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WO2000006699A9 - Nouvelles molecules de la famille des proteines liees au tango 175 et utilisations de ces dernieres - Google Patents

Nouvelles molecules de la famille des proteines liees au tango 175 et utilisations de ces dernieres

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Publication number
WO2000006699A9
WO2000006699A9 PCT/US1999/017289 US9917289W WO0006699A9 WO 2000006699 A9 WO2000006699 A9 WO 2000006699A9 US 9917289 W US9917289 W US 9917289W WO 0006699 A9 WO0006699 A9 WO 0006699A9
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seq
tango
nucleic acid
polypeptide
protein
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PCT/US1999/017289
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English (en)
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WO2000006699A1 (fr
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Publication of WO2000006699A1 publication Critical patent/WO2000006699A1/fr
Publication of WO2000006699A9 publication Critical patent/WO2000006699A9/fr

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  • protease inhibitors are secreted into blood, mucous, salivary gland secretions, tear fluid and skin and can act systemically or locally. Their secretion by cells at sites of protease action may help localize degradation by proteases to specific areas within affected tissues.
  • a number of protease inhibitors are members of the "four- disulfide core” family of proteins. The conserved pattern of cysteines found in members of this family predicts a related tertiary structure and is suggestive of protease inhibitory activity.
  • One group of locally-acting protease inhibitors within the " four-disulfide core” family are the anti- leukoproteinases . These protease inhibitors have been shown to be involved in a variety of cell processes and disorders.
  • rat WDNM-1 (Dear & Kefford (1991) Biochem . & Biophys . Res . Comm . 176:247-254) is downregulated in metastatic versus non-metastatic rat mammary adenocarcinoma and may function as a metastasis inhibitor (Dear et al . (1988) Cancer Res . 48:5203-5209).
  • murine WDNM-1 has been identified as a genetic marker for murine mammary tumors transformed by the oncogenes neu or ras (Morrison & Leder (1994) Oncogene 9:3417-3426).
  • human anti -leukoproteinase has been shown to promote hematopoiesis by inhibiting degradation of cytokines, growth factor receptors and other proteins involved in blood cell growth and differentiation (Goselink et al . (1996) J. Exp . Med . 184:1305-1313), while experiments with porcine anti- leukoproteinase demonstrate a function in the maintenance and progression of pregnancy (Simmen et al . (1991) Biol . Reprod . 44:191-200). In rats, anti-leukoproteinase has been shown to be depleted in arthritic cartilage (Burkhardt et al . (1997) J. Rheumatol . 24:1145-1154.
  • a murine anti-leukoproteinase, MALP (SLPI) , inhibits bacterial lipopolysaccharide and may be useful in the treatment of septic shock (Jin et al . (1997) Cell 88:417-26).
  • SLPI has also been implicated in chronic respiratory diseases such as chronic bronchitis, emphysema, cystic fibrosis (Mitsuhashi, et al . (1997) J “ . Pharmacol . Exp . Ther. 282:1005-1010) and asthma (Fath et al. (1998) J. Biol . Chem . 273:13563-13569).
  • SLPI may also have a broad spectrum antibiotic activity that includes antiretroviral , bactericidal, and antifungal activity (Tomee et al . (1998) Thorax 53:114-116) .
  • a human skin-derived anti-leukoproteinase, SKALP is elevated in psoriasis and wound healing (Schalkwijk et al . (1991) Biochem. Biophys . Acta 1096:148-154) and is differentially expressed in epidermal carcinomas (Alkemade et al . (1993) Am. J. Path . 143:1679-1687).
  • anti-leukoproteinases have been identified, including one from trout (GenBankTM Accession Number U03890) , and it is believed that additional anti- leukoproteinases with different or related functions are yet to be identified. Active peptides derived from anti- leukoproteinases have been proposed as therapies for the treatment of conditions in which anti-leukoproteinases play a role. Thus, these molecules, peptides derived from them, and modulators thereof may have utility in the treatment and prevention of such conditions and as markers for specific disease states.
  • integrins Cellular interactions with the extracellular matrix are mediated, in part, through the family of cell- surface molecules known as integrins .
  • a subfamily of integrins recognizes and binds to the peptide sequence Arginine-Glycine-Aspartate (RGD) found in extracellular matrix proteins such as fibronectin.
  • RGD Arginine-Glycine-Aspartate
  • the interaction of cells with matrix RGD is important in normal processes such as wound healing, blood clotting and hematopoiesis and plays a role in abnormal states, such as metastasis. Secreted proteins that contain RGD have potential clinical value in modulating these interactions.
  • the disintegrins a family of secreted snake venom proteins that bind integrins, contain RGD and act as potent platelet aggregation inhibitors (Perutelli (1995) decent! Progressi in Medicina 86:168-74).
  • RGD-containing peptides may have utility as antithrombotic agents and in the prevention of arterial thrombosis (Schafer (1996) Am. J. Med . 101:199-209).
  • the present invention is based, at least in part, on the discovery of a gene encoding TANGO-175, a secreted protein that is related to several proteins in the four disulfide core family.
  • the present invention is also based, at least in part, on the discovery of a gene encoding murine WDNM-2, a protein that, like TANGO0175, is related to several proteins in the four disulfide core family.
  • the mouse TANGO-175 cDNA described below has a 189 nucleotide open reading frame (nucleotides 18-206 of SEQ ID NO:l; SEQ ID NO:3) which encodes a 63 amino acid protein (SEQ ID NO: 2) .
  • This protein includes a predicted signal sequence of about 24 amino acids (from amino acid 1 to about amino acid 24 of SEQ ID NO: 2; SEQ ID NO: 21) and a predicted mature protein of about 39 amino acids (from about amino acid 25 to amino acid 63 of SEQ ID NO: 2; SEQ ID NO: 22) .
  • Murine TANGO-175 protein possesses six cysteine residues, C1-C6, which occur at amino acid 35, 39, 45, 51, 56 and 60 of SEQ ID NO: 2, respectively. These cysteine residues are expected to form interdomain disulfide bonds which stabilize the TANGO-175 protein. Cysteines C1-C5, C2-C4 and C3-C6 are expected to form disulfide bonds.
  • Murine TANGO-175 protein has some sequence similarity to murine WDNM-1 protein (SEQ ID NO: 16; Dear & Kefford (1991) Biochem & Biophy. Res . Co m . 176:247; EMBL database accession no.
  • trout anti -leukoproteinase (Genbank accession no. U03890) , rat WDNM-1 (SEQ ID NO:17; Genbank accession no. P14730) , human antileukoproteinasse (Goselink et al . (1996) J. Exp Med 184:1305-12), and murine anti-leukoproteinase (SLPI) (SEQ ID NO: 19; Jin et al . (1997) Cell 88:417-26; Genbank accession no. P97430) .
  • SLPI murine anti-leukoproteinase
  • nucleotide sequences encoding human TANGO-175 are described below (SEQ ID NO: 4, 5, 6, and 7) . Each of these sequences has a 183 nucleotide open reading frame (nucleotides 23-205 of SEQ ID NO : 4 , 5, 6, and 7; SEQ ID NO: 8, 9, 10, and 11) which encodes a 61 amino acid protein (SEQ ID NO:12). The four sequences differ only at nucleotide 52 (the third nucleotide in the codon encoding Valine at residue 10) .
  • the human TANGO-175 protein includes a predicted signal sequence of about 24 amino acids (from amino acid 1 to about amino acid 24 of SEQ ID NO: 12; SEQ ID NO: 23) and a predicted mature protein of about 37 amino acids (from about amino acid 25 to amino acid 61 of SEQ ID NO: 12; SEQ ID NO: 24) .
  • Human TANGO-175 protein possesses six cysteine residues, cysteines C1-C6, which occur at amino acids 33, 37, 43, 49, 54 and 58 of SEQ ID NO: 12, respectively. These cysteine residues are expected to form interdomain disulfide bonds which stabilize the human TANGO-175 protein. Cysteines C1-C5, C2-C4 and C3-C6 are expected to form disulfide bonds. Like murine TANGO-175, human TANGO-175 protein has some sequence similarity to murine WDNM-1 protein (SEQ ID N0:16; Dear & Kefford (1991) Biochem & Biophy. Res . Comm . 176:247; EMBL database accession no.
  • trout anti-leukoproteinase (Genbank accession no. U03890) , rat WDNM-1 (SEQ ID NO:17; Genbank accession no. P14730) , human antileukoproteinasse (Goselink et al.(1996) J. Exp Med 184:1305-12), and murine anti-leukoproteinase (SLPI) (SEQ ID NO:19; Jin et al . (1997) Cell 88:417-26; Genbank accession no. P97430) .
  • SLPI murine anti-leukoproteinase
  • Both murine and human TANGO-175 have six cysteines that are spaced identically to cysteines 2, 3, 4, 5, 7, and 8 of murine WDNM-1, a four-disulfide core protein. However, murine and human TANGO-175 lack equivalents of cysteines 1 and 6 present in murine WDNM-1. Thus, rather than following the 1-6, 2-7, 3-5, and 4-8 disulfide bonding pattern found in the four-disulfide core proteins, TANGO-175 likely follows a 1-5, 2-4, and 3-6 disulfide bonding pattern (corresponding to the 2-7, 3-5, and 4-8 disulfide bonds of WDNM-1) .
  • the nucleotide sequence of murine WDNM-2 ( Figure 3; SEQ ID NO: 13, SEQ ID NO: 15 open reading frame only) is predicted to encode a 75 amino acid protein (SEQ ID NO: 14) having a four-disulfide core sequence.
  • the protein is predicted to have a signal sequence extending from acid 1 to amino acid 17 of SEQ ID NO: 4 (SEQ ID NO: 27) .
  • the mature protein is predicted to extend from amino acid 18 to amino acid 75 of SEQ ID NO: 12 (SEQ ID NO: 28) .
  • WDNM-2 is likely a serine protease inhibitor.
  • Murine WDNM-2 contains a four-disulfide core pattern of cysteines found in WDNM-1 and related proteins.
  • murine WDNM-2 protein possesses eight cysteine residues, cysteines C1-C8, which occur at amino acids 35, 46, 50, 56, 62, 63, 67, and 71 of SEQ ID NO: 14, respectively.
  • a ninth cysteine residue occurs at amino acid 25.
  • Cysteine residues CI to C8 are expected to form four interdomain disulfide bonds which stabilize murine WDNM-2 protein.
  • Cysteines C1-C6, C2-C7, C3-C5, and C4-C8 are expected to form disulfide bonds .
