WO2001000673A9 - Membrane-associated and secreted proteins and uses thereof - Google Patents

Membrane-associated and secreted proteins and uses thereof

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Publication number
WO2001000673A9
WO2001000673A9 PCT/US2000/018198 US0018198W WO0100673A9 WO 2001000673 A9 WO2001000673 A9 WO 2001000673A9 US 0018198 W US0018198 W US 0018198W WO 0100673 A9 WO0100673 A9 WO 0100673A9
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WO
WIPO (PCT)
Prior art keywords
seq
amino acid
polypeptide
atcc
acid sequence
Prior art date
Application number
PCT/US2000/018198
Other languages
French (fr)
Other versions
WO2001000673A1 (en
WO2001000673A8 (en
Inventor
Thomas M Barnes
Christopher C Fraser
Nicholas Wrighton
Paul Myers
Samantha J Busfield
John D Sharp
Original Assignee
Millennium Pharm Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Millennium Pharm Inc filed Critical Millennium Pharm Inc
Priority to AU59078/00A priority Critical patent/AU5907800A/en
Publication of WO2001000673A1 publication Critical patent/WO2001000673A1/en
Publication of WO2001000673A8 publication Critical patent/WO2001000673A8/en
Publication of WO2001000673A9 publication Critical patent/WO2001000673A9/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • cytokines Many secreted proteins, for example, cytokines, play a vital role in the regulation of cell growth, cell differentiation, and a variety of specific cellular responses.
  • membrane-associated proteins are receptors which bind a ligand and transduce an intracellular signal, leading to a variety of cellular responses.
  • the identification and characterization of such a receptor enables one to identify both the ligands which bind to the receptor and the intracellular molecules and signal transduction pathways associated with the receptor, permitting one to identify or design modulators of receptor activity, e.g., receptor agonists or antagonists and modulators of signal transduction.
  • the present invention is based, at least in part, on the discovery of cDNA molecules encoding INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, and TANGO 378 all of which are either wholly secreted or transmembrane proteins. These proteins, fragments, derivatives, and variants thereof are collectively referred to as “polypeptides of the invention” or “proteins of the invention.” Nucleic acid molecules encoding the polypeptides or proteins of the invention are collectively referred to as "nucleic acids of the invention.”
  • nucleic acids and polypeptides of the present invention are useful as modulating agents in regulating a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding a polypeptide of the invention or a biologically active portion thereof. The present invention also provides nucleic acid molecules which are suitable for use as primers or hybridization probes for the detection of nucleic acids encoding a polypeptide of the invention.
  • the invention features nucleic acid molecules which are at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequence of SEQ ID NOs:l, 3, 4, 6, 1, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, the nucleotide sequence of the cDNA insert of a clone deposited with ATCC ® as Accession Number 207178 (the "cDNA of ATCC ® Accession Number 207178"), the nucleotide sequence of the cDNA insert of a clone deposited with ATCC ® as Accession Number PTA-249 (the "cDNA of ATCC ® Accession Number PTA-249"), or the nucleotide sequence of the cDNA insert of a clone deposited with ATCC ® as Accession Number PTA-250 (the "cDNA of ATCC ® Accession Number PTA-250”), or a complement thereof.
  • the invention features nucleic acid molecules which include a fragment of at least
  • 10 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, or 4000) nucleotides of the nucleotide sequence of SEQ ID NOs:l, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, the nucleotide sequence of the cDNA of ATCC ® Accession Number 207178, the nucleotide sequence of the cDNA of ATCC ® Accession Number PTA-249, or
  • the invention also features nucleic acid molecules which include 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 NOs:2, 5, 8,
  • the nucleic acid molecules have the nucleotide sequence
  • nucleotide sequence of the cDNA of ATCC ® Accession Number 207178 the nucleotide sequence of the cDNA of ATCC ® Accession Number PTA-249, or the nucleotide sequence of the cDNA of ATCC ® Accession Number PTA-250, or a complement thereof.
  • nucleic acid molecules which encode a fragment of a
  • polypeptide having the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, or a fragment including at least 15 (25, 30, 50, 100, 150, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, or 1400) contiguous amino acids of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, the amino acid sequence encoded by the cDNA of ATCC ® Accession Number 207178, the amino acid sequence encoded by the cDNA of ATCC ®
  • the invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, the amino acid sequence encoded by the cDNA of ATCC ® Accession Number 207178, the amino acid sequence encoded by the cDNA of ATCC ® Accession Number PTA-249, or the amino acid sequence encoded by the cDNA of ATCC ® Accession Number PTA-250, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule consisting of a nucleic acid sequence encoding SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, the nucleotide sequence of the cDNA of ATCC ® Accession Number 207178, the nucleotide sequence of the cDNA of ATCC ® Accession Number PTA-249, or the
  • isolated polypeptides or proteins having an amino acid sequence that is at least about 60%, preferably 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, the amino acid sequence encoded by the cDNA of ATCC ® Accession Number 207178, the amino acid sequence encoded by the cDNA of ATCC ® Accession Number PTA-249, or the amino acid sequence encoded by the cDNA of ATCC ® Accession Number PTA-250.
  • isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 60% o , preferably
  • nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ
  • polypeptides which are naturally occurring allelic variants of a polypeptide that includes the amino acid sequence of SEQ ID NOs:2, 5, 8, 11,
  • polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule having the sequence of SEQ ID NO: 1
  • allelic variant differ at 1%, 2%, 3%, 4%, or 5% of the amino acid residues.
  • the invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NOs: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, the cDNA of ATCC ® Accession Number 207178, the cDNA of ATCC ® Accession Number PTA-249, or the cDNA of ATCC ® Accession Number PTA-250, or a complement thereof.
  • the nucleic acid molecules are at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, or 4200) nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NOs.T, 3, 4, 6, 1, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, the cDNA of ATCC ® Accession Number 207178, the cDNA of ATCC ® Accession Number PTA-249, or the cDNA of ATCC ® Accession Number PTA-250, or a complement thereof.
  • the isolated nucleic acid molecules encode an extracellular, transmembrane, or cytoplasmic domain of a polypeptide of the invention.
  • the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a nucleic acid of the invention.
  • vectors e.g., recombinant expression vectors, comprising a nucleic acid molecule of the invention.
  • the invention provides host cells containing such a vector or a nucleic acid molecule of the invention.
  • the invention also provides methods for producing a polypeptide of the invention by culturing, in a suitable medium, a host cell of the invention containing a recombinant expression vector such that a polypeptide is produced.
  • Another aspect of this invention features isolated or recombinant proteins and polypeptides of the invention.
  • Preferred proteins and polypeptides possess at least one biological activity possessed by the corresponding naturally-occurring human polypeptide.
  • An activity, a biological activity, or a functional activity of a polypeptide or nucleic acid of the invention refers to an activity exerted by a protein, polypeptide or nucleic acid molecule of the invention on a responsive cell as determined in vivo, or in vitro, according to standard techniques.
  • activities can be a direct activity, such as an association with or an enzymatic activity on a second protein or an indirect activity, such as a cellular signaling activity mediated by interaction of the protein with a second protein.
  • the isolated polypeptide of the invention lacks both a transmembrane and a cytoplasmic domain. In another embodiment, the polypeptide lacks both a transmembrane domain and a cytoplasmic domain and is soluble under physiological conditions.
