular proliferation disorder, can be used as a marker of the immune responsiveness of a particular cell.
For example, and not by way of limitation, genes, including those of the invention, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates activity or expression of a polypeptide of the invention (e.g., as identified in a screening assay described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of a gene of the invention and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of a gene of the invention or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of the polypeptide or nucleic acid of the invention in the preadministration sample; (iii) obtaining one or more post- administration samples from the subject; (iv) detecting the level the of the polypeptide or nucleic acid of the invention in the post-administration samples; (v) comparing the level of the polypeptide or nucleic acid of the invention in the pre-administration sample with the level of the polypeptide or nucleic acid of the invention in the post-administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of the polypeptide to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of the polypeptide to lower levels than detected, i.e., to decrease the effectiveness of the agent.
C. Methods of Treatment
The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant expression or activity of a polypeptide of the invention. For example, disorders characterized by aberrant expression or activity of the poljφeptides of the invention include atherosclerosis and obesity-related disorders. In addition, the polypeptides of the invention can be used to bind Ox- LDL, to modulate the alternative complement pathway, to treat immune related disorders (e.g., autoimmune disorders (e.g., arthritis and T cell autoimmune disorders (e.g., AIDS)) and inflammatory disorders (e.g., bacterial infection, and arthritis (e.g., rheumatoid arthritis, osteoarthritis)), and obesity related disorders (e.g., hypertension and energy balance regulation), as well as other uses described herein. Moreover, the TANGO 269 and TANGO 298 polypeptides of the invention can be used to modulate hepatocyte and bone marrow cell proliferation and differentiation (e.g., to treat cirrhosis and leukemia).
1. Prophylactic Methods
In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant expression or activity of a polypeptide of the invention, by administering to the subject an agent which modulates expression or at least one activity of the polypeptide. Subjects at risk for a disease which is caused or contributed to by aberrant expression or activity of a polypeptide of the invention can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of aberrancy, for example, an agonist or antagonist agent can be used for treating the subject. For example, an antagonist of a TANGO 269 protein can be used to treat a blood related disorder, e.g., acute myeloid leukemia, hemophilia, leukemia, anemia (e.g., sickle cell anemia), and thalassemia, associated with aberrant TANGO 269 expression or activity, and an antagonist of a TANGO 298 protein may be used to treat an obesity related disorder, e.g., hyperglycemic shock or poor regulation of energy balance, associated with aberrant TANGO 269 expression or activity. The appropriate agent can be determined based on screening assays described herein.
2. Therapeutic Methods
Another aspect of the invention pertains to methods of modulating expression or activity of a polypeptide of the invention for therapeutic puφoses. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of the polypeptide. An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of the polypeptide, a peptide, a peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more of the biological activities of the polypeptide. Examples of such stimulatory agents include the active polypeptide of the invention and a nucleic acid molecule encoding the polypeptide of the invention that has been introduced into the cell. In another embodiment, the agent inhibits one or more of the biological activities of the polypeptide of the invention. Examples of such inhibitory agents include antisense nucleic acid molecules and antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a poljφeptide of the invention. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) expression or activity. In another embodiment, the method involves administering a polypeptide of the invention or a nucleic acid molecule of the invention as therapy to compensate for reduced or aberrant expression or activity of the polypeptide.
Stimulation of activity is desirable in situations in which activity or expression is abnormally low or downregulated and/or in which increased activity is likely to have a beneficial effect, e.g., in treatment of immune disorders. Conversely, inhibition of activity is desirable in situations in which activity or expression is abnormally high or upregulated and/or in which decreased activity is likely to have a beneficial effect, e.g., in treatment of a obesity related disorder.
This invention is further illustrated by the following examples, which should not be construed as limiting.