  • murine WDNM-2 protein has some sequence similarity to murine WDNM-1 (mWDNM-1; SEQ ID NO:16), rat WDNM-1 (rWDNM; SEQ ID NO: 17), and murine anti- leukoproteinase (mALP; SEQ ID NO: 19) ( Figure 5)
  • TANGO-175, human TANGO-175, and murine WDNM-2 bear homology to the amino acid sequences of murine anti-leukoproteinase and WDNM-1. This suggests that TANGO-175 and WDNM-2 have activities similar to that of anti-leukoproteinase and WDNM-1. Thus, TANGO-175 and WDNM-2 may play a functional role similar to that proposed for WDNM-1 by inhibiting proteinases associated with metastasis. TANGO-175 and WDNM-2 may, like murine anti-leukoproteinase, be LPS- induced IFN-gamma suppressible proteins that can inhibit LPS response.
  • TANGO-175 and WDNM-2 may play a role in regulating inflammation.
  • a functional role for TANGO- 175 in inflammation is further suggested by the fact that murine TANGO-175 is highly expressed in the liver during inflammation.
  • TANGO-175 and WDNM-2 like human anti- leukoproteinase (Goselink et al . (1996) J. Exp . Med . 184:1305-1312), may also play a role in the growth of hematopoietic stem cells by neutralizing proteinases produced by bone marrow accessory cells.
  • TANGO-175 and WDNM-2 polypeptides and nucleic acid molecules, anti-TANGO- 175 and WDNM-2 antibodies, and modulators of TANGO-175 and WDNM-2 expression or activity may be useful in the treatment and diagnosis of cancer, inflammation, and hematopoietic disorders.
  • Murine TANGO-175 and WDNM-2 include an Arg-Gly-Asp (RGD) motif.
  • the RGD is present in many proteins which bind to integrins, a group of cell surface receptor proteins which mediate cell attachment. Because integrin-mediated cell attachment influences cell migration, growth, differentiation and apoptosis, among other things, TANGO-175 and WDNM-2 may play a role in such events. More particularly, the presence of the RGD motif in TANGO-175 and WDNM-2 suggests that TANGO-175 and WDNM- 2 may play a role in blood coagulation. For example, TANGO-175 or WDNM-2 (or an RGD motif-containing fragment thereof) may act as an inhibitor of coagulation.
  • this invention provides isolated nucleic acid molecules encoding TANGO- 175 or WDNM-2 proteins or biologically active portions thereof, as well as nucleic acid fragments suitable for use as primers or hybridization probes for the detection of TANGO-175-encoding nucleic acids or WDNM-2 -encoding nucleic acids.
  • TANGO-175", "TANGO-175 protein” and “TANGO-175 polypeptide” refers to either or both of the human and murine gene products described above as well as homologues of these proteins in other species.
  • WDNM-2 refers to the murine gene product described herein as well as homologues in other species.
  • the invention features a nucleic acid molecule which includes a nucleotide sequence encoding a protein having an amino acid sequence that is at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NO: 2, 12, 14, 22, 24, or 28.
  • a nucleic acid molecule has the nucleotide sequence shown SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO : 6 , SEQ ID NO: 7, SEQ ID NO : 8 , SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.
  • nucleic acid molecule which encodes a fragment of a polypeptide having the amino acid sequence of SEQ ID NO: 2, 12, 14, 22, 24, or 28, the fragment including at least 15 (25, 30, 50, 60, or 63) contiguous amino acids of SEQ ID NO : 2 , 12, 14, 22, 24, or 28.
  • the invention includes a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 24, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO: 8, SEQ ID NO : 9 , SEQ ID NO: 10 or SEQ ID NO: 11 or the complement thereof under stringent conditions.
  • an isolated TANGO- 175 protein having an amino acid sequence that is at least about 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NO -.24 (mature human TANGO-175) or the amino acid sequence of SEQ ID NO: 12 (immature human TANGO-175) .
  • an isolated WDNM-2 protein having an amino acid sequence that is at least about 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NO: 4 (immature WDNM- 2) or SEQ ID NO: 28 (mature WDNM-2) .
  • an isolated TANGO- 175 protein which is encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 65%, preferably 75%, 85%, or 95% identical to SEQ ID NO : 4 , SEQ ID NO: 5, SEQ ID NO : 6 , SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11; and an isolated TANGO- 175 protein which is 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 : 4 , SEQ ID NO: 5, SEQ ID NO : 6 , SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or the complement thereof .
  • polypeptide which is a naturally occurring allelic variant of a polypeptide that includes the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 24, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO : 9 , SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO : 9 , SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO : 9 , SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO : 9 , SEQ ID
  • SEQ ID NO: 10 SEQ ID NO: 10 or the complement thereof under stringent conditions.
  • Another embodiment of the invention features TANGO-175 nucleic acid molecules which specifically detect TANGO-175 nucleic acid molecules relative to nucleic acid molecules encoding other members of the three disulfide core superfamily.
  • a vector e.g., a recombinant expression vector, comprising a TANGO-175 nucleic acid molecule of the invention.
  • the invention provides a host cell containing such a vector.
  • the invention also provides a method for producing TANGO- 175 protein by culturing, in a suitable medium, a host cell of the invention containing a recombinant expression vector such that a TANGO-175 protein is produced.
  • TANGO-175 proteins and polypeptides possess at least one biological activity possessed by naturally occurring human TANGO-175, e.g., (1) the ability to form protein:protein interactions with a protein that naturally binds TANGO-175; (2) the ability to bind a TANGO-175 receptor, e.g., an integrin; (3) the ability to inhibit a proteinase activity; (4) the ability to modulate cell -cell interactions; (5) the ability to modulate hematopoiesis (e.g., the ability to modulate proliferation of hematopoietic stem cells); (6) the ability to modulate inflammation, and (7) the ability to modulate cell intravasation and/or extravasation; (8) the ability to modulate clotting; and (a) the ability to modulate cell proliferation.
  • TANGO-175 e.g., (1) the ability to form protein:protein interactions with a protein that naturally binds TANGO-175
  • the ability to bind a TANGO-175 receptor e.g.
  • the TANGO-175 proteins of the present invention can be operably linked to a non-TANGO-175 polypeptide (e.g., heterologous amino acid sequences) to form TANGO-175 fusion proteins.
  • the invention further features antibodies that specifically bind TANGO-175 proteins, such as monoclonal or polyclonal antibodies.
  • the TANGO-175 proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
  • the present invention provides a method for detecting the presence of TANGO-175 activity or expression in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of TANGO-175 activity such that the presence of TANGO-175 activity is detected in the biological sample.
  • the invention provides a method for modulating TANGO-175 or WDNM-2 activity comprising contacting a cell with an agent that modulates (inhibits or stimulates) TANGO-175 or WDNM-2 activity or expression such that TANGO-175 or WDNM-2 activity or expression in the cell is modulated.
  • the agent is an antibody that specifically binds to TANGO-175 protein or WDNM-2 protein.
  • the agent modulates expression of TANGO-175 or WDNM-2 by modulating transcription of a TANGO-175 or WDNM-2 gene, splicing of a TANGO-175 or WDNM-2 mRNA, or translation of a TANGO-175 or WDNM-2 mRNA.
  • the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of the TANGO-175 or WDNM-2 mRNA or the TANGO-175 or WDNM-2 gene.
  • the methods of the present invention are used to treat a subject having a disorder characterized by aberrant TANGO-175 or WDNM-2 protein activity or nucleic acid expression by administering an agent which is a TANGO-175 or WDNM-2 modulator to the subject.
  • the TANGO-175 or WDNM-2 modulator is a TANGO-175 or WDNM-2 protein.
  • the TANGO-175 or WDNM-2 modulator is a TANGO- 175 or WDNM-2 nucleic acid molecule.
  • the TANGO-175 or WDNM-2 modulator is a peptide, peptidomimetic, or other small molecule.
  • the disorder characterized by aberrant TANGO-175 or WDNM-2 protein or nucleic acid expression is a coagulation disorder, a proliferative disorder (e.g., cancer), an inflammatory disorder, or a hematopoietic disorder.
  • the present invention also provides a diagnostic assay 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 TANGO-175 protein; (ii) mis-regulation of a gene encoding a TANGO-175 protein; and (iii) aberrant post-translational modification of a TANGO-175 protein, wherein a wild-type form of the gene encodes a protein with a TANGO-175 activity.
  • the invention provides a method for identifying a compound that binds to or modulates the activity of a TANGO-175 protein. In general, such methods entail measuring a biological activity of a TANGO-175 protein in the presence and absence of a test compound and identifying those compounds which alter the activity of the TANGO-175 protein.
  • the invention also features methods for identifying a compound which modulates the expression of TANGO-175 by measuring the expression of TANGO-175 in the presence and absence of a compound.
  • the present invention also provides a diagnostic assay 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 WDNM-2 protein; (ii) mis-regulation of a gene encoding a WDNM-2 protein; and (iii) aberrant post- translational modification of a WDNM-2 protein, wherein a wild-type form of the gene encodes a protein with a WDNM- 2 activity.
  • the invention provides a method for identifying a compound that binds to or modulates the activity of a WDNM-2 protein.
  • such methods entail measuring a biological activity of a WDNM-2 protein in the presence and absence of a test compound and identifying those compounds which alter the activity of the WDNM-2 protein.
  • the invention also features methods for identifying a compound which modulates the expression of WDNM-2 by measuring the expression of WDNM-2 in the presence and absence of a compound.
  • Figure 1 depicts the cDNA sequence (SEQ ID NO:l) and predicted amino acid sequence (SEQ ID NO: 2) of murine TANGO-175 (also referred to as "murine T175").
  • the open reading frame of SEQ ID NO : 1 extends from nucleotide 18 to nucleotide 206 (SEQ ID NO: 3) .
  • Figures 2a, 2b, 2c, and 2d depict nucleic acid sequences (SEQ ID NOS: 4-7) encoding human TANGO-175 (also referred to as "human T175") .
  • the open reading frame of SEQ ID NOS: 4 -7 extends from nucleotide 23 to nucleotide 204 (SEQ ID NO:8-ll).
  • Figures 2a, 2b, 2c, and 2d also depict the amino acid sequence of human TANGO-175 (SEQ ID NO: 12) .
  • the open reading frame of each of SEQ ID NOS: 4 -7 (SEQ ID NOS: 8-11) encode the same amino acid sequence and differ only in the codon for amino acid 10.
  • Figure 3 depicts the cDNA sequence (SEQ ID NO: 13) and predicted amino acid sequence (SEQ ID NO: 14) of murine WDNM-2.
  • the open reading frame of SEQ ID NO: 3 extends from nucleotide 37 to 264 (SEQ ID NO: 15) .