  • biological activities include, e.g., (1) the ability to form protein-protein interactions with proteins in the signaling pathway of the naturally- occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; (3) the ability to interact with an INTERCEPT 340 receptor, e.g., a cell surface receptor (e.g., an integrin); (4) the ability to modulate the activity of an intracellular molecule that participates in a signal transduction pathway, e.g., an intracellular molecule in the integrin signalling (e.g., a cdk2 inhibitor); (5) the ability to assemble into fibrils; (6) the ability to strengthen and organize the extracellular matrix; (7) the ability to modulate the shape of tissues and cells; (8) the ability to interact with (e.g., bind to) components of the extracellular matrix; and (9) the ability to modulate cell migration.
  • an INTERCEPT 340 receptor e.g., a cell surface receptor (e.g., an integr
  • INTERCEPT 340 Other activities include the ability to modulate function, survival, morphology, migration, proliferation and/or differentiation of cells of tissues in which it is expressed (e.g., splenic cells).
  • additional biological activities of INTERCEPT 340 include: (1) the ability to modulate splenic cell activity; (2) the ability to modulate skeletal morphogenesis; and/or (3) the ability to modulate smooth muscle cell proliferation and differentiation.
  • biological activities include, e.g., (1) the ability to form protein- protein (e.g., protein-ligand) interactions with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to interact with (e.g., bind to) a ligand of the naturally-occurring polypeptide; (3) the ability to interact with a MANGO 003 receptor, e.g., a cell surface receptor; (4) the ability to modulate cell surface recognition; (5) the ability to transduce an extracellular signal (e.g., by interacting with a ligand and/or a cell- surface receptor); (6) the ability to modulate a signal transduction pathway; and (7) the ability to modulate signal transmission at a chemical synapse.
  • protein- protein e.g., protein-ligand
  • biological activities include, e.g., (1) the ability to form protein- protein (e.g., protein-ligand) interactions with proteins in the signaling pathway of the naturally-occurring polypeptide;
  • Other activities include the ability to modulate function, survival, morphology, proliferation and/or differentiation of cells of tissues in which it is expressed (e.g., thyroid, liver, skeletal muscle, kidney, heart, lung, testis and brain).
  • the activities of MANGO 003 can include modulation of endocrine, hepatic, skeletal muscular, renal, cardiovascular, reproductive and/or brain function.
  • biological activities include, e.g., (1) the ability to form protein- protein interactions with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to interact with a ligand of the naturally-occurring polypeptide;
  • MANGO 347 the ability to interact with a MANGO 347 receptor; and (4) the ability to modulate a developmental process, e.g., morphogenesis, cellular migration, adhesion, proliferation, differentiation, and/or survival.
  • Other activities include the ability to modulate function, survival, morphology, proliferation and/or differentiation of cells of tissues in which it is expressed (e.g., brain cells).
  • the activities of MANGO 347 can include modulation of neural (e.g., CNS) function.
  • biological activities include, e.g., (1) the ability to form protein- protein interactions with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; (3) the ability to interact with a TANGO 272 receptor, e.g., a cell surface receptor (e.g., an integrin); (4) the ability to modulate cell-cell contact; (5) the ability to modulate cell attachment; (6) the ability to modulate cell fate; and (7) the ability to modulate tissue repair and/or wound healing.
  • a TANGO 272 receptor e.g., a cell surface receptor (e.g., an integrin)
  • a cell surface receptor e.g., an integrin
  • Other activities include the ability to modulate function, survival, morphology, proliferation and/or differentiation of cells of tissues in which it is expressed (e.g., microvascular endothelial cells).
  • the activities of MANGO 347 can include modulation of cardiovascular function.
  • biological activities include, e.g., (1) the ability to form protein- protein interactions with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; (3) the ability to interact with a TANGO 295 receptor; (4) the ability to interact with (e.g., bind to) a nucleic acid; and (5) the ability to elicit pyrimidine-specific endonuclease activity.
  • Other activities include the ability to modulate function, survival, morphology, proliferation and/or differentiation of cells of tissues in which it is expressed (e.g., mammary epithelium).
  • biological activities include, e.g., (1) the ability to fomi protein- protein interactions with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; (3) the ability to interact with (e.g., bind to) a TANGO 354 receptor, e.g., a cell surface receptor; (4) the ability to modulate cell surface recognition; (5) the ability to modulate cellular motility, e.g., chemotaxis and/or chemokinesis; (6) the ability to transduce an extracellular signal (e.g., by interacting with a ligand and/or a cell-surface receptor); and (7) the ability to modulate a signal transduction pathway.
  • TANGO 354 biological activities can further include: (1) regulation of hematopoiesis; (2) modulation (e.g., increasing or decreasing) of haemostasis; (3) modulation of an inflammatory response; (4) modulation of neoplastic growth, e.g., inhibition of tumor growth; and (5) modulation of thrombolysis.
  • biological activities include, e.g., (1) the ability to form protein- protein interactions with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; (3) the ability to interact with a TANGO 378 receptor; (4) the ability to transduce an extracellular signal; and (5) the ability to modulate a signal transduction pathway (e.g., adenylate cyclase, or phosphatidylinositol 4,5-bisphosphate (PrP 2 ), inositol 1,4,5-triphosphate (IP 3 )).
  • a signal transduction pathway e.g., adenylate cyclase, or phosphatidylinositol 4,5-bisphosphate (PrP 2 ), inositol 1,4,5-triphosphate (IP 3 )
  • TANGO 378 biological activities can further include the ability to modulate an immune response in a subject, for example, (1) by modulating immune cytotoxic responses against pathogenic organisms, e.g., viruses, bacteria, and parasites; (2) by modulating organ rejection after transplantation; and (3) by modulating immune recognition and lysis of normal and malignant cells.
  • a polypeptide of the invention has an amino acid sequence sufficiently identical to an identified domain of a polypeptide of the invention.
  • the term "sufficiently identical" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity.
  • amino acid or nucleotide sequences which contain a common structural domain having about 60% identity, preferably 65%> identity, more preferably 75%, 85%, 95%>, 98% or more identity are defined herein as sufficiently identical.
  • TANGO 354, or TANGO 378 polypeptide of the invention includes a signal peptide.
  • nucleic acid molecule of the invention encodes a
  • 378 polypeptide of the invention includes one or more of the following domains: (1) a signal peptide; (2) an N-terminal extracellular domain; (3) a C-terminal transmembrane domain; and (4) a cytoplasmic domain.
  • polypeptides of the present invention can be operably linked to a heterologous amino acid sequence to form fusion proteins.
  • the fusion protein consists of a chimeric protein assembled from portions of the protein from different species.
  • the isolated polypeptide of the invention lacks both a transmembrane and a cytoplasmic domain. In another embodiment, the polypeptide lacks both a transmembrane domain and a cytoplasmic domain and is soluble under physiological conditions.
  • the invention further features antibodies that specifically bind a polypeptide of the invention such as monoclonal or polyclonal antibodies.
  • a polypeptide of the invention such as monoclonal or polyclonal antibodies.
  • the polypeptides of the invention or biologically active portions thereof, or antibodies of the invention can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
  • the present invention provides methods for detecting the presence of the activity or expression of a polypeptide of the invention in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of activity such that the presence of activity is detected in the biological sample.