EXAMPLES Gene Expression Analysis (Experiment I)
Total RNA was prepared from various human tissues by a single step extraction method using RNA STAT-60 according to the manufacturer's instructions (TelTest, Inc). Each RNA preparation was treated with DNase I (Ambion) at 37°C for 1 hour. DNAse I treatment was determined to be complete if the sample required at least 38 PCR amplification cycles to reach a threshold level of fluorescence using β-2 microglobulin as an internal amplicon reference. The integrity of the RNA samples following DNase I treatment was confirmed by agarose gel electrophoresis and ethidium bromide staining. After phenol extraction cDNA was prepared from the sample using the SUPERSCRIPT™ Choice System following the manufacturer's instructions (GibcoBRL). A negative control of RNA without reverse transcriptase was mock reverse transcribed for each RNA sample.
Human TANGO 298 expression was measured by TaqMan® quantitative PCR (Perkin Elmer Applied Biosystems) in cDNA prepared from the following normal human tissues or cell lines: lymph node, spleen, kidney, brain, lung, skeletal muscle, fetal liver, tonsil, colon, heart, and liver from two adult donors; fibrotic liver samples prepared from seven different donors; transformed human cell lines K562, an erythroleukemia; Hep3B hepatocellular liver carcinoma cells cultured in reduced oxygen tension (Hep3B hjφoxia); CD34+ cells from mobilized peripheral blood (mPB CD34+), adult resting bone marrow (ABM CD34+), and G-CSF mobilized bone marrow (mBM CD34+); CD34" cells purified from mPB leukocytes (mPB CD34 ); G-CSF mobilized peripheral blood leukocytes (mPB leukocytes); CD34" cells from mobilized bone marrow (MBM CD34 ); Thl and Th2 cells stimulated for six or 48 hours with anti-CD3 antibody; CD4+, and CD8+ T cells; resting CD19+ B cells; resting and phytohemaglutinin activated peripheral blood mononuclear cells (PBMC); CD14+ cells; granulocytes; HEK 293, epithelial cells from embryonic kidney transformed with adenovirus 5 DNA; and Jurkat, a T cell leukemia.
Probes were designed by PrimerExpress software (PE Biosystems) based on the sequence of the human TANGO 298 gene. The primers and probes for expression analysis of human TANGO 298 were as follows: human TANGO 298 Forward Primer: 5'-AGCAGGTGTTTGGCATCGAT-3' (SEQ ID NO:35) human TANGO 298 Reverse Primer: 5'-GTCGTTGGCGTGGGTCAT-3' (SEQ ID NO:36) human TANGO 298 TaqMan Probe: FAM-TCACCACGCACCCCGACTACCAC-TAMRA
(SEQ ID NO:37)
Each human TANGO 298 gene probe was labeled using FAM (6-carboxyfluorescein), and the β2-microglobulin reference probe was labeled with a different fluorescent dye, VIC. The differential labeling of the target gene and internal reference gene thus enabled measurement in same well. Forward and reverse primers and the probes for both β2 -microglobulin and target gene were added to the TaqMan® Universal PCR Master Mix (PE Applied Biosystems). Although the final concentration of primer and probe could vary, each was internally consistent within a given experiment. A typical experiment contained 200nM of forward and reverse primers plus lOOnM probe for β-2 microglobulin and 600 nM forward and reverse primers plus 200 nM probe for the target gene. TaqMan matrix experiments were carried out on an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems). The thermal cycler conditions were as follows: hold for 2 min at 50°C and 10 min at 95°C, followed by two-step PCR for 40 cycles of 95°C for 15 sec followed by 60°C for 1 min. The following method was used to quantitatively calculate human TANGO 298 gene expression in the various tissues relative to β-2 microglobulin expression in the same tissue. The threshold cycle (Ct) value is defined as the cycle at which a statistically significant increase in fluorescence is detected. A lower Ct value is indicative of a higher mRNA concentration. The Ct value of the human TANGO 298 gene is normalized by subtracting the Ct value of the β-2 microglobulin gene to obtain a ΔCt value using the following formula: ΔCt=Cthuman TANG0298 - Ct p.2 micro lobulin- Expression is then calibrated against a cDNA sample showing a comparatively low level of expression of the human TANGO 298 gene. The ΔCt value for the calibrator sample is then subtracted from ΔCt for each tissue sample according to the following formula: ΔΔCt=ΔCt- sampie " _Ct-caljbrator. Relative expression is then calculated using the arithmetic formula given by 2" ά&Ct. Expression of the target human TANGO 298 gene in each of the tissues tested is then graphically represented as discussed in more detail below.