  • Figure 4 depicts an alignment of the amino acid sequence of murine TANGO-175 (SEQ ID NO: 2) with human TANGO-175 (SEQ ID NO: 12) .
  • Murine and human TANGO-175 display 66.7% sequence identity in this alignment.
  • Figure 5 depicts an alignment of the amino acid sequence of murine WDNM-2 (SEQ ID NO: 14) with murine
  • WDNM-1 (mWDNM-1; SEQ ID NO:16), rat WDNM-1 (rWDNM; SEQ ID NO:17), etmM031 (SEQ ID NO:18), murine TANGO-175 (mT.175orf; SEQ ID NO:2), human TANGO-175 (hT.175prot; SEQ ID NO: 12), and murine anti-leukoproteinase (mALP; SEQ ID NO: 19) .
  • Figure 6 is a hydropathy plot of murine TANGO-175.
  • Figure 7 is a hydropathy plot of human TANGO-175.
  • Figure 8 is a hydropathy plot of murine WDNM-2.
  • Figure 9 depicts the complete cDNA sequence of the clone corresponding to IMAGE EST W52431 (SEQ ID NO: 20) .
  • Figure 10 depicts an alignment of the nucleic acid sequence of murine TANGO-175 (SEQ ID N0:1) with EST W52431 (SEQ ID NO : 2 ) .
  • the present invention is based, in part, on the discovery of genes encoding murine TANGO-175, human TANGO-175, and murine WDNM-2.
  • a nucleotide sequence encoding a murine TANGO-175 protein is shown in Figure 1 (SEQ ID NO : 1 ; SEQ ID NO : 3 includes the open reading frame only) .
  • the predicted amino acid sequence of murine TANGO-175 is also shown in Figure 1 (SEQ ID NO:2) .
  • a nucleotide sequence encoding a human TANGO-175 protein is shown in Figures 2a-2d (SEQ ID NO: 4 -7; SEQ ID NO: 8-11 includes the open reading frame only) .
  • the predicted amino acid sequence of human TANGO-175 is also shown in Figures 2a-2d (SEQ ID NO:12).
  • a nucleotide sequence encoding murine WDNM-2 is shown in Figure 3 (SEQ ID NO: 13; SEQ ID NO: 15 includes the open reading frame only) .
  • the predicted amino acid sequence of murine WDNM-2 is also shown in Figure 3.
  • a cDNA encoding a portion of murine TANGO-175 was identified in a subtraction library created using stimulated and unstimulated bone marrow cells. The sequence of this partial clone was used to search the IMAGE EST database. This search led to the ??? of a clone encoding full-length murine TANGO-175.
  • the murine TANGO-175 nucleic sequence was used search the IMAGE EST database in an effort to identify an EST having homology to murine TANGO-175. This search led to the identification of EST W52431.
  • the IMAGE clone corresponding to EST W52431 was obtained and sequenced (SEQ ID NO: 20; Figure 9) . The resulting sequence was translated using all three possible reading frames, and the clone does not appear to encode a human homologue of murine TANGO-175.
  • Figures 2a-2d depict nucleotide sequences (SEQ ID NOS: 4-7; SEQ ID NO: 8-11, the open reading frame encoding human TANGO-175 protein.
  • This 501 nucleotide sequence encodes a 61 amino protein having a molecular weight of approximately 4 kDa (excluding post-translational modifications) .
  • Murine WDNM-2 was identified by searchind the IMAGE EST database using a composite sequence based on the nucleotide sequences of murine TANGO-175, human TANGO-175, and rat WDNM-1.
  • Murine TANGO-175 protein (SEQ ID NO:2), human TANGO-175 protein (SEQ ID NO: 12) and murine WDNM-2 bear some similarity to WDNM-1 and anti-leukoproteinase.
  • a sequence alignment of murine TANGO-175 (SEQ ID NO:2), human TANGO-175 (SEQ ID NO:12), murine WDNM-2 (SEQ ID NO:14), murine WDNM-1 (SEQ ID NO:16), murine anti- leukoproteinase (SEQ ID NO: 19), and rat WDNM-1 (SEQ ID NO: 17) is depicted in Figure 5.
  • An approximate 0.5 kb murine TANGO-175 mRNA is expressed at a very high level in liver. Much lower level expression of this mRNA is observed in spleen, heart, skeletal muscle, and kidney. An approximate 0.5 kb human TANGO-175 was identified in lymph node, spleen, thymus, uterus, and lung.
  • Human TANGO-175 is one member of a family of molecules (the "TANGO-175 family") having certain conserved structural and functional features.
  • family when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain, the three disulfide core, and having sufficient amino acid or nucleotide sequence identity as defined herein.
  • family members can be naturally occurring and can be from either the same or different species.
  • a family can contain a first protein of human origin and a homologue of that protein of murine origin, as well as a second, distinct protein of human origin and a murine homologue of that protein.
  • Members of a family may also have common functional characteristics as described herein.
  • Preferred TANGO-175 polypeptides of the present invention have an amino acid sequence sufficiently identical to the human TANGO-175 amino acid sequence (SEQ ID NO: 12) .
  • 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., an amino acid residue which has 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 65% identity, preferably 75% identity, more preferably 85%, 95%, or 98% identity are defined herein as sufficiently identical.
  • TANGO-175 activity refers to an activity exerted by a TANGO-175 protein, polypeptide or nucleic acid molecule on a TANGO-175 responsive cell as determined in vivo, or in vi tro, according to standard techniques.
  • a TANGO-175 activity 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 adhesion activity mediated by interaction of the TANGO-175 protein with a second protein.
  • Another aspect of this invention features isolated or recombinant TANGO-175 proteins and polypeptides.
  • Preferred TANGO-175 proteins and polypeptides possess at least one biological activity possessed by naturally occurring human TANGO-175, e.g., (1) the ability to form protein: protein interactions with a protein that naturally binds TANGO-175; (2) the ability to bind a TANGO-175 receptor, e.g., an integrin; (3) the ability to inhibit a proteinase activity; (4) the ability to modulate cell-cell interactions; (5) the ability to modulate hematopoiesis (e.g., the ability to modulate proliferation, differentiation or function of hematopoietic cells, e.g., stem cells); (6) the ability to modulate of inflammation, and (7) the ability to modulate intravasation and/or extravasation; (8) the ability to modulate clotting; and (a) the ability to modulate cell proliferation.
  • another embodiment of the invention features isolated TANGO-175 proteins and polypeptides having at least one TANGO-175 activity.
  • TANGO-175 molecules which contain a signal sequence.
  • a signal sequence is a peptide containing approximately 15-30 (e.g., 20-30) amino acids which occurs at the extreme N-terminal end of secretory and integral membrane proteins and which contains large numbers of hydrophobic amino acid residues and serves to direct a protein containing such a sequence to a lipid bilayer.
  • the native human TANGO-175 signal sequence or signal peptide (SEQ ID NO: 23) can be removed and replaced with a signal sequence from another protein.
  • expression and/or secretion of TANGO-175 can be increased through use of a heterologous signal sequence.
  • the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence in expression systems, e.g., to facilitate the secretion of a protein of interest.
  • Human WDNM-2 is one member of a family of molecules (the "WDNM-2 family") having certain conserved structural and functional features.
  • family when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain, the three disulfide core, and having sufficient amino acid or nucleotide sequence identity as defined herein.
  • family members can be naturally occurring and can be from either the same or different species.
  • a family can contain a first protein of human origin and a homologue of that protein of murine origin, as well as a second, distinct protein of human origin and a murine homologue of that protein.
  • Members of a family may also have common functional characteristics as described herein.
  • Preferred WDNM-2 polypeptides of the present invention have an amino acid sequence sufficiently identical to the human WDNM-2 amino acid sequence (SEQ ID NO:12).
  • 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., an amino acid residue which has 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 65% identity, preferably 75% identity, more preferably 85%, 95%, or 98% identity are defined herein as sufficiently identical.
  • WDNM-2 activity refers to an activity exerted by a WDNM-2 protein, polypeptide or nucleic acid molecule on a WDNM-2 responsive cell as determined in vivo, or in vi tro, according to standard techniques.
  • a WDNM-2 activity 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 adhesion activity mediated by interaction of the WDNM-2 protein with a second protein.
  • Another aspect of this invention features isolated or recombinant WDNM-2 proteins and polypeptides.
  • Preferred WDNM-2 proteins and polypeptides possess at least one biological activity possessed by naturally occurring human WDNM-2, e.g., (1) the ability to form protein: protein interactions with a protein that naturally binds WDNM-2; (2) the ability to bind a WDNM-2 receptor, e.g., an integrin; (3) the ability to inhibit a proteinase activity; (4) the ability to modulate cell- cell interactions; (5) the ability to modulate hematopoiesis (e.g., the ability to modulate proliferation of hematopoietic stem cells) ; (6) the ability to modulate of inflammation, and (7) the ability to modulate intravasation and/or extravasation; (8) the ability to modulate clotting; and (a) the ability to modulate cell proliferation.
  • another embodiment of the invention features isolated WDNM-2 proteins and polypeptides having at least one WDNM-2 activity.
  • Yet another embodiment of the invention features WDNM-2 molecules which contains a signal sequence.
  • a signal sequence is a peptide containing approximately 15-30 (e.g., 20-30) amino acids which occurs at the extreme N-terminal end of secretory and integral membrane proteins and which contains large numbers of hydrophobic amino acid residues and serves to direct a protein containing such a sequence to a lipid bilayer.
  • the native human WDNM-2 signal sequence or signal peptide (SEQ ID NO: 23) can be removed and replaced with a signal sequence from another protein.
  • expression and/or secretion of WDNM-2 can be increased through use of a heterologous signal sequence.
  • the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence in expression systems, e.g., to facilitate the secretion of a protein of interest.
  • Table 1 Summary of Murine TANGO-175, Human TANGO-175, and Murine WDNM-2 Sequence Information
  • 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.
  • an “isolated” nucleic acid 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 TANGO-175 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.
  • nucleic acid molecules comprising an open reading frame encoding a TANGO-175 protein, preferably a mammalian TANGO-175 protein.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO : 4 , SEQ ID NO: 5, SEQ ID NO : 6 , SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO : 9 , SEQ ID NO: 10, SEQ ID NO: 11, or a complement of any of these nucleotide sequences, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • TANGO- 175 nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sa brook et al . , eds., Molecular Cloning: A Laboratory Manual , 2nd ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) .
  • WDNM-2 sequence SEQ ID NO: 13
  • a nucleic acid 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 TANGO-175 nucleotide sequences 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 shown in SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO : 9 , SEQ ID NO: 10, SEQ ID NO: 11, 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.