  • the invention provides methods for modulating activity of a polypeptide of the invention comprising contacting a cell with an agent that modulates (inhibits or stimulates) the activity or expression of a polypeptide of the invention such that activity or expression in the cell is modulated.
  • the agent is an antibody that specifically binds to a polypeptide of the invention.
  • the agent modulates expression of a polypeptide of the invention by modulating transcription, splicing, or translation of an mRNA encoding a polypeptide of the invention.
  • the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an mRNA encoding a polypeptide of the invention.
  • the present invention also provides methods to treat a subject having a disorder characterized by aberrant activity of a polypeptide of the invention or aberrant expression of a nucleic acid of the invention by administering an agent which is a modulator of the activity of a polypeptide of the invention or a modulator of the expression of a nucleic acid of the invention to the subject.
  • the modulator is a protein of the invention.
  • the modulator is a nucleic acid of the invention.
  • the modulator is a peptide, peptidomimetic, or other small organic molecule.
  • the present invention also provides diagnostic assays for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of: (i) aberrant modification or mutation of a gene encoding a polypeptide of the invention, (ii) mis- regulation of a gene encoding a polypeptide of the invention, and (iii) aberrant post- translational modification of the invention wherein a wild-type form of the gene encodes a protein having the activity of the polypeptide of the invention.
  • the invention provides a method for identifying a compound that binds to or modulates the activity of a polypeptide of the invention.
  • such methods entail measuring a biological activity of the polypeptide in the presence and absence of a test compound and identifying those compounds which alter the activity of the polypeptide.
  • the invention also features methods for identifying a compound which modulates the expression of a polypeptide or nucleic acid of the invention by measuring the expression of the polypeptide or nucleic acid in the presence and absence of the compound.
  • the invention provides substantially purified antibodies or fragments thereof including human and non-human antibodies or fragments thereof which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC ® as Accession Number 207178, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC ® as Accession Number PTA-249, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC ® as Accession Number PTA-250; a fragment of at least 15 amino acid residues of the amino acid sequence of
  • any of the antibodies of the invention can be conjugated to a therapeutic moiety or to a detectable substance.
  • detectable substances that can be conjugated to the antibodies of the invention are an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, and a radioactive material.
  • the invention also provides a kit containing an antibody of the invention conjugated to a detectable substance, and instructions for use.
  • Still another aspect of the invention is a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition contains an antibody of the invention, a therapeutic moiety, and a pharmaceutically acceptable carrier.
  • Figures 1A-1B depict the cDNA sequence of human INTERCEPT 340 (SEQ ID NO:l) and the predicted amino acid sequence of INTERCEPT 340 (SEQ ID NO:2).
  • the open reading frame of SEQ ID NO.T extends from nucleotide 1222 to nucleotide 1944 of SEQ ID NO.T (SEQ ID NO:3).
  • Figure 2 depicts a hydropathy plot of human INTERCEPT 340. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) and potential N- glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of INTERCEPT 340 are indicated. The amino acid sequence of each of the fibrillar collagen C-terminal domains are indicated by underlining and the abbreviation "COLF".
  • Figure 3 depicts an alignment of each of the fibrillar collagen C-terminal domains (also referred to herein as "COLF domains") of human INTERCEPT 340 with consensus hidden Markov model COLF domains.
  • the upper sequence is the consensus amino acid sequence (SEQ ID NOs:31, 32, and 33), while the lower sequence amino acid sequence corresponds to amino acid 58 to amino acid 116 of SEQ ID NO:2 (SEQ ID NO:34), amino acid 126 to amino acid 151 of SEQ ID NO:2 (SEQ ID NO:35), and amino acid 186 to amino acid 217 of SEQ ID NO:2 (SEQ ID NO:36).
  • Figures 4A-4C depict the cDNA sequence of human MANGO 003 (SEQ ID NO:4) and the predicted amino acid sequence of MANGO 003 (SEQ ID NO:5).
  • the open reading frame of SEQ ID NO:4 extends from nucleotide 57 to nucleotide 1568 of SEQ ID NO:4 (SEQ ED NO:6).
  • Figure 5 depicts a hydropathy plot of human MANGO 003. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) and potential N-glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of MANGO 003 are indicated. The amino acid sequence of each of the immunoglobulin domains, and the neurotransmitter gated ion channel domain are indicated by underlining and the abbreviations "ig” and "neur chan", respectively.
  • Figure 6 depicts an alignment of each of the immunoglobulin domains (also referred to herein as "Ig domains") of human MANGO 003 with the consensus hidden Markov model immunoglobulin domains.
  • the upper sequence is the consensus sequence (SEQ ID NO:37), while the lower sequence corresponds to amino acid 44 to amino acid 101 of SEQ ID NO:5 (SEQ ID NO:38), amino acid 165 to amino acid 223 of SEQ ID NO:5 (SEQ ID NO:39), and amino acid 261 to amino acid 340 of SEQ ID NO:5 (SEQ ID NO:40).
  • Figure 7 depicts an alignment of the neurotransmitter gated ion channel domain of human MANGO 003 with the consensus hidden Markov model neurotransmitter gated ion channel domain.
  • the upper sequence is the consensus sequence (SEQ ID NO:42), while the lower sequence corresponds to amino acid 388 amino acid 397 of SEQ ID NO: 5 (SEQ ID NO:43).
  • Figure 8 depicts the cDNA sequence of mouse MANGO 003 (SEQ ID NO:7) and the predicted amino acid sequence of MANGO 003 (SEQ ID NO:8).
  • the open reading frame of SEQ ID NO:7 extends from nucleotide 1 to nucleotide 626 of SEQ ID NO:4 (SEQ ID NO:9).
  • Figure 9 depicts a hydropathy plot of mouse MANGO 003. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) and potential N-glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of mouse MANGO 003 are indicated.
  • Figure 10 depicts the cDNA sequence of human MANGO 347 (SEQ ID NO: 10) and the predicted amino acid sequence of MANGO 347 (SEQ ID NOT 1).
  • the open reading frame of SEQ ID NO:10 extends from nucleotide 31 to nucleotide 444 of SEQ ID NO:10 (SEQ ID NO: 12).
  • Figure 11 depicts a hydropathy plot of human MANGO 347. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of MANGO 347 are indicated. The amino acid sequence of the CUB domain is indicated by underlining and the abbreviation "CUB".
  • Figure 12 depicts an alignment of the CUB domain of human MANGO 347 with a consensus hidden Markov model CUB domain.
  • the upper sequence is the consensus amino acid sequence (SEQ ID NO:44), while the lower sequence corresponds to amino acid 40 to amino acid 136 of SEQ ID NO: 11 (SEQ ID NO:45).
  • Figures 13A-13D depict the cDNA sequence of human TANGO 272 (SEQ ID NO:13) and the predicted amino acid sequence of TANGO 272 (SEQ ID NO:14).
  • the open reading frame of SEQ ID NO:13 extends from nucleotide 230 to nucleotide 3379 of SEQ ID NO:13 (SEQ ID O:15).
  • Figure 14 depicts a hydropathy plot of human TANGO 272. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line.
  • the cysteine residues (cys) and potential N- glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of TANGO 272 are indicated.
  • the amino acid sequence of each of the fourteen EGF-like domains and the delta serrate ligand domain is indicated by underlining and the abbreviation "EGF-like" and "DSL", respectively.