Figure 15 shows expression of human TANGO 298 in various tissues and cell lines as described above, relative to expression in tonsil cells. The results indicate significant expression in CD34+ cells from mobilized peripheral blood (mPB CD34+), adult resting bone marrow (ABM CD34+), G-CSF mobilized bone marrow (mBM CD34+), and G-CSF mobilized peripheral blood leukocytes (mPB leukocytes); and moderate expression in fetal liver, cell line K526 (an ei throleukemia), and CD34" cells from mobilized bone marrow (MBM CD34 ).
Gene Expression Analysis (Experiment II) Total RNA was prepared from various human tissues as described above, and human
TANGO 298 expression was measured by TaqMan® quantitative PCR (Perkin Elmer Applied Biosystems) in cDNA prepared from the following normal human tissues or cell lines: lung; brain; colon; heart; spleen; kidney; fetal liver; skeletal muscle; mononuclear cells from mobilized bone marrow (mBM MNC); mononuclear cells from mobilized bone marrow (mBM MNC); CD34+ cells from mobilized bone marrow (CD34 is a progenitor cell marker) (mBM CD34+); CD34+ cells from mobilized peripheral blood (mPB CD34+); CD34+ cells from mobilized peripheral blood (mPB CD34+); CD34+ cells from mobilized peripheral blood (mPB CD34+); CD34+ cells from adult resting bone marrow (ABM CD34+); CD34+ cells from adult resting bone marrow (ABM CD34+); CD34+ cells from umbilical cord blood (cord blood CD34+); CD34+ cells from umbilical cord blood (cord blood CD34+); CD34+ cells from fetal liver (fetal liver CD34+); CD34+ cells from fetal liver (fetal liver CD34+); GPA+ cells from bone marrow (BM GPA+)(GPA is an eiythroid progenitor cell marker)(cells from two sources); GPAlo and CD71+ bone marrow cells (GPAlo is an earlier version of the erythroid cell marker GPA, e.g., used to detect erythroid progenitor cells at an earlier stage of development than those possessing the GPA marker)(CD71 is an erythroid progenitor cell marker)(BM GPAlo CD71+)(cells from 2 sources); CD417CD14" mobilized peripheral blood cells (CD41 is a megakaryocyte marker, and CD 14 is a marker for monocytes and macrophages, and neutrophils, to a lesser extent)(mPB CD41 CD14 ); and CD417CD14" bone marrow cells (BM CD4l7CD14 )(cells from 2 sources); CD15+ cells from mobilized bone marrow (CD15 is a marker for granulocytes)(mBM CD15+); CD157CD1 lb' cells from mobilized bone marrow (CDl 1 is a marker for leukocytes, including granulocytes)(mBM CD157CD1 lb")(cells from three sources); CD157CD1 lb+ cells from mobilized bone marrow (mBM CD157CD1 lb+)(cells from 2 sources); CD157CD34" cells from bone marrow (BM CD157CD34')(cells from 2 sources); erythroblasts, cultured for 24 hours; erythroblasts, cultured for 48 hours (cells from 2 sources); erythroblasts, cultured for 6 days (cells from 2 sources); erythroblasts, cultured for 10 days; erythroblasts, cultured for 12 days (cells from 2 sources); CD36+ erythroblasts, cultured for 14 days (CD36 is a platelet marker); megakaryocytes, cultured for 24 hours; megakaryocytes, cultured for 44 hours; megakaryocytes, cultured for 7 days; megakaryocytes, cultured for 12 days; megakaryocytes, cultured for 14 days; neutrophils, cultured for 4 days (cells from 2 sources); neutrophils, cultured for 6 days (cells from 2 sources); neutrophils, cultured for 7 days; neutrophils, cultured for 12 days (cells from 2 sources); neutrophils, 14 days stimulated with LP3T; and a negative control (water).