  • the nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence encoding TANGO-175, for example, a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of TANGO-175.
  • the nucleotide sequence determined from the cloning of the human TANGO-175 gene allows for the generation of probes and primers designed for use in identifying and/or cloning TANGO-175 homologues in other cell types, e.g., from other tissues, as well as TANGO-175 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, or 350 consecutive nucleotides of the sense or anti -sense sequence of SEQ ID NO:4, SEQ ID NO : 5 , SEQ ID NO: 6, SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO : 9 , SEQ ID NO: 10, SEQ ID NO: 11, or of a naturally occurring mutant Of SEQ ID NO: 4, SEQ ID NO : 5 , SEQ ID NO : 6 , SEQ ID NO : 7 , SEQ ID NO: 8, SEQ ID NO : 9 , SEQ ID NO: 10 or SEQ ID NO: 11.
  • Probes based on the human TANGO-175 nucleotide sequence can be used to detect transcripts or genomic sequences encoding similar or identical proteins.
  • 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 a part of a diagnostic test kit for identifying cells or tissues which mis-express a TANGO-175 protein, such as by measuring a level of a TANGO- 175-encoding nucleic acid in a sample of cells from a subject, e.g., detecting TANGO- 175 mRNA levels or determining whether a genomic TANGO- 175 gene has been mutated or deleted.
  • a nucleic acid fragment encoding a "biologically active portion of TANGO-175" can be prepared by isolating a portion of SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO : 6 , SEQ ID NO: 7, SEQ ID NO : 8 , SEQ ID NO : 9 , SEQ ID NO: 10 or SEQ ID NO: 11, which encodes a polypeptide having a TANGO-175 biological activity, expressing the encoded portion of TANGO-175 protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of TANGO-175.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of SEQ ID NO : 4 , SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO : 8 , SEQ ID NO : 9 , SEQ ID NO: 10, or SEQ ID NO: 11, due to degeneracy of the genetic code and thus encode the same TANGO-175 protein as that encoded by the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO : 9 , SEQ ID NO: 10 or SEQ ID NO: 11.
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of TANGO-175 may exist within a population (e.g., the human population) .
  • Such genetic polymorphism in the TANGO-175 gene 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.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the TANGO-175 gene.
  • Alternative alleles can be identified by 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 polymorphisms in TANGO-175 that are the result of natural allelic variation and that do not alter the functional activity of TANGO-175 are intended to be within the scope of the invention.
  • nucleic acid molecules encoding TANGO- 175 proteins from other species which have a nucleotide sequence which differs from that of a human TANGO-175, are intended to be within the scope of the invention.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the TANGO- 175 cDNA of the invention can be isolated based on their identity to the human TANGO-175 nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • splice variants of human and mouse TANGO-175 cDNA can be isolated based on identity to human and mouse TANGO-175.
  • 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 coding or non-coding (or "sense” or “anti-sense") sequence of SEQ ID NO : 4 , SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO : 9 , SEQ ID NO: 10, or SEQ ID NO: 11 corresponds to a naturally- occurring nucleic acid molecule.
  • 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).
  • allelic variants of the TANGO-175 sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO : 9 , SEQ ID NO: 10 or SEQ ID NO: 11, thereby leading to changes in the amino acid sequence of the encoded TANGO-175 protein, without altering the biological ability of the TANGO-175 protein.
  • Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • 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
  • a predicted nonessential amino acid residue in Tango- 175 is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a Tango- 175 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for Tango-175 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.
  • amino acid residues that are conserved among the TANGO-175 proteins of various species are not altered (except by conservative substitution) .
  • Both murine and human TANGO-175 protein have a conserved pattern of six cysteine residues. Such conserved domains and cysteine residues are less likely to be amenable to mutation.
  • Other amino acid residues e.g., those that are not conserved or only semi-conserved among TANGO-175 of various species e.g., between murine and human TANGO-175) may not be essential for activity and thus are likely to be amenable to alteration.
  • nucleic acid molecules encoding TANGO-175 proteins that contain changes in amino acid residues that are not essential for activity.
  • Such TANGO-175 proteins differ in amino acid sequence from SEQ ID NO: 5 or SEQ ID NO: 8 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: 12, or SEQ ID NO: 24.
  • An isolated nucleic acid molecule encoding a TANGO-175 protein having a sequence which differs from that of SEQ ID NO: 12 or SEQ ID NO: 24 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 4-11, 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).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g.
  • a predicted nonessential amino acid residue in TANGO-175 is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a TANGO-175 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for TANGO-175 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.
  • Preferred TANGO-175 proteins and polypeptides possess at least one biological activity possessed by naturally occurring human TANGO-175, e.g., (1) the ability to form protein:protein interactions with a protein that naturally binds TANGO-175; (2) the ability to bind a TANGO-175 receptor, e.g., an integrin; (3) the ability to inhibit a proteinase activity; (4) the ability to modulate cell -cell interactions; (5) the ability to modulate hematopoiesis (e.g., the ability to modulate proliferation of hematopoietic stem cells) ; (6) the ability to modulate of inflammation, and (7) the ability intravasation and/or extravasation; (8) the ability to modulate clotting; and (a) the ability to modulate cell proliferation.
  • TANGO-175 e.g., (1) the ability to form protein:protein interactions with a protein that naturally binds TANGO-175
  • the ability to bind a TANGO-175 receptor e.g
  • the present invention encompasses antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid encoding a protein, 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 TANGO-175 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 a noncoding region of the coding strand of a nucleotide sequence encoding TANGO-175.
  • the noncoding regions (“5' and 3' untranslated regions") are the 5' and 3' sequences which flank the coding region and are not translated into amino acids .
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of TANGO-175 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of human TANGO-175 mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of TANGO-175 mRNA, e.g., an oligonucleotide having the sequence TCGTCGTACTTCGATCCT (SEQ ID NO: 25) or TACTTCGATCCTCGGAGG (SEQ ID NO: 26) .
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides 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-
  • 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 si tu such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a TANGO-175 protein to thereby inhibit expression of the protein, 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 /3-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)
  • ribozymes can be used to catalytically cleave TANGO-175 mRNA transcripts to thereby inhibit translation of TANGO-175 mRNA.
  • a ribozyme having specificity for a TANGO-175-encoding nucleic acid can be designed based upon the nucleotide sequence of a TANGO-175 cDNA disclosed herein (e.g., SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO: 9, SEQ ID NO:10, and SEQ ID NO:ll).
  • 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 TANGO-175- encoding mRNA. See, e .
  • TANGO-175 mRNA 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.
  • TANGO-175 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the TANGO-175 (e.g., the TANGO-175 promoter and/or enhancers) to form triple helical structures that prevent transcription of the TANGO-175 gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the TANGO-175 e.g., the TANGO-175 promoter and/or enhancers
  • 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) Bioorganic & Medicinal Chemistry 4(1) : 5-23) .
  • peptide nucleic acids 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 of TANGO-175 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 of TANGO-175 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. Na tl . Acad . Sci . USA 93: 14670-675) .
  • PNAs of TANGO-175 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 of TANGO-175 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 Research 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) .
  • 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 . Ac
  • 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 TANGO-175 proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti -TANGO-175 antibodies.
  • native TANGO-175 proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • TANGO-175 proteins are produced by recombinant DNA techniques.
  • a TANGO-175 protein or polypeptide 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 TANGO-175 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 TANGO-175 protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • TANGO-175 protein that is substantially free of cellular material includes preparations of TANGO-175 protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of non-TANGO-175 protein (also referred to herein as a "contaminating protein”) .
  • TANGO-175 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.
  • TANGO-175 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 TANGO-175 protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or non-TANGO-
  • Biologically active portions of a TANGO-175 protein include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the TANGO-175 protein (e.g., the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 23 and SEQ ID NO: 24), which include fewer amino acids than the full length TANGO-175 proteins, and exhibit at least one activity of a TANGO-175 protein.
  • biologically active portions comprise a domain or motif with at least one activity of the TANGO-175 protein.
  • a biologically active portion of a TANGO-175 protein can be a polypeptide which is, for example, 10, 25, 50, 60, 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 a native TANGO- 175 protein.
  • Preferred TANGO-175 protein has the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 24.
  • Other useful TANGO-175 proteins are substantially identical to SEQ ID NO: 12 or SEQ ID NO: 24 and retain the functional activity of the protein of SEQ ID NO: 12, SEQ ID NO:24, yet differ in amino acid sequence due to natural allelic variation or mutagenesis.
  • a useful TANGO-175 protein is a protein which includes an amino acid sequence at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 24 and retains the functional activity of the TANGO-175 proteins of SEQ ID NO: 12 or SEQ ID NO: 24.
  • the TANGO-175 protein retains a functional activity of the TANGO-175 protein of SEQ ID NO: 12.
  • the sequences are aligned for optimal comparison purposes (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 two sequences are the same length.
  • the determination of percent homology 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
  • Gapped BLAST can be utilized as described in Altschul et al .
  • 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, only exact matches are counted.
  • the invention also provides TANGO-175 chimeric or fusion proteins.
  • a TANGO-175 "chimeric protein" or “fusion protein” comprises a TANGO-175 polypeptide operably linked to a non-TANGO-175 polypeptide.
  • TANGO-175 polypeptide refers to a polypeptide having an amino acid sequence corresponding to TANGO-175, whereas a “non-TANGO-175 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially identical to the TANGO-175 protein, e.g., a protein which is different from the TANGO-175 protein and which is derived from the same or a different organism.
  • the TANGO-175 polypeptide can correspond to all or a portion of a TANGO-175 protein, preferably at least one biologically active portion of a TANGO-175 protein.
  • operably linked is intended to indicate that the TANGO- 175 polypeptide and the non-TANGO-175 polypeptide are fused in- frame to each other.
  • the non-TANGO-175 polypeptide can be fused to the N-terminus or C-terminus of the TANGO-175 polypeptide.
  • One useful fusion protein is a GST-TANGO-175 fusion protein in which the TANGO-175 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant TANGO-175.
  • the fusion protein is a TANGO-175 protein containing a heterologous signal sequence at its N-terminus.
  • the native TANGO-175 signal sequence i.e., about amino acids 1 to 25 of SEQ ID NO: 12; SEQ ID NO: 23
  • expression and/or secretion of TANGO-175 can be increased through use of a heterologous signal sequence.
  • 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 .
  • 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 ⁇ Molecular cloning, Sambrook et al . , supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey) .
  • the fusion protein is an TANGO-175-immunoglobulin fusion protein in which all or part of TANGO-175 is fused to sequences derived from a member of the immunoglobulin protein family.