  • Figures 15A-15C depict an alignment of each of the EGF-like domains of human TANGO 272 with consensus hidden Markov model EGF-like domains.
  • the upper sequence is the consensus amino acid sequence (SEQ ID NO:46), while the lower sequence corresponds to amino acid 151 to amino acid 181 of SEQ ID NO: 14 (SEQ ID NO:49); amino acid 200 to amino acid 229 of SEQ ID NO: 14 (SEQ ID NO:50); amino acid 242 to amino acid 272 of SEQ ID NO: 14 (SEQ ID NO:51); amino acid 285 to amino acid 315 of SEQ ID NO: 14 (SEQ ID NO:52); amino acid 328 to amino acid 358 of SEQ ID NO: 14
  • SEQ ID NO:53 amino acid 378 to amino acid 404 of SEQ ID NO: 14 (SEQ ID NO:54); amino acid 417 to amino acid 447 of SEQ ID NO:14 (SEQ ID NO:55); amino acid 460 to amino acid 490 of SEQ ID NO: 14 (SEQ ID NO:56); amino acid 503 to amino acid 533 of SEQ ID NO:14 (SEQ ID NO:57); amino acid 546 to amino acid 576 of SEQ ID NO:14 (SEQ ID NO:58); amino acid 589 to amino acid 619 of SEQ ID NO: 14 (SEQ ID NO:59);
  • Figures 16A-16B depict the cDNA sequence of mouse TANGO 272 (SEQ ID NO:16) and the predicted amino acid sequence of TANGO 272 (SEQ ID NO:17).
  • the open reading frame of SEQ ID NO: 16 extends from nucleotide 1 to nucleotide 1492 of SEQ ID NO:16 (SEQ ID NO:18).
  • Figure 17 depicts a hydropathy plot of mouse TANGO 272. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) and potential N-glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of mouse TANGO
  • Figure 18 depicts the cDNA sequence of human TANGO 295 (SEQ ID NO:22) and the predicted amino acid sequence of TANGO 295 (SEQ ID NO:23).
  • the open reading frame of SEQ ID NO:22 extends from nucleotide 217 to nucleotide 684 of SEQ ID NO:28 (SEQ ID NO:24).
  • Figure 19 depicts a hydropathy plot of human TANGO 295. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line.
  • the cysteine residues (cys) and potential N- glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace.
  • Below the hydropathy plot the numbers corresponding to the amino acid sequence of human TANGO 295 are indicated.
  • the amino acid sequence of the pancreatic ribonuclease domain is indicated by underlining and the abbreviation "RNase A".
  • Figure 20 depicts an alignment of the pancreatic ribonuclease domain of human TANGO 295 with a consensus hidden Markov model pancreatic ribonuclease domain.
  • the upper sequence is the consensus amino acid sequence (SEQ ID NO:96), while the lower sequence corresponds to amino acid 32 to amino acid 156 of SEQ ID NO:23 (SEQ ID NO:97).
  • Figures 21A-21B depict the cDNA sequence of human TANGO 354 (SEQ ID NO:25) and the predicted amino acid sequence of TANGO 354 (SEQ ID NO:26).
  • the open reading frame of SEQ ID NO:25 extends from nucleotide 62 to nucleotide 976 of SEQ ID NO:25 (SEQ ID NO:27).
  • Figure 22 depicts a hydropathy plot of human TANGO 354. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line.
  • the cysteine residues (cys) and potential N- glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of human TANGO 354 are indicated.
  • the amino acid sequence of the immunoglobulin domain is indicated by underlining and the abbreviation "ig".
  • Figure 23 depicts an alignment of the immunoglobulin domain of human TANGO 354 with a consensus hidden Markov model immunoglobulin domains.
  • the upper sequence is the consensus amino acid sequence (SEQ ID NO: 37), while the lower sequence corresponds to amino acid 33 to amino acid 110 of SEQ ID NO:26 (SEQ ID NO:41).
  • Figures 24A-24C depict the cDNA sequence of human TANGO 378 (SEQ ID NO:28) and the predicted amino acid sequence of TANGO 378 (SEQ ID NO:29).
  • the open reading frame of SEQ ID NO:28 extends from nucleotide 42 to nucleotide 1625 of SEQ ID NO:28 (SEQ ID NO:30).
  • Figure 25 depicts a hydropathy plot of human TANGO 378. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) and potential N- glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of human TANGO 378 are indicated. The amino acid sequence of the seven transmembrane domain is indicated by underlining and the abbreviation "7tm”.
  • Figure 26 depicts an alignment of the seven transmembrane receptor domain of human TANGO 378 with a consensus hidden Markov model of this domain. The upper sequence is the consensus amino acid sequence (SEQ ID NO:98), while the lower sequence corresponds to amino acid 187 to amino acid 515 of SEQ ID NO:29 (SEQ ID NO:99).
  • Figures 27A-27C depict a global alignment between the nucleotide sequence of the open reading frame (ORF) of human MANGO 003 (SEQ ID NO:6) and the nucleotide sequence of the open reading frame of mouse MANGO 003 (SEQ ID NO:9).
  • the upper sequence is the human MANGO 003 ORF nucleotide sequence, while the lower sequence is the mouse MANGO 003 ORF nucleotide sequence.
  • These nucleotides sequences share a 31.1% identity.
  • the global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of -1212; Myers and Miller, 1989, CABIOS 4:11-1).
  • Figures 28A-28B depict a local alignment between the nucleotide sequence of human MANGO 003 (SEQ ID NO:4) and the nucleotide sequence of mouse MANGO 003 (SEQ ID NO:7).
  • the upper sequence is the human MANGO 003 nucleotide sequence
  • the lower sequence is the mouse MANGO 003 nucleotide sequence.
  • These nucleotides sequences share a 62.8 % identity over nucleotide 970 to nucleotide 2080 of the human MANGO 003 sequence (nucleotide 10 to nucleotide 1070 of mouse MANGO 003).
  • Figure 29 depicts a global alignment between the amino acid sequence of human MANGO 003 (SEQ ID NO:5) and the amino acid sequence of mouse MANGO 003 (SEQ ID NO:8).
  • the upper sequence is the human MANGO 003 amino acid sequence, while the lower sequence is the mouse MANGO 003 amino acid sequence. These amino acid sequences share a 30.1% identity.
  • the global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of -488; Myers and Miller, 1989, C4R/OS4.T 1-7).
  • Figures 30A-30E depict a global alignment between the nucleotide sequence of the open reading frame ( ⁇ RF) of human TANGO 272 (SEQ ID NO: 15) and the nucleotide sequence of the open reading frame of mouse TANGO 272 (SEQ ID NO:18).
  • the upper sequence is the mouse TANGO 272 ORF nucleotide sequence
  • the lower sequence is the human TANGO 272 ORF nucleotide sequence.
  • These nucleotides sequences share a 39.1% identity.
  • the global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of -79; Myers and Miller, 1989, CABIOS 4:11-1).
  • Figures 31A-31D depict a local alignment between the nucleotide sequence of human TANGO 272 (SEQ ID NO: 13) and the nucleotide sequence of mouse TANGO 272 (SEQ ID NO: 16).
  • the upper sequence is the human TANGO 272 nucleotide sequence
  • the lower sequence is the mouse TANGO 272 nucleotide sequence.