Probes were designed by PrimerExpress software (PE Biosystems) based on the sequence of the human TANGO 298 gene. The primers and probes for expression analysis of human TANGO 298 were as follows:
human TANGO 298 Forward Primer: 5*-AGCAGGTGTTTGGCATCGAT-3' (SEQ ID NO:35) human TANGO 298 Reverse Primer: 5'-GTCGTTGGCGTGGGTCAT-3' (SEQ ID NO:36) human TANGO 298 TaqMan Probe: FAM-TCACCACGCACCCCGACTACCAC-TAMRA
(SEQ ID NO:37)
The method as described in Experiment I was used to quantitatively calculate human TANGO 298 gene expression in the various tissues relative to β-2 microglobulin expression in the same tissue.
Figures 16A-16B show expression of human TANGO 298 in various tissues and cell lines as described above, relative to expression in CDl 57CD1 lb+ cells from mobilized bone marrow (mBMCF157CDl lb+). [The figures are from the same experiment, but are shown on two pages for ease of display.] In general, expression is revealed to be highest in the erythroblast samples, the megakaryocyte samples, the neutrophil samples, and in cells populations positive for CD34+ marker (including cells from mobilized bone marrow, adult resting bone marrow, umbilical cord blood, and fetal liver). In particular, the samples from each group manifest higher levels of TANGO 298 expression in the earlier stages of development, and expression tapers off after a certain point in time. For example, in the erythroblast cell samples tested, levels of TANGO 298 expression increase in proportion to the time the cells were cultured, from 24 hours to 48 hours, then decrease with culture times of 6 days, 10 days, and 12 days.
Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:
1. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 55% identical to the nucleotide sequence of SEQ ID NO: l, 2, 11, 12, 24, 25, the cDNA insert of the
5 plasmid deposited with the ATCC as Accession Number 207218, 207216, or a complement thereof; b) a nucleic acid molecule comprising a fragment of at least 300 nucleotides of the nucleotide sequence of SEQ ID NO:l, 2, 11, 12, 24, 25, the cDNA insert of the plasmid deposited with the ATCC as Accession Number 207218, 207216, or a complement thereof; 0 c) a nucleic acid molecule which encodes a poljφeptide comprising the amino acid sequence of SEQ ID NO:3, 13, 26, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number 207218 or 207216; d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 13, 26, or the amino acid sequence encoded by the cDNA 5 insert of the plasmid deposited with the ATCC as Accession Number 207218, 207216, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:3, 13, 26, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number 207218 or 207216. e) a nucleic acid molecule which encodes a naturally occurring allelic variant of a 0 polypeptide comprising the amino acid sequence of SEQ ID NO:3, 13, 26, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number 207218 or 207216, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:2, 12, 25, or a complement thereof under stringent conditions.
5 2. The isolated nucleic acid molecule of claim 1, which is selected from the group consisting of: a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, 2, 11, 12, 24, 25, the cDNA insert of the plasmid deposited with the ATCC as Accession Number 207218, 207216, or a complement thereof; and b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 13, 26, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number 207218 or 207216.
3. The nucleic acid molecule of claim 1 further comprising vector nucleic acid sequences.
4. The nucleic acid molecule of claim 1 further comprising nucleic acid sequences encoding a heterologous poljφeptide.
5. A host cell which contains the nucleic acid molecule of claim 1.
6. The host cell of claim 5 which is a mammalian host cell.
7. A non-human mammalian host cell containing the nucleic acid molecule of claim
1.