  • 175-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a TANGO-175 ligand and a TANGO-175 protein on the surface of a cell, to thereby suppress TANGO-175-mediated signal transduction in vivo .
  • the TANGO-175- immunoglobulin fusion proteins can be used to affect the bioavailability of a TANGO-175 cognate ligand. Inhibition of the TANGO-175 ligand/TANGO-175 interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as for modulating (e.g. promoting or inhibiting) cell survival.
  • TANGO-175-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-TANGO-175 antibodies in a subject, to purify TANGO-175 ligands and in screening assays to identify molecules which inhibit the interaction of TANGO-175 with a TANGO-175 ligand.
  • a TANGO-175 chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in- frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • 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. , Current Protocols in Molecular Biology, Ausubel et al . eds., John Wiley & Sons: 1992).
  • 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
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide) .
  • An TANGO-175-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the TANGO-175 protein.
  • the present invention also pertains to variants of the TANGO-175 proteins (i.e., proteins having a sequence which differs from that of the TANGO-175 amino acid sequence) .
  • variants can function as either TANGO- 175 agonists (mimetics) or as TANGO-175 antagonists.
  • Variants of the TANGO-175 protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the TANGO-175 protein.
  • An agonist of the TANGO-175 protein can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the TANGO-175 protein.
  • An antagonist of the TANGO-175 protein can inhibit one or more of the activities of the naturally occurring form of the TANGO- 175 protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the TANGO-175 protein.
  • 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 TANGO- 175 proteins.
  • Variants of the TANGO-175 protein which function as either TANGO-175 agonists (mimetics) or as TANGO-175 antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the TANGO-175 protein for TANGO-175 protein agonist or antagonist activity.
  • a variegated library of TANGO-175 variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of TANGO-175 variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential TANGO-175 sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of TANGO-175 sequences therein.
  • a degenerate set of potential TANGO-175 sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of TANGO-175 sequences therein.
  • Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector.
  • Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential TANGO-175 sequences.
  • 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 TANGO- 175 protein coding sequence can be used to generate a variegated population of TANGO-175 fragments for screening and subsequent selection of variants of a T7ANGO-175 protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a TANGO-175 coding sequence 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 SI 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 TANGO-175 protein.
  • 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. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of T7ANGO-175 proteins.
  • 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 TANGO-175 variants (Arkin and Yourvan (1992) Proc. Natl . Acad . Sci . USA 89:7811-7815; Delgrave et al . (1993) Protein Engineering 6(3) :327-331) .
  • An isolated TANGO-175 protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind TANGO-175 using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length TANGO-175 protein can be used or, alternatively, the invention provides antigenic peptide fragments of TANGO-175 for use as immunogens .
  • the antigenic peptide of TANGO-175 comprises at least 8 (preferably 10, 15, 20, or 30) amino acid residues of the amino acid sequence shown in SEQ ID NO: 12 and encompasses an epitope of TANGO-175 such that an antibody raised against the peptide forms a specific immune complex with TANGO-175.
  • Preferred epitopes encompassed by the antigenic peptide are regions of TANGO-175 that are located on the surface of the protein, e.g., hydrophilic regions, and lack cysteines of n-glycosylation sites.
  • a TANGO-175 immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed TANGO-175 protein or a chemically synthesized TANGO-175 polypeptide.
  • the preparation can further include an adjuvant, such as Freund' s complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic TANGO-175 preparation induces a polyclonal anti-TANGO- 175 antibody response.
  • 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 TANGO-175.
  • a molecule which specifically binds to TANGO-175 is a molecule which binds TANGO-175, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains TANGO-175.
  • 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 that bind TANGO-175.
  • 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 of TANGO-175.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular TANGO-175 protein with which it immunoreacts .
  • Polyclonal anti -TANGO-175 antibodies can be prepared as described above by immunizing a suitable subject with a TANGO-175 immunogen.
  • the anti-TANGO- 175 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 TANGO-175.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against TANGO-175 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.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line, e.g., a myeloma cell line that is sensitive to culture medium containing hypoxanthine , aminopterin and thymidine ("HAT medium”) .
  • HAT medium hypoxanthine , aminopterin and thymidine
  • any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3- NSl/l-Ag4-l, P3 -x63 -Ag8.653 or Sp2/0-Agl4 myeloma lines. These myeloma lines are available from ATCC .
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed) .
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind TANGO-175, e.g., using a standard ELISA assay.
  • a monoclonal anti-TANGO-175 antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with TANGO-175 to thereby isolate immunoglobulin library members that bind TANGO-175.
  • 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 Surf ZAPTM 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.
  • recombinant anti-TANGO- 175 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 .
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of TANGO-175.
  • 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.
  • 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) .
  • a selected non-human monoclonal antibody e.g., a murine antibody
  • a non-human monoclonal antibody which binds a selected antigen (epitope), e.g., an antibody which inhibits Tango-69 activity is identified.
  • the heavy chain and the light chain of the non-human antibody are cloned and used to create phage display Fab fragments .
  • the heavy chain gene can be cloned into a plasmid vector so that the heavy chain can be secreted from bacteria.
  • the light chain gene can be cloned into a phage coat protein gene so that the light chain can be expressed on the surface of phage.
  • a repertoire (random collection) of human light chains fused to phage is used to infect the bacteria which express the non-human heavy chain.
  • the resulting progeny phage display hybrid antibodies (human light chain/non-human heavy chain) .
  • the selected antigen is used in a panning screen to select phage which bind the selected antigen. Several rounds of selection may be required to identify such phage.
  • human light chain genes are isolated from the selected phage which bind the selected antigen. These selected human light chain genes are then used to guide the selection of human heavy chain genes as follows.
  • the selected human light chain genes are inserted into vectors for expression by bacteria. Bacteria expressing the selected human light chains are infected with a repertoire of human heavy chains fused to phage. The resulting progeny phage display human antibodies (human light chain/human heavy chain) .
  • the selected antigen is used in a panning screen to select phage which bind the selected antigen.
  • the phage selected in this step display a completely human antibody which recognizes the same epitope recognized by the original selected, non-human monoclonal antibody.
  • the genes encoding both the heavy and light chains are readily isolated and can be further manipulated for production of human antibody. This technology is described by Jespers et al . (1994, Bio/technology 12:899-903) .
  • An anti-TANGO-175 antibody (e.g., monoclonal antibody) can be used to isolate TANGO-175 by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-TANGO-175 antibody can facilitate the purification of natural TANGO-175 from cells and of recombinantly produced TANGO-175 expressed in host cells.
  • an anti-TANGO-175 antibody can be used to detect TANGO-175 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the TANGO-175 protein.
  • Anti-TANGO-175 antibodies can 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.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, /3-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;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 1 , 35 S or 3 H.
  • vectors preferably expression vectors, containing a nucleic acid encoding TANGO-175 (or a portion thereof) .
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • 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 wherein additional DNA segments can be ligated into the viral genome .
  • 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
  • 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 (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, which 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.
  • "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 (e.g., TANGO-175 proteins, mutant forms of TANGO-175, fusion proteins, etc.).
  • proteins or peptides including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., TANGO-175 proteins, mutant forms of TANGO-175, fusion proteins, etc.).
  • the recombinant expression vectors of the invention can be designed for expression of TANGO-175 in prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. coli , insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) .
  • the recombinant expression vector can be transcribed and translated in vi tro, 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 purposes: 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
  • E. coli expression vectors examples include pTrc (Amann et al . , (1988) Gene 69:301-315) and pET lid (Studier et al . , Gene Expression Technology: Methods in Enzymology 185,
  • 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 lid 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.
  • 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 .
  • nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the TANGO-175 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 Corporation, San Diego, CA) , and pPicZ (InVitrogen Corp, San Diego, CA) .
  • TANGO-175 can be expressed in insect cells using baculovirus expression vectors.
  • 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.
  • 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.
  • commonly used 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) EMBO J.
  • 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 TANGO- 175 mRNA.
  • 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.
  • 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 or eukaryotic cell.
  • TANGO-175 protein can be expressed in bacterial cells such as E. coli , insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells) . Other suitable host cells are known to those skilled in the art.
  • 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 (e.g., DNA) 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.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding TANGO-175 or can be introduced on a separate vector.
  • Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated 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 (i.e., express) TANGO-175 protein.
  • the invention further provides methods for producing TANGO-175 protein using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding TANGO-175 has been introduced) in a suitable medium such that TANGO-175 protein is produced.
  • the method further comprises isolating TANGO-175 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 TANGO- 175-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous TANGO-175 sequences have been introduced into their genome or homologous recombinant animals in which endogenous TANGO-175 sequences have been altered.
  • Such animals are useful for studying the function and/or activity of TANGO-175 and for identifying and/or evaluating modulators of TANGO-175 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 TANGO-175 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 TANGO- 175 -encoding nucleic acid 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.
  • the TANGO-175 cDNA sequence e.g., that of (SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO : 6 , SEQ ID NO : 7 , SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO: 11) can be introduced as a transgene into the genome of a non-human animal.
  • a nonhuman homologue of the human TANGO-175 gene can be isolated based on hybridization to the human TANGO-175 cDNA and used as a transgene.
  • 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 can be operably linked to the TANGO-175 transgene to direct expression of TANGO-175 protein to particular cells.
  • a transgenic founder animal can be identified based upon the presence of the TANGO-175 transgene in its genome and/or expression of TANGO-175 mRNA 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 a transgene encoding TANGO-175 can further be bred to other transgenic animals carrying other transgenes.
  • a vector which contains at least a portion of a TANGO-175 gene (e.g., a human or a non-human homolog of the TANGO-175 gene, e.g., a murine TANGO-175 gene) into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the TANGO-175 gene.
  • the vector is designed such that, upon homologous recombination, the endogenous TANGO-175 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
  • TANGO-175 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 TANGO-175 protein) .
  • the altered portion of the TANGO'- 175 gene is flanked at its 5' and 3' ends by additional nucleic acid of the TANGO-175 gene to allow for homologous recombination to occur between the exogenous TANGO-175 gene carried by the vector and an endogenous TANGO-175 gene in an embryonic stem cell.
  • the additional flanking TANGO-175 nucleic acid is 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 TANGO-175 gene has homologously recombined with the endogenous TANGO-175 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.
  • a system is the cre/loxP recombinase system of bacteriophage PI .
  • cre/loxP recombinase system of bacteriophage PI .
  • a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (0' Gorman et al . (1991) Science 251:1351-1355.
  • mice 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 ⁇ os . WO 97/07668 and WO 97/07669.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal .
  • the offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • compositions suitable for administration can be incorporated 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 absorption 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 incorporated into the compositions.
  • 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, disposable syringes or multiple dose vials made of glass or plastic.
  • 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 absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a TANGO-175 protein or anti-TANGO-175 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 incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • 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.
  • the active compound can be incorporated 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.
  • 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 Corporation 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.
  • 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.
  • 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 isolated nucleic acid molecules of the invention can be used to express TANGO-175 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect TANGO-175 mRNA (e.g., in a biological sample) or a genetic lesion in a TANGO-175 gene, and to modulate TANGO-175 activity.
  • the TANGO-175 proteins can be used to screen drugs or compounds which modulate the TANGO-175 activity or expression as well as to treat disorders characterized by insufficient or excessive production of TANGO-175 protein or production of TANGO-175 protein forms which have decreased or aberrant activity compared to TANGO-175 wild type protein.
  • the anti-TANGO-175 antibodies of the invention can be used to detect and isolate TANGO- 175 proteins and modulate TANGO-175 activity.
  • 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 TANGO-175 proteins or have a stimulatory or inhibitory effect on, for example, TANGO- 175 expression or TANGO-175 activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to TANGO-175 proteins or have a stimulatory or inhibitory effect on, for example, TANGO- 175 expression or TANGO-175 activity.
  • an assay of the present invention is a cell-free assay comprising contacting a TANGO-175 protein or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to the TANGO-175 protein or biologically active portion thereof. Binding of the test compound to the TANGO-175 protein can be determined either directly or indirectly as described above.
  • the assay includes contacting the TANGO-175 protein or biologically active portion thereof with a known compound which binds TANGO-175 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a TANGO-175 protein, wherein determining the ability of the test compound to interact with a TANGO-175 protein comprises determining the ability of the test compound to preferentially bind to TANGO-175 or biologically active portion thereof as compared to the known compound.
  • an assay is a cell-free assay comprising contacting TANGO-175 protein 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 TANGO-175 protein or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of TANGO-175 can be accomplished, for example, by determining the ability of the TANGO-175 protein to bind to a TANGO-175 target molecule by one of the methods described above for determining direct binding.
  • determining the ability of the test compound to modulate the activity of TANGO-175 can be accomplished by determining the ability of the TANGO-175 protein to further modulate a TANGO-175 target molecule.
  • the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as previously described.
  • the cell -free assay comprises contacting the TANGO-175 protein or biologically active portion thereof with a known compound which binds TANGO-175 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a TANGO-175 protein, wherein determining the ability of the test compound to interact with a TANGO-175 protein comprises determining the ability of the TANGO-175 protein to preferentially bind to or modulate the activity of a TANGO-175 target molecule.
  • TANGO-175 it may be desirable to immobilize either TANGO-175 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 TANGO-175, or interaction of TANGO-175 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/ TANGO-175 fusion proteins or glutathione-S-transferase/target 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 TANGO-175 protein, 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 T7ANGO-175 binding or activity determined using standard techniques.
  • TANGO-175 or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated TANGO-175 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 TANGO-175 or target molecules but which do not interfere with binding of the TANGO-175 protein to its target molecule can be derivatized to the wells of the plate, and unbound target or TANGO-175 trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the TANGO-175 or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the TANGO-175 or target molecule.
  • determining the ability of the test compound to modulate the activity of TANGO-175 or a biologically active portion thereof can be accomplished, for example, by determining the ability of the TANGO-175 protein to bind to or interact with a TANGO-175 target molecule.
  • a "target molecule” is a molecule with which a TANGO-175 protein binds or interacts in nature, for example, a molecule on the surface of a cell, e.g., an integrin or a extracellular.
  • a TANGO-175 target molecule can be a non-TANGO-175 molecule or a TANGO-175 protein or polypeptide of the present invention.
  • the target for example, can be a extracellular protein which has catalytic activity e.g., a proteinase particularly a serine proteinase.
  • Determining the ability of the TANGO-175 protein to bind to or interact with a T7ANGO-175 target molecule can be accomplished by one of the methods described above for determining direct binding. In a preferred embodiment, determining the ability of the TANGO-175 protein to bind to or interact with a TANGO-175 target molecule can be accomplished by determining the activity of the target molecule.
  • the activity of the target molecule can be determined by detecting catalytic/enzymatic activity of the target (e.g., a proteinase) on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element responsive to a TANGO-175 generated signal operatively linked to a nucleic acid encoding a detectable marker, e.g. luciferase) , or detecting a cellular response.
  • a reporter gene e.g., a regulatory element responsive to a TANGO-175 generated signal operatively linked to a nucleic acid encoding a detectable marker, e.g. luciferase
  • modulators of TANGO-175 expression are identified in a method in which a cell is contacted with a candidate compound and the expression of TANGO-175 mRNA or protein in the cell is determined.
  • the level of expression of TANGO-175 mRNA or protein in the presence of the candidate compound is compared to the level of expression of TANGO-175 mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of TANGO- 175 expression based on this comparison. For example, when expression of TANGO-175 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 TANGO-175 mRNA or protein expression.
  • the candidate compound when expression of TANGO-175 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 T7ANGO-175 mRNA or protein expression.
  • the level of TANGO-175 mRNA or protein expression in the cells can be determined by methods described herein for detecting TANGO-175 mRNA or protein.
  • the TANGO- 175 proteins 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 .
  • TANGO-175-binding proteins bind to or interact with TANGO-175 ( "TANGO-175-binding proteins" or "TANGO-175-bp" ) and modulate TANGO-175 activity.
  • TANGO-175-binding proteins are also likely to be involved in the propagation of signals by the TANGO-175 proteins as, for example, upstream or downstream elements of the TANGO-175 pathway.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for TANGO-175 is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4) .
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA- binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with TANGO-175.
  • a reporter gene e.g., LacZ
  • 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.
  • TANGO-175 nucleic acid molecules described herein or fragments thereof can be used to map the location of TANGO-175 genes on a chromosome.
  • the mapping of the TANGO-175 sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
  • TANGO-175 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the TANGO-175 sequences.
  • Computer analysis of TANGO-175 sequences 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 TANGO-175 sequences will yield an amplified fragment.
  • Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells) . As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow (because they lack a particular enzyme) , but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D'Eustachio et al . (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
  • 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 TANGO-175 sequences 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 TANGO-175 sequence to its chromosome include in si tu hybridization (described in Fan et al . (1990) Proc. Natl . Acad . Sci .
  • FISH Fluorescence in situ hybridization
  • Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical, e.g., colcemid that disrupts the mitotic spindle.
  • the chromosomes can be treated briefly with trypsin, and then stained with Giemsa.
  • the FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. 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 purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with the TANGO-175 gene 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 polymorphisms .
  • TANGO-175 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 polymorphism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymorphism
  • 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 TANGO-175 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 TANGO-175 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 purposes.
  • SEQ ID NO: 4 SEQ ID NO: 5
  • SEQ ID NO: 6 SEQ ID NO: 7
  • SEQ ID NO: 7 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: 8-11 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • a panel of reagents from TANGO-175 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 perpetrator 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 of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO : 7 are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique.
  • polynucleotide reagents include the TANGO-175 sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 having a length of at least 20 or 30 bases.
  • the TANGO-175 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 si tu 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 TANGO- 175 probes can be used to identify tissue by species and/or by organ type .
  • polynucleotide reagents e.g., labeled or labelable probes which can be used in, for example, an in si tu 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 TANGO- 175 probes can be used to identify tissue by species and/or by organ type .
  • these reagents e.g., TANGO- 175 primers or probes can be used to screen tissue culture for contamination (i.e., screen for the presence of a mixture of different types of cells in a culture) .
  • 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) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining TANGO-175 protein and/or nucleic acid expression as well as TANGO-175 activity, 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 TANGO-175 expression or activity.
  • 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 TANGO-175 protein, nucleic acid expression or activity. For example, mutations in a TANGO-175 gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with TANGO- 175 protein, nucleic acid expression or activity.
  • Another aspect of the invention provides methods for determining TANGO-175 protein, nucleic acid expression or TANGO-175 activity 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 TANGO-175 in clinical trials.
  • An exemplary method for detecting the presence or absence of TANGO-175 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 TANGO-175 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes TANGO-175 protein such that the presence of TANGO-175 is detected in the biological sample.
  • a preferred agent for detecting TANGO-175 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to TANGO-175 mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, a full-length TANGO-175 nucleic acid, such as the nucleic acid of SEQ ID NO: 1 or 3 , or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 400 nucleotides in length and sufficient to specifically hybridize under stringent conditions to TANGO-175 mRNA or genomic DNA.
  • a full-length TANGO-175 nucleic acid such as the nucleic acid of SEQ ID NO: 1 or 3
  • a portion thereof such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 400 nucleotides in length and sufficient to specifically hybridize under stringent conditions to TANGO-175 mRNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays of the invention are described herein.
  • a preferred agent for detecting TANGO-175 protein is an antibody capable of binding to TANGO-175 protein, 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 TANGO-175 mRNA, protein, or genomic DNA in a biological sample in vi tro as well as in vivo .
  • vi tro techniques for detection of TANGO- 175 mRNA include Northern hybridizations and in si tu hybridizations.
  • vi tro techniques for detection of TANGO-175 protein include enzyme linked immunosorbent assays (ELISAs) , Western blots, immunoprecipitations and immunofluorescence.
  • vi tro techniques for detection of TANGO-175 genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of TANGO- 175 protein include introducing into a subject a labeled anti-TANGO-175 antibody.
  • 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 TANGO-175 protein, mRNA, or genomic DNA, such that the presence of TANGO-175 protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of TANGO-175 protein, mRNA or genomic DNA in the control sample with the presence of TANGO-175 protein, mRNA or genomic DNA in the test sample .
  • kits for detecting the presence of TANGO-175 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 TANGO-175 (e.g., an immunological disorder).
  • the kit can comprise a labeled compound or agent capable of detecting TANGO-175 protein or mRNA in a biological sample and means for determining the amount of TANGO-175 in the sample (e.g., an anti-TANGO-175 antibody or an oligonucleotide probe which binds to DNA encoding TANGO- 175, e.g., SEQ ID NO:4, SEQ ID NO : 5 , SEQ ID NO : 6 , SEQ ID NO: 7, SEQ ID NO : 8 , SEQ ID NO : 9 , SEQ ID NO: 10 or SEQ ID NO: 11) .
  • Kits may also include instruction for observing that the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of T7ANGO-175 if the amount of TANGO-175 protein or mRNA is above or below a normal level.