  • These nucleotides sequences share a 67.6 % identity over nucleotide 1890 to nucleotide 4610 of the human TANGO 272 sequence (nucleotide 10 to nucleotide 2560 of mouse TANGO 272).
  • Figures 32A-32B depict a global alignment between the amino acid sequence of human TANGO 272 (SEQ ID NO: 14) and the amino acid sequence of mouse TANGO 272 (SEQ ID NO: 17).
  • the upper sequence is the human TANGO 272 amino acid sequence, while the lower sequence is the mouse TANGO 272 amino acid sequence. These amino acid sequences share a 38.2% identity.
  • the global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of -19; Myers and Miller, 1989, CABIOS 4:11-7).
  • Figures 33A-33D depict the cDNA sequence of rat TANGO 272 (SEQ ID NO: 19) and the predicted amino acid sequence of TANGO 272 (SEQ ID NO:20).
  • the open reading frame of SEQ ID NO:19 extends from nucleotide 925 to nucleotide 2832 of SEQ ID NO:19
  • Figures 34A-34H depict a global alignment between the nucleotide sequence of human TANGO 272 (SEQ ID NO:13) and the nucleotide sequence of rat TANGO 272
  • the upper sequence is the human TANGO 272 nucleotide sequence, while the lower sequence is the rat TANGO 272 nucleotide sequence. These nucleotides sequences share a 55.7% identity.
  • the global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of 8635; Myers and Miller, 1989, G4R/OS 4:l l-7).
  • Figures 35A-35F depict a global alignment between the nucleotide sequence of mouse TANGO 272 (SEQ ID NO:16) and the nucleotide sequence of rat TANGO 272
  • the upper sequence is the mouse TANGO 272 nucleotide sequence, while the lower sequence is the rat TANGO 272 nucleotide sequence. These nucleotides sequences share a 43.1% identity.
  • the global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of 2827; Myers and Miller, 1989, CABIOS 4:11-7).
  • Figure 36 depicts a global alignment of the human TANGO 295 and GenPept
  • the upper sequence is the human TANGO 295 sequence
  • GenPept AF037081 encodes a ribonuclease k6 protein.
  • the global alignment revealed a 53.2% identity between these two sequences (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of 405; Myers and Miller, 1989, CABIOS 4:11-7).
  • Figures 37A-37C depict a global alignment of the human TANGO 295 (SEQ ID NO:22) and GenPept AF037081 (SEQ ID NO: 100) nucleotide sequences.
  • the upper sequence is the human TANGO 295 sequence, while the lower sequence is the GenPept AF037081 sequence.
  • the global alignment revealed a 22.6% identity between these two sequences (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of -2718; Myers and Miller, 1989, CABIOS 4:11-1).
  • Figures 38A-38B depict a local alignment of the human TANGO 295 (SEQ ID NO:22) and GenPept AF037081 (SEQ ID NO: 100) nucleotide sequences.
  • the upper sequence is the human TANGO 295 sequence, while the lower sequence is the GenPept AF037081 sequence.
  • the local alignment revealed a 62.7% identity between nucleotide 235 to nucleotide 687 of human TANGO 295, and nucleotide 3 to nucleotide 453 of
  • Figures 39A-39B depict an alignment of each of the EGF-like domains and laminin- EGF-like domains of mouse TANGO 272 with consensus hidden Markov model EGF-like domains.
  • the upper sequence is the consensus amino acid sequence (SEQ ID NO:46), while the lower sequence corresponds to amino acids 37-67 of SEQ ID NO:17 (SEQ ID NO:64); amino acid 80 to amino acid 110 of SEQ
  • SEQ ID NO:69 amino acid 83 to amino acid 123 of SEQ ID NO: 17 (SEQ ID NO:70); and amino acid 127 to amino acid 172 of SEQ ID NO:17 (SEQ ID NO:71).
  • the upper sequence is the consensus hidden Markov model domain (SEQ ID NO:47), while the lower sequence corresponds to amino acid 10 to amino acid 67 of SEQ ID NO: 17 (SEQ ID NO:72).
  • Figure 40 depicts a hydropathy plot of rat TANGO 272. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) and potential N-glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of rat TANGO 272 are indicated.
  • Figures 41A-41D depict an alignment of each of the EGF-like domains and laminin- EGF-like domains of rat TANGO 272 with consensus hidden Markov model of EGF-like domains.
  • the upper sequence is the consensus amino acid sequence (SEQ ID NO:46), while the lower sequence corresponds to amino acid 18 to amino acid 48 of SEQ ID NO:20 (SEQ ED NO:73); amino acid 61 to amino acid 91 of SEQ ID NO:20 (SEQ ID NO:74); amino acids 105-137 of SEQ ID NO:20 (SEQ ID NO:46).
  • amino acids 150-180 of SEQ ID NO:20 amino acids 193-223 of SEQ ID NO:20 (SEQ ID NO:77); amino acids 236-266 of SEQ ID NO:20 (SEQ ID NO:78); amino acids 279-309 of SEQ ID NO:20 (SEQ ID NO:79); amino acids 322-352 of SEQ ID NO:20 (SEQ ID NO:80); amino acids 365-394 of SEQ ID NO:20 (SEQ ID NO:81); amino acids 407-437 of SEQ ID NO:20 (SEQ ID NO:82); and amino acids 450-
  • the upper sequence is the consensus hidden Markov model domain (SEQ ID NO:48), while the lower sequence corresponds to amino acids 22-61 of SEQ ID NO:20 (SEQ ID NO:84); amino acids 65-105 of SEQ ID NO:20 (SEQ ID NO:85); amino acids 109-150 of SEQ ID NO:20 (SEQ ID NO:86); amino acids 154-193 of SEQ ID NO:20 (SEQ ID NO:87); amino 0 acids 197-236 of SEQ ID NO:20 (SEQ ID NO:88); amino acids 240-279 of SEQ ID NO:20 (SEQ ID NO:89); amino acids 283-322 of SEQ ID NO:20 (SEQ ID NO:90); amino acids 326-365 of SEQ ID NO:20 (SEQ ID NO:91); amino acids 368-407 of SEQ ID NO:20 (SEQ ID NO:92); amino acids 411-450 of
  • the present invention is based, at least in part, on the discovery of cDNA molecules encoding INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, and TANGO 378, all of which are either wholly secreted or transmembrane proteins.
  • the proteins and nucleic acid molecules of the present invention comprise a family 5 of molecules having certain conserved structural and functional features.
  • family is intended to mean two or more proteins or nucleic acid molecules having a common structural domain and having sufficient amino acid or nucleotide sequence identity as defined herein.
  • Family members can be from either the same or different species.
  • a family can comprise two or more proteins of human origin, or can comprise one or more proteins of human origin and one or more of non- human origin. Members of the same family may also have common structural domains.
  • INTERCEPT 340 family members can include at least one, preferably two, and more preferably three fibrillar collagen C-terminal domains (also referred to herein as "COLF domains").
  • a "fibrillar collagen C-terminal domain” refers to an amino acid sequence of about 15 to 65, preferably about 20-60, more preferably about 25, 31-58 amino acids in length.
  • Consensus hidden Markov model COLF domains contain the sequence of SEQ ID NOs:31, 32, and 33 ( Figure 3). The more conserved residues in the consensus sequence are indicated by uppercase letters and the less conserved residues in the consensus sequence are indicated by lowercase letters.
  • a comparison of the C-terminal sequences of fibrillar collagens, collagens X, NIII, and the collagen Clq revealed a conserved cluster of amino acid residues having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) that exhibited marked similarities in hydrophilicity profiles between the different collagens, despite a low level of sequence similarity.
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the COLF domains of INTERCEPT 340 extend from about amino acids 58 to 116, 126 to 151, and 186 to 217 of SEQ ID NO:2 (SEQ ID NOs:34, 35, and 36, respectively) ( Figure 3).
  • conserved amino acid residues having aromatic side chains can be found. For example, conserved tyrosine, tryptophan and phenylalanine residues can be found at amino acid 87, 88 and 133 of SEQ
  • MANGO 003 and TANGO 354 family members can include at least one, preferably two, and more preferably three immunoglobulin domains.
  • an immunoglobulin domain As used herein, an
  • immunoglobulin domain refers to an amino acid sequence of about 45 to 85, preferably about 55-80, more preferably about 57, 58, or 78, 79 amino acids in length.
  • the immunoglobulin domains have a bit score for the alignment of the sequence to the Ig family Hidden Markov Model (HMM) of at least 10, preferably
  • the Ig family HMM has been assigned the PFAM Accession PF00047. Consensus hidden
  • Immunoglobulin domains are present in a variety of proteins (including secreted and membrane-associated proteins). Membrane-associated proteins may be involved in protein-protein, and protein-ligand interaction at the cell surface, and thus may influence diverse activities including cell surface recognition and or signal transduction.
  • the immunoglobulin domains of MANGO 003 extend from about amino acids 44 to 101, 165 to 223, and 261 to 240 of SEQ ED NO:5 (SEQ ID NOs:38, 39, and 40, respectively) ( Figure 6).
  • the immunoglobulin domain of TANGO 354 extend from about amino acids 33 to 110 of SEQ ID NO:26 (SEQ ED NO:41) ( Figure 23).
  • MANGO 003 family member can include a neurotransmitter-gated ion channel domain.
  • a "neurotransmitter-gated ion channel domain” refers to an amino acid sequence of about 5 to 20, preferably about 7 to 12, more preferably about 9 to 10 amino acids in length.
  • the neurotransmitter-gated ion channel domain HMM has been assigned the PFAM Accession PF00065.
  • a consensus hidden Markov model neurotransmitter-gated ion chaimel domain contain the sequence of SEQ ED NO:42 shown in Figure 7. The more conserved residues in the consensus sequence are indicated by uppercase letters and the less conserved residues in the consensus sequence are indicated by lowercase letters.
  • the neurotransmitter-gated ion channel domains of MANGO 003 extend from about amino acids 388 to 397 of SEQ ID NO:5 (SEQ ID NO:43).
  • TANGO 272 family members can include at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, preferably thirteen, and more preferably fourteen
  • EGF-like domains Preferably, the EGF-like domains are found in the extracellular domain of a TANGO 272 protein.
  • an "EGF-like domain” refers to an amino acid sequence of about 25 to 50, preferably about 30 to 45, and more preferably 30 to 40 amino acid residues in length.
  • An EGF domain further contains at least about 2 to 10, preferably,
  • EGF-like domain sequence includes six cysteines, all of which are thought to be involved in disulfide bonds having the following amino acid sequence: Cys-Xaa(5, 7)-Cys-Xaa(4, 5,
  • Xaa is any amino acid.
  • the region between the fifth and the sixth cysteine typically contains two conserved glycines of which at least one is present in most EGF-like domains.
  • TANGO 272 includes at least one EGF-like domain having the sequences selected from the group consisting of: amino acids 151-181 of SEQ ID NO: 14
  • SEQ ID NO:14 SEQ ID NO:51
  • amino acids 285-315 of SEQ ID NO:14 SEQ ID NO:52
  • amino acids 328-358 of SEQ ID NO.T4 SEQ ID NO:53
  • amino acids 378-404 of SEQ ID NO: 14 SEQ ID NO:54
  • amino acids 417-447 of SEQ ID NO: 14 SEQ ID NO:55
  • amino acids 460-490 of SEQ ID NO:14 SEQ ED.NO:56
  • amino acids 503-533 of SEQ ID NO: 14 SEQ ID NO:57
  • amino acids 546-576 of SEQ ID NO: 14 SEQ ID NO:58
  • amino acids 589-619 of SEQ ID NO:14 SEQ ID NO:59
  • amino acids 632-661 of SEQ ID NO: 14 SEQ ID NO:60
  • amino acids 674-704 of SEQ ID NO: 14 SEQ ID NO:61
  • amino acids 717-747 of SEQ ED NO: 14 SEQ ID NO:62
  • TANGO 272 includes at least one EGF-like domain having the sequences selected from the group consisting of: 37-67 of SEQ ID NO: 17 (SEQ ID NO:64); amino acids 80-110 of SEQ ID NO:17 (SEQ ID NO:65); amino acids 123-153 of SEQ ID NO: 17 (SEQ ID NO:66); and amino acids 166-196 of SEQ ED NO: 17 (SEQ ID NO:67).
  • TANGO 272 includes at least one EGF-like domain having the sequences selected from the group consisting of: amino acids 18-48 of SEQ ID NO:20 (SEQ ID NO:73); amino acids 61-91 of SEQ ID NO:20 (SEQ ID NO:74); amino acids 105-137 of SEQ ID NO:20 (SEQ ID NO:75); amino acids 150-180 of SEQ ID NO.20 (SEQ ID NO:76); amino acids 193-223 of SEQ ID NO:20 (SEQ ID NO:77); amino acids 15 236-266 of SEQ ID NO:20 (SEQ ID NO:78); amino acids 279-309 of SEQ ID NO:20 (SEQ ID NO:79); amino acids 322-352 of SEQ ID NO:20 (SEQ ID NO:80); amino acids 365-394 of SEQ ID NO:20 (SEQ ID NO:81); amino acids 407-437 of SEQ ID NO:20 (SEQ ID NO:82); and amino acids 450-480 of SEQ ID NO:20 (SEQ ID NO:73
  • cysteine residues can be found. For example, conserved cysteine residues can be found at amino acid 151, 159, 164, 167, 200, 206, 211, 218, 220, 229, 242, 249, 263, 264, 272, 285,
  • TANGO 272 family members can include at least one delta serrate ligand domain.
  • a “delta serrate ligand domain” (also referred to herein as a “DSL domain”) refers to an amino acid sequence of about 30-70, more preferably 45-60, and most preferably 58 amino acids in length typically found in transmembrane signaling molecules
  • human TANGO 272 includes a delta serrate ligand domain from about amino acids 518 to 576 of SEQ ID NO: 14 (SEQ ID NO:63); and about amino acids 246 to 309 of SEQ ID NO:20 (SEQ ID NO:95).
  • Figure 15B depicts an alignment of the consensus hidden Markov model delta serrate ligand domain (SEQ ID NO:47) with this domain in human TANGO 272 at amino acids 518 to 576 of SEQ ED NO: 14 (SEQ ED NO:63).
  • Figures 39A-39B depict an alignment of the consensus hidden Markov model delta serrate ligand domain (SEQ ID NO:47) with this domain in mouse TANGO 272 at amino acids 10 to 67 of SEQ ID NO:17 (SEQ ID NO:72).
  • Figures 41A- 41B depict an alignment of the consensus hidden Markov model delta serrate ligand domain (SEQ ID NO:47) with this domain in rat TANGO 272 at amino acids 246 to 309 of SEQ ID
  • TANGO 272 family members can include at least one RGD cell attachment site.
  • RGD cell attachment site refers to a cell adhesion sequence consisting of amino acids Arg-Gly-Asp typically found in extracellular matrix proteins such as collagens, laminin and fibronectin, among others (reviewed in Ruoslahti, 1996, Annu.
  • the RGD cell attachment site is located in the extracellular domain of a TANGO 272 protein and interacts (e.g. , binds to) a cell surface receptor, such as an integrin receptor.
  • a cell surface receptor such as an integrin receptor.
  • integrin refers to a family of receptors comprising ⁇ / ⁇ heterodimers that mediate cell attachment to extracellular matrices and cell-cell adhesion events.
  • the subunits vary in size between 120 and 180 kDa and are each noncovalently associated with a ⁇ subunit (90-110 kDa) (reviewed by
  • integrins are expressed in a wide variety of cells, and most cells express several integrins. There are at least 8 known subunits and 14 known ⁇ subunits. The majority of the integrin ligands are extracellular matrix proteins involved in substratum cell adhesion such as collagens, laminin, fibronectin among others.
  • the RGD cell attachment site is located at about amino acid residues 177-179 of SEQ ID NO: 14.
  • MANGO 347 family members can include a CUB domain sequence.
  • CUB domain includes an amino acid sequence having at least about 80-
  • a CUB domain further includes at least one, preferably two, three, and most preferably four conserved cysteine residues.
  • the conserved cysteine residues form at least one, and preferably two disulfide bridges (e.g., Cysl-Cys2, and Cys3-
  • the CUB domain of MANGO 347 extends from about amino acid 40 to amino acid 136 of SEQ ID NO.T 1 (SEQ ID NO:45).
  • Figure 12 depicts an alignment of the consensus hidden Markov model CUB domain (SEQ ID NO:44) with this domain in human MANGO 347 at amino acids 40 to 136 of SEQ ED NO: 11 (SEQ ID NO:45).
  • TANGO 295 family members can include a pancreatic ribonuclease domain sequence.
  • pancreatic ribonuclease domain includes an amino acid sequence having at least about 100 to 150, preferably 110-140, more preferably 120- 130, and most preferably 124 amino acids in length.
  • a pancreatic ribonuclease domain further includes at least one, preferably two, three, four and most preferably five conserved cysteine residues and an amino acid residue, e.g., a lysine, which is involved in catalytic activity.
  • at least one cysteine residue is involved in a disulfide bond, a lysine residue is involved in catalytic activity, and three other residues involved in substrate binding.
  • Proteins having the pancreatic ribonuclease domain are pyrimidine-specific endonucleases present in high quantities in the pancreas of a number of mammalian taxa and of a few reptiles.
  • the pancreatic ribonuclease domain of TANGO 295 extends from about amino acid 32 to amino acid 156 of SEQ ID NO:23 (SEQ ID NO:97).
  • Figure 20 depicts an alignment of the consensus hidden Markov model pancreatic ribonuclease domain (SEQ ID NO:96) with this domain in human TANGO 295 at amino acids 32 to 156 of SEQ ID NO:23 (SEQ ID NO:97).
  • G protein-coupled receptor refers to a family of proteins that preferably comprise an N-terminal extracellular domain, seven transmembrane domains (also referred to as membrane-spanning domains), three extracellular domains (also referred to as extracellular
  • cytoplasmic loops also referred to as cytoplasmic loops
  • C-terminal cytoplasmic domain also referred to as a cytoplasmic tail
  • TANGO 378 family members can include at least one, two, three, four, five, six, or preferably, seven transmembrane domains, and thus has a
  • 7 transmembrane receptor profile includes an amino acid sequence having at least about 10-300, preferably about 15-
  • HMM Hidden Markov Model
  • the seven transmembrane domains of TANGO 378 extend from about amino acids 245 to about amino acid 269 of SEQ ID NO:29 (SEQ ID NO: 135), about amino acids 287 to about amino acid 306 of SEQ ED NO:29 (SEQ ID NO: 136), about amino acids 323 to about amino acid 343 of SEQ ID NO:29 (SEQ ID NO: 137), about amino acids 358 to about amino acid 376 of SEQ ID NO:29 (SEQ ID NO.138), about amino acids 414 to about amino acid 438 of SEQ ID NO:29 (SEQ ID NOT39), about amino acids 457 to about amino acid 477 of SEQ ID NO:29 (SEQ ED NO: 140), and about amino acids 485 to about amino acid 504 of SEQ ID NO:29 (SEQ ID NO: 141); and a C-terminal cytoplasmic domain which extends from
  • the amino acid sequence of the protein is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters
  • the hmmsf program which is available as part of the HMMER package of search programs, is a family specific default program for PF00001 and score of 15 is the default threshold score for determining a hit.
  • the seven transmembrane domain can be predicted based on stretches of hydrophobic amino acids forming ⁇ -helices (SOUSI server). Accordingly, proteins having at least 50-60%> identity, preferably about 60-70%, more preferably about 10-80%, or about 80-90% identity with the 7 transmembrane receptor profile of human TANGO 378 are within the scope of the invention.
  • TANGO 378 family members can include at least one, preferably two, and most preferably three extracellular loops.
  • the term "loop” includes an amino acid sequence having a length of at least about 4, preferably about 5-10, preferably about 10-20, and more preferably about 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, or 100-150 amino acid residues, and has an amino acid sequence that connects two transmembrane domains within a protein or polypeptide.
  • the N-terminal amino acid of a loop is adjacent to a C-terminal amino acid of a transmembrane domain in a naturally-occurring TANGO 378 or TANGO 378-like molecule
  • the C-terminal amino acid of a loop is adjacent to an N-terminal amino acid of a transmembrane domain in a naturally-occurring TANGO 378 or TANGO 378-like molecule.
  • an "extracellular loop" includes an amino acid sequence located outside of a cell, or extracellularly. For example, an extracellular loop can be found at about amino acids 307- 322, 377-413, and 478-484 of SEQ ID NO:29.
  • TANGO 378 family members can include at least one, preferably two, and most preferably three cytoplasmic loops.
  • a "cytoplasmic loop” includes an amino acid sequence located within a cell or within the cytoplasm of a cell.
  • a cytoplasmic loop is found at about amino acids 270-286, 344-357, and 439-456 of SEQ ID NO:29.
  • a MANGO 003, a TANGO 272, a TANGO 354 or a TANGO 378 family member can include one or more of the following domains: (1) an N-terminal extracellular domain, (2) a transmembrane domain, or (3) a C-terminal cytoplasmic domain.
  • MANGO 003, a TANGO 272, a TANGO 354 or a TANGO 378 family member can include an extracellular domain.
  • the extracellular domain When located at the N-terminal domain the extracellular domain is referred to herein as an "N-terminal extracellular domain” or an "extracellular domain".
  • an "N-terminal extracellular domain” includes an amino acid sequence having about 1-800, preferably about 1-746, more preferably about 1-650, more preferably about 1-550, more preferably about 1-369, about 150 amino acid residues in length and is located outside of a cell or extracellularly.
  • the C-terminal amino acid residue of a "N-terminal extracellular domain” is adjacent to an N-terminal amino acid residue of a transmembrane domain in a naturally-occurring MANGO 003, TANGO 272, TANGO 354 or TANGO 378 protein.
  • the N-terminal extracellular domain is capable of interacting (e.g., binding to) with an extracellular signal, for example, a ligand (e.g., a glycoprotein hormone) or a cell surface receptor (e.g., an integrin receptor).
  • an N-terminal extracellular domain mediates a variety of biological processes, for example, protein-protein interactions, signal transduction and/or cell adhesion.
  • an N-terminal cytoplasmic domain is located at about amino acids 25-374 of
  • SEQ ID NO:5 SEQ ID NO: 103
  • amino acids 1-73 of SEQ ID NO:8 SEQ ID NO:
  • SEQ ID NO:20 (SEQ ID NO: 122); at about amino acids 20-169 of SEQ ID NO:26 (SEQ ID NO.T29); and at about amino acids 22-244 of SEQ ID NO:29 (SEQ ID NO.T34).
  • TANGO 378 family member can include a transmembrane domain.
  • transmembrane domain includes an amino acid sequence of about 15 amino acid residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 20, 25, 30, 35, 40, or 45 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an -helical structure. In a preferred embodiment, at least 50%o, 60%, 70%>,
  • transmembrane domains are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans.
  • a MANGO 003, TANGO 272, TANGO 354 or TANGO 378 family member can include a C-terminal cytoplasmic domain.
  • a "C-terminal cytoplasmic domain" includes an amino acid sequence having a length of at least about 10, preferably about 10-25, more preferably about 25-50, more preferably about 50-75, even more preferably about 75-100, 100-133, 133-150, 150-200, 200-250, 250-300, 300-400, 400-500, or 500-600 amino acid residues and is located within a cell or within the cytoplasm of a cell.
  • the N-terminal amino acid residue of a "C-terminal cytoplasmic domain" is adjacent to a C-terminal amino acid residue of a transmembrane domain in a naturally- occurring MANGO 003, TANGO 272, TANGO 354 or TANGO 378 protein.
  • a C-terminal cytoplasmic domain is found at about amino acid residues 399-504 of SEQ ID NO:5, 97-208 of SEQ ID NO:8, 792-1050 of SEQ ID NOT4, 241-497 of SEQ ID NO:17, 525-636 of SEQ ID NO:20; 194-305 of SEQ ID NO:26, and 505-528 of SEQ ID NO:29.
  • a "signal peptide” includes a peptide of at least about 15 amino acid residues in length which occurs at the N- terminus of secretory and membrane-bound proteins and which contains at least about 70% hydrophobic amino acid residues such as alanine, leucine, isoleucine, phenylalanine, proline, tyrosine, tryptophan, or valine.
  • the sequence can contain about 15 to 45 amino acid residues or about 17-22 amino acid residues, and has at least about 60-80%, 65-75%, or about 70% hydrophobic residues.
  • a signal peptide serves to direct a protein containing such a sequence to a lipid bilayer.
  • a MANGO 003 protein contains a signal peptide of about amino acids 1-22, 1-23, 1-24, 1-25, or 1-26 of SEQ ID
  • a MANGO 347 protein contains a signal peptide of about amino acids 1-33, 1-34, 1-35, 1-36, or 1-37 of SEQ ID NO:l l (SEQ ID NO:
  • a TANGO 272 protein contains a signal peptide of amino acids 1-18, 1-19, 1-20, 1-21, or 1-22 of SEQ ID NO: 14 (SEQ ID NOT 12).
  • a TANGO 295 protein contains a signal peptide of amino acids 1-26, 1-27, 1-
  • a TANGO 378 protein contains a signal peptide of amino acids 1-17, 1-18, 1-19, 1-20, or 1-21 of SEQ ID NO:26 (SEQ ID NO: 127).
  • a TANGO 378 protein contains a signal peptide of amino acids 1-19, 1-20, 1-21, 1-22, or 1-23 of SEQ ID NO:29 (SEQ ID NO: 132).
  • the signal peptide is cleaved during processing of the mature protein.
  • the amino acid sequence of the mature MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 protein starts at the next amino acid after the signal peptide is cleaved.
  • the amino acid sequence of MANGO 003 may start at amino acids 23, 24, 25, 26, or 27 depending on the exact location of the cleavage of the signal peptide.
  • a MANGO 003 protein does not contain a signal peptide or an initial methionine residue and begins from residue 2 of SEQ ID NO: 102.
  • a MANGO 347 protein does not contain a signal peptide or an initial methionine residue and begins from residue 2 of SEQ ID NO 11.
  • a TANGO 272 protein does not contain a signal peptide or an initial methionine residue and begins from residue 2 of SEQ ID NOT 13.
  • a TANGO 295 protein does not contain a signal peptide or an initial methionine residue an begins from residue 2 of SEQ ID NOT26.
  • a TANGO 354 protein does not contain a signal peptide or an initial methionine residue an begins from residue 2 of SEQ ED NO: 128.
  • a TANGO 378 protein does not contain a signal peptide or an initial methionine residue an begins from residue 2 of SEQ ED NO: 133.
  • a MANGO 003 family member includes three immunoglobulin domains and a neurotransmitter-gated ion channel domain. In another embodiment, a
  • MANGO 003 family member includes three immunoglobulin domains, a neurotransmitter- gated ion channel domain and a transmembrane domain.
  • a MANGO 003 family member includes three immunoglobulin domains, a neurotransmitter- gated ion channel domain, a transmembrane domain and an N-terminal extracellular domain.
  • a MANGO 003 family member includes three immunoglobulin domains, a neurotransmitter-gated ion channel domain, a transmembrane domain, an N-terminal extracellular domain and a C-terminal cytoplasmic domain.
  • a MANGO 003 family member includes three immunoglobulin domains, a neurotransmitter-gated ion channel domain, a transmembrane domain, an N- terminal extracellular domain, a C-terminal cytoplasmic domain, and a signal peptide.
  • a MANGO 354 family member includes at least one immunoglobulin domain and a transmembrane domain. In another embodiment, a
  • MANGO 354 family member includes at least one immunoglobulin domain, a transmembrane domain and a signal peptide.
  • a TANGO 272 family member includes fourteen EGF-like domains and a delta serrate ligand domain. In another embodiment, a TANGO 272 family member includes fourteen EGF-like domains, a delta serrate ligand domain and an RGD cell attachment site. In yet another embodiment, a TANGO 272 family member includes fourteen EGF-like domains, a delta serrate ligand domain, an RGD cell attachment site, and a transmembrane domain. In another embodiment, a TANGO 272 family member includes fourteen EGF-like domains, a delta serrate ligand domain, an RGD cell attachment site, a transmembrane domain, and an extracellular N-terminal domain.
  • a TANGO 272 family member includes fourteen EGF-like domains, a delta serrate ligand domain, an RGD cell attachment site, a transmembrane domain, an extracellular N-terminal domain and a C-terminal cytoplasmic domain.
  • a TANGO 272 family member includes fourteen EGF-like domains, a delta serrate ligand domain, an RGD cell attachment site, a transmembrane domain, an extracellular N-terminal domain, a C- terminal cytoplasmic domain, and a signal peptide.
  • a TANGO 