8. An isolated polypeptide selected from the group consisting of: a) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 13, or 26, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:3,
13, or 26; b) a naturally occurring allelic variant of a poljφeptide comprising the amino acid sequence of SEQ ID NO:3, 13, 26, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number 207218, 207216, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:2, 12, 25, or a complement thereof under stringent conditions; and c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 55% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2, 12, 25, or a complement thereof.
9. The isolated poljφeptide of claim 8 comprising the amino acid sequence of SEQ ID NO:3, 13, or 26.
10. The polypeptide of claim 8 further comprising heterologous amino acid sequences.
11. An antibody which selectively binds to a polypeptide of claim 8.
12. A method for producing a poljφeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 14, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number 207218 or 207216; b) a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:3, 13, 26, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the
ATCC as Accession Number 207218 or 207216, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:3, 13, 26, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number 207218 or 207216; and c) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 13, 26, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number 207218, 207216, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO: 1, 11, or 24, or a complement thereof under stringent conditions; comprising culturing the host cell of claim 5 under conditions in which the nucleic acid molecule is expressed.
13. A method for detecting the presence of a polypeptide of claim 8 in a sample, comprising: a) contacting the sample with a compound which selectively binds to a polypeptide of claim 8; and b) determining whether the compound binds to the polypeptide in the sample.
14. The method of claim 13, wherein the compound which binds to the polypeptide is an antibody.
15. A kit comprising a compound which selectively binds to a polypeptide of claim 8 and instructions for use.
16. A method for detecting the presence of a nucleic acid molecule of claim 1 in a sample, comprising the steps of: a) contacting the sample with a nucleic acid probe or primer which selectively hybridizes to the nucleic acid molecule; and b) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample.
17. The method of claim 16, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.
18. A kit comprising a compound which selectively hybridizes to a nucleic acid molecule of claim 1 and instructions for use.
19. A method for identifying a compound which binds to a polypeptide of claim 8 comprising the steps of: a) contacting a polypeptide, or a cell expressing a polypeptide of claim 8 with a test compound; and b) determining whether the poljφeptide binds to the test compound.
20. The method of claim 19, wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of: a) detection of binding by direct detecting of test compound/polypeptide binding; b) detection of binding using a competition binding assay; c) detection of binding using an assay for TANGO 269 or TANGO 298-mediated signal transduction.
21. A method for modulating the activity of a poljφeptide of claim 8 comprising contacting a polypeptide or a cell expressing a polypeptide of claim 8 with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the poljφeptide.
22. A method for identifying a compound which modulates the activity of a polypeptide of claim 8, comprising: a) contacting a poljφeptide of claim 8 with a test compound; and b) determining the effect of the test compound on the activity of the polypeptide to thereby identify a compound which modulates the activity of the polypeptide.
23. A method for modulating the function of hematopoietic cells by contacting the cells with an agent which modulates the function of a poljφeptide having the amino acid sequence of SEQ ID NO:26.
I l l -
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002079433A2 (en) * 2001-03-29 2002-10-10 Deltagen, Inc. Transgenic mice containing tmem3 beta-1,3-n-acetylglucosaminyltransferase gene disruptions

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US5686597A (en) * 1995-06-06 1997-11-11 Incyte Pharmaceuticals, Inc. Thrombin receptor homolog

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5686597A (en) * 1995-06-06 1997-11-11 Incyte Pharmaceuticals, Inc. Thrombin receptor homolog

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002079433A2 (en) * 2001-03-29 2002-10-10 Deltagen, Inc. Transgenic mice containing tmem3 beta-1,3-n-acetylglucosaminyltransferase gene disruptions
WO2002079433A3 (en) * 2001-03-29 2003-05-08 Deltagen Inc Transgenic mice containing tmem3 beta-1,3-n-acetylglucosaminyltransferase gene disruptions

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