  • the kit may comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to TANGO-175 protein; and, optionally, (2) a second, different antibody which binds to TANGO-175 protein 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 TANGO-175 protein or the first antibody and is conjugated to a detectable agent.
  • the kit may comprise, for example: (1) an oligonucleotide, e.g., a detectably labelled oligonucleotide, which hybridizes to a TANGO-175 nucleic acid sequence or (2) a pair of primers useful for amplifying a TANGO-175 nucleic acid molecule;
  • an oligonucleotide e.g., a detectably labelled oligonucleotide, which hybridizes to a TANGO-175 nucleic acid sequence or (2) a pair of primers useful for amplifying a TANGO-175 nucleic acid molecule;
  • the kit may also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent.
  • the kit may also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate) .
  • the kit may 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 TANGO-175.
  • 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 TANGO-175 expression or activity.
  • 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 TANGO- 175 protein, nucleic acid expression or activity.
  • 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 TANGO-175 expression or activity.
  • 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 TANGO-175 activity)
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant TANGO-175 expression or activity in which a test sample is obtained and TANGO-175 protein or nucleic acid is detected (e.g., wherein the presence of TANGO-175 protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant TANGO- 175 expression or activity) .
  • the methods of the invention can also be used to detect genetic lesions or mutations in a TANGO-175 gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation.
  • 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 a TANGO- 175-protein, or the mis-expression of the TANGO-175 gene.
  • 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 a TANGO-175 gene; 2) an addition of one or more nucleotides to a TANGO-175 gene; 3) a substitution of one or more nucleotides of a T7ANGO-175 gene; 4) a chromosomal rearrangement of a TANGO-175 gene; 5) an alteration in the level of a messenger RNA transcript of a TANGO-175 gene; 6) an aberrant modification of a TANGO-175 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 a TANGO-175 gene; 8) a non-wild type level of a TANGO-175-protein; 9) allelic loss of a TANGO-175 gene; and 10) an inappropriate post-translational modification of a TANGO-1
  • 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 .
  • 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 a TANGO-175 gene under conditions such that hybridization and amplification of the TANGO-175-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.
  • 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. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers .
  • mutations in a TANGO-175 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 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 ribozyme cleavage site.
  • genetic mutations in TANGO- 175 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 in T7ANGO-175 can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin et al . supra.
  • 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 TANGO-175 gene and detect mutations by comparing the sequence of the sample TANGO-175 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) .
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in the T7ANGO-175 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 TANGO-175 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.
  • 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 TANGO-175 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/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at
  • a probe based on a TANGO-175 sequence e.g., a wild-type TANGO- 175 sequence
  • 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 TANGO-175 genes.
  • single strand conformation polymorphism 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 D ⁇ A fragments of sample and control TANGO-175 nucleic acids will be denatured and allowed to renature.
  • SSCP single strand conformation polymorphism
  • 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
  • D ⁇ A 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 D ⁇ A by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample D ⁇ A (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 . Acad . Sci . USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target D ⁇ A or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target D ⁇ A.
  • 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 TANGO-175 gene.
  • any cell type or tissue preferably peripheral blood leukocytes, in which TANGO-175 is expressed may be utilized in the prognostic assays described herein.
  • Pharmacogenomics Agents, or modulators which have a stimulatory or inhibitory effect on TANGO-175 activity (e.g., TANGO-175 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g., proliferative) associated with aberrant TANGO-175 activity.
  • 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 TANGO-175 protein, expression of TANGO-175 nucleic acid, or mutation content of TANGO-175 genes 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 polymorphisms.
  • 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.
  • the discovery of genetic polymorphisms of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • NAT 2 N-acetyltransferase 2
  • 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 polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM) .
  • EM extensive metabolizer
  • PM poor metabolizer
  • the prevalence of PM is different among different populations.
  • the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6.
  • the activity of TANGO-175 protein, expression of TANGO-175 nucleic acid, or mutation content of TANGO-175 genes 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 polymorphic 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 TANGO-175 modulator, such as a modulator identified by one of the exemplary screening assays described herein.
  • the effectiveness of an agent, as determined by a screening assay as described herein, to increase TANGO-175 gene expression, protein levels, or protein activity can be monitored in clinical trials of subjects exhibiting decreased TANGO-175 gene expression, protein levels, or protein activity.
  • the effectiveness of an agent, as determined by a screening assay, to decrease TANGO-175 gene expression, protein levels, or protein activity can be monitored in clinical trials of subjects exhibiting increased TANGO-175 gene expression, protein levels, or protein activity.
  • the expression or activity of TANGO-175 and, preferably, other genes 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 TANGO-175, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates TANGO-175 activity (e.g., as identified in a screening assay described herein) can be identified.
  • an agent e.g., compound, drug or small molecule
  • TANGO-175 activity 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 TANGO-175 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 TANGO-175 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 expression of a TANGO-175 protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the TANGO-175 protein, mRNA, or genomic
  • 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
  • decreased administration of the agent may be desirable to decrease expression or activity of TANGO- 175 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 TANGO-175 expression or activity.
  • disorders include cancer, inflammatory disorders, and hematopoietic disorders.
  • the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant TANGO-175 expression or activity, by administering to the subject an agent which modulates TANGO-175 expression or at least one TANGO-175 activity.
  • Subjects at risk for a disease which is caused or contributed to by aberrant TANGO-175 expression or activity 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 TANGO-175 aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • a TANGO-175 agonist or TANGO-175 antagonist agent can be used for treating the subject. 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 TANGO-175 protein activity associated with the cell.
  • An agent that modulates TANGO-175 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a TANGO-175 protein, a peptide, a TANGO-175 peptidomimetic, or other small molecule.
  • the agent stimulates one or more of the biological activities of TANGO-175 protein.
  • stimulatory agents include active TANGO-175 protein and a nucleic acid molecule encoding TANGO-175 that has been introduced into the cell.
  • the agent inhibits one or more of the biological activities of TANGO-175 protein.
  • inhibitory agents include antisense TANGO-175 nucleic acid molecules and anti-TANGO-175 antibodies.
  • 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 TANGO-175 protein or nucleic acid molecule .
  • 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) TANGO-175 expression or activity.
  • the method involves administering a TANGO-175 protein or nucleic acid molecule as therapy to compensate for reduced or aberrant TANGO-175 expression or activity. Stimulation of TANGO-175 activity is desirable in situations in which TANGO-175 is abnormally downregulated and/or in which increased TANGO-175 activity is likely to have a beneficial effect. Conversely, inhibition of TANGO-175 activity is desirable in situations in which TANGO-175 is abnormally upregulated and/or in which decreased TANGO-175 activity is likely to have a beneficial effect.
  • a partial cDNA encoding murine TANGO-175 was identified by subtractive cDNA hybridization using stimulated and unstimulated bone marrow cells.
  • the bone marrow cells were obtained from the femurs of adult C57BL/6 female mice following the procedure of StemCell Technologies, Inc. (StemCell Technologies, Inc., Vancouver, Canada) with minor changes. Briefly, bone marrow was flushed from the femurs using phosphate buffered saline (PBS), pH 7.4, supplemented with 5% heat- inactivated fetal calf serum (PBS/5% HIFCS) .
  • PBS phosphate buffered saline
  • HIFCS heat- inactivated fetal calf serum
  • the cells were washed once in PBS/5% HIFCS, and the red blood cells were lysed by incubation with 3M ammonium chloride for 3 minutes on ice. Following termination of lysis by addition of PBS/5%
  • HIFCS human myeloid long-term culture medium
  • MyeloCultTM M5300 StemCell Technologies, Inc., Vancouver, Canada
  • the cultures were incubated at 33 °C in a 5% C0 2 humidified chamber for three weeks. Half the medium was replaced weekly with fresh medium.
  • the bone marrow cultures were stimulated for 2 hours at 33 °C with 50 ng/ml phorbol 12 -myristate 13- acetate (TPA; Sigma, Inc.) and 1 ⁇ M ionomycin (Sigma, Inc . ) .
  • RNA was then isolated from stimulated bone marrow cells, and from unstimulated sister cultures, using Qiagen RNeasy Maxi Kit (Qiagen, Inc.) .
  • the polyA+ RNA was isolated from each total RNA pool using the Oligotex mRNA Kit (Qiagen, Inc.) and then treated with RNase-free DNase (Boehringer Mannheim) .
  • the DNase-treated, polyA+ RNA was subjected to "PCR select" using the PCR-Select cDNA Subtraction Kit (Clontech, Inc.) .
  • the cDNA of unstimulated bone marrow cells was obtained and subtracted from that of stimulated bone marrow cells.
  • the PCR-amplified, differentially expressed cDNA was subcloned using TA Cloning Kit (Invitrogen, Inc.), transformed into ElectroMAX DH10B cells (Gibco BRL) and plated onto LB/amp plates.
  • the DNA from individual transformant colonies was isolated and sequenced using an automated sequencer.
  • the clone sequences were analyzed by comparison to available protein databases using the BLAST algorithm.
  • the nucleotide sequence of clone etmM013 was used to search the IMAGE EST database. This search led to the identification of EST W11247. A clone corresponding to this EST was fully sequenced ( Figure 1; SEQ ID N0:1) and found to encode full-length murine TANGO-175. This clone was used to search the human IMAGE EST database in an effort to identify an EST having homology to the murine TANGO-175 cDNA described above. This search led to the identification of EST W52431. A clone corresponding to EST W52431 was fully sequenced ( Figure 9; SEQ ID NO: 20) .
  • the cDNAs are 501 nucleotides long, including untranslated regions, and have a 183 nucleotide open reading frame (nucleotides 23-204 of SEQ ID NOS:4-7, SEQ ID NOS: 8-11) which encodes a 61 amino acid protein (SEQ ID NO: 12) .
  • Figure 2a is thought most likely to represent a naturally occurring cDNA encoding human Tango-175.
  • Human TANGO-175 protein is predicted to be a 4 kDa protein (excluding post-translational modifications) .
  • the expression patterns of murine and human TANGO- 175 were analyzed using Northern blot hybridization.
  • liver An approximately 0.5 kb murine TANGO-175 mRNA transcript was identified in liver, spleen, heart, kidney, and skeletal muscle. The expression in liver was far higher than in spleen, heart, kidney, or skeletal muscle
  • Endogenous murine TANGO-175 gene expression was determined using the Perkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMan technology. Briefly, TaqMan technology relies on standard RT-PCR with the addition of a third gene-specific oligonucleotide (referred to as a probe) which has a fluorescent dye coupled to its 5' end (typically 6-F7 ⁇ M) and a quenching dye at the 3' end (typically TAMRA) . When the fluorescently tagged oligonucleotide is intact, the fluorescent signal from the 5' dye is quenched.
  • a probe a third gene-specific oligonucleotide
  • TAMRA quenching dye
  • PCR As PCR proceeds, the 5' to 3 ' nucleolytic activity of taq polymerase digest the labeled primer, producing a free nucleotide labeled with 6-FAM, which is now detected as a fluorescent signal.
  • the PCR cycle where fluorescence is first released and detected is directly proportional to the starting amount of the gene of interest in the test sample, thus providing a way of quantitating the initial template concentration.
  • Samples can be internally controlled by the addition of a second set of primers/probe specific for a housekeeping gene such as GAPDH which has been labeled with a different fluor on the 5' end (typically JOE) .
  • RNA was prepared from a series of murine tissues using an RNeasy kit from Qiagen.
  • First strand cDNA was prepared from one ug total RNA using an oligo dT primer and Superscript II reverse transcriptase (Gibco/BRL) .
  • cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. Normal tissues tested include mouse brain, heart, liver, lung, spleen, testis, kidney and megakaryocytes .
  • TANGO-175 was expressed weakly in testis, heart and kidney and absent in total brain.
  • si tu hybridization analysis in mice revealed that TANGO-175 is expressed hepatocytes .
  • Within the liver expression was not detected in vascular endothelium and associated muscle cells, mesenchymal cells of the capsule, and areas of extramedullary hematopoesis .
  • This same analysis revealed that TANGO-175 appears to be ubiquitously expressed in adult thymus .
  • si tu expression analysis in mice revealed that TANGO-175 is expressed in fetal liver beginning at day E14.5.
  • Expression in this tissue increases to a maximum at day E16.5 and stays at that level at least through post-natal day 1.5.
  • expression was not detected in pancreas, placenta, eye, heart, thymus, spleen, kidney, lung, brain, colon, small intestine, skeletal muscle, and smooth muscle.
  • the IMAGE EST database was searched in an effort to identify clones which might encode unknown proteins having homology to human and murine
  • the predicted amino acid sequence of the TANGO-175 proteins and murine WDNM-2 are compared to amino acid sequences of known proteins and various motifs are identified.
  • the murine TANGO-175 cDNA (SEQ ID N0:1) has a 189 nucleotide open reading frame (nucleotides 18-206 of SEQ ID NO:l; SEQ ID NO : 3 ) which encodes a 63 amino acid protein (SEQ ID NO: 2) .
  • This protein includes a predicted signal sequence of about 24 amino acids (from amino acid 1 to about amino acid 24 of SEQ ID NO: 2; SEQ ID NO: 21) and a predicted mature protein of about 39 amino acids (from about amino acid 25 to amino acid 63 of SEQ ID NO:2; SEQ ID NO: 22) .
  • Murine TANGO-175 protein possesses six cysteine residues which form interdomain bonds which stabilize the protein and are likely to be essential for biological activity.
  • Murine TANGO-175 also includes an RGD motif, which likely mediates cell attachment to the TANGO-175 protein.
  • Murine TANGO-175 protein has some sequence similarity to the amino acid sequence of murine WDNM-1 (mWDNM-1; SEQ ID NO: 16), rat WDNM-1 (rWDNM; SEQ ID NO: 17), and murine anti-leukoproteinase (mALP; SEQ ID NO: 19) ( Figure 5) .
  • Hidden Markov Motifs refer to Sonnhammer et al . (1997 Protein 28:405-420) and http: //www.psc . edu/general/software/packages/ pfam/pfam. html .
  • the nucleotide sequences encoding human TANGO-175 ( Figures 2a, 2b, 2c and 2d; SEQ ID NO:4-7) encode a 61 amino acid protein ( Figures 2a-2d; SEQ ID NO: 12) .
  • the signal peptide prediction program SIGNALP Optimized Tool (Nielsen et al . (1997) Protein Engineering 10:1-6) predicted that TANGO-175 includes a 24 amino acid signal peptide (amino acid 1 to about amino acid 24 of SEQ ID NO: 12; SEQ ID NO: 23) preceding the mature protein (about amino acid 25 to amino acid 61; SEQ ID NO: 12; SEQ ID NO: 24) .
  • Human TANGO-175 contains a three-disulfide core pattern of cysteines found in murine TANGO-175.
  • human TANGO-175 protein possesses six cysteine residues, cysteines C1-C6, which occur at amino acids 33, 37, 43, 49, 54 and 58 of SEQ ID NO: 12, respectively. These cysteine residues form interdomain disulfide bonds which stabilize the human TANGO-175 protein. Cysteines C1-C5, C2-C4 and C3-C6 pair to form disulfide bonds.
  • human TANGO-175 protein has some sequence similarity to murine WDNM-1 (mWDNM-1; SEQ ID NO:16), rat WDNM-1 (rWDNM; SEQ ID NO:17), and murine anti-leukoproteinase (mALP; SEQ ID NO: 19) ( Figure 5) .
  • Figure 5 is an alignment of the amino acid sequence of murine WDNM-2 (SEQ ID NO: 14) with murine WDNM-1 (mWDNM-1; SEQ ID NO:16), rat WDNM-1 (rWDNM; SEQ ID NO:17), etmM031 (SEQ ID NO:18), murine TANGO-175 (mT.175orf; SEQ ID NO : 2 ) , human TANGO-175 (hT.175prot; SEQ ID NO: 12) , and murine anti-leukoproteinase (mALP; SEQ ID NO: 19) .
  • murine WDNM-2 SEQ ID NO: 14
  • murine WDNM-1 mWDNM-1; SEQ ID NO:16
  • rat WDNM-1 rat WDNM-1
  • SEQ ID NO:17 rat WDNM-1
  • etmM031 SEQ ID NO:18
  • murine TANGO-175 mT.175orf
  • Murine TANGO-175 has 15 residues identical to rat WDNM-1; 16 residues identical to murine WDNM-1; and 19 residues identical to murine anti-leukoproteinase.
  • human Tango- 175 has 19 residues identical to rat WDNM- - I ll -
  • Figure 5 also shows that WDNM-2 has 37 residues identical to rat WDNM- 1; 51 residues identical to murine WDNM-1; and 25 residues identical to murine anti-leukoproteinase.
  • Example 5 Assay Confirming that TANGO-175 is Secreted Secretion assays reveal that human TANGO-175 is secreted when expressed in 293T cells.
  • the secretion assay was performed as follows. Approximately 8xl0 5 293T cells were plated per well in a 6-well plate, and the cells were incubated in growth medium (DMEM, 10% fetal bovine serum, penicillin/strepomycin) at 37°, 5% C0 2 overnight. The 293T cells were transfected with 2 ⁇ g of full-length human TANGO 175 inserted in the pMET7 vector/well and 10 ⁇ g LipofectAMINE (GIBCO/BRL Cat.
  • DMEM 10% fetal bovine serum
  • penicillin/strepomycin penicillin/strepomycin
  • Recombinant TANGO-175 can be produced in a variety of expression systems.
  • the mature TANGO-175 peptide can be expressed as a recombinant glutathione-S- transferase (GST) fusion protein in E. coli and the fusion protein can be isolated and characterized.
  • GST glutathione-S- transferase
  • TANGO-175 can be fused to GST and this fusion protein can be expressed in E. coli strain PEB199.
  • Expression of the GST-TANGO- 175 fusion protein in PEB199 can be induced with IPTG.
  • the recombinant fusion protein can be purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads.
  • Example 7 Assaying the Expression of TANGO-175 in a Murine Model of Mice with Septic Shock
  • TANGO-175 is expressed in response to septic shock a mouse model of septic shock was used. Mice were injected intravenously with either 20 mg/kg lipolysaccharide (LPS) or, as a control, PBS, and sacrificed at 2 , 8 or 24 hours post-injection. Organs were harvested and cDNA was prepared for use in TaqMan as described above. The level of TANGO-175 gene expression was significantly upregulated in liver, heart and spleen by 8 hours post-LPS compared to PBS controls.
  • LPS lipolysaccharide
  • Example 8 Measurement of TANGO-175 or WDNM-2 Activity
  • a TANGO-175 or WDNM-2 polypeptide or a variant thereof to modulate hematopoiesis can be measured using the assay described by Goselink et al . ⁇ J. Exp Med . 184:1305-12, 1996). Alternatively, a colony formation assay can be used. Briefly, a single cell suspension of washed, RBC-free bone marrow cells is obtained as described above and diluted to 5 x 10 4 cells/ml in methylcellulose (StemCell Technologies, Inc.) .
  • 0.1 ml of diluted bone marrow cells in methylcellulose are added to the wells of a 96-well round bottom tissue culture plate (Corning) containing 11 ul of supernatant.
  • the plates are incubated at 37°C in a 5% C0 2 humidified chamber for 7 days at which time the number of colonies in each well are counted.
  • TANGO-175 or WDNM-2 polypeptide or a variant thereof can be measured using the assay described by Jin et al . ⁇ Cell 88:417-26, 1997) .
  • the ability to modulate the effect of septic shock in mice is evaluated using the mouse septic shock model. Briefly, the protein being tested is administered to mice prior to or simultaneously with administration of 20 mg/kg LPS or or PBS (which serves as a control) . The mice are then sacrificed at 2 , 8 or 24 hours post-injection of the mixture. The modulatory effect of TANGO-175 on LPS-induced septic shock in mice is evaluated.
  • Kits for performing coagulation assays are available from American Bioproducts Company (New Jersey) and Helene Laboratories (San Rafeal, CA) .

Abstract

L'invention concerne de nouvelles molécules d'acide nucléique, de nouvelles protéines et de nouveaux polypeptides TANGO 175 et WDNM-2. Outre les protéines TANGO-175 et WDNM-2 de pleine longueur et isolées, l'invention concerne également des protéines de fusion TANGO-175 et WDNM-2 isolées, des peptides antigènes et des anticorps anti-TANGO-175 et WDNM-2. L'invention traite aussi de molécules d'acide nucléique TANGO-175 et WDNM-2, de vecteurs d'expression de recombinaison contenant une molécule d'acide nucléique selon l'invention, des cellules hôtes dans lesquelles les vecteurs d'expression ont été introduits et des animaux transgéniques non humains dans lesquels un gène TANGO-175 et WDNM-2 a été introduit ou interrompu. L'invention traite aussi de procédés de diagnostique, de détection et de procédés thérapeutiques utilisant les compositions selon l'invention.
PCT/US1999/017289 1998-07-29 1999-07-29 Nouvelles molecules de la famille des proteines liees au tango 175 et utilisations de ces dernieres WO2000006699A1 (fr)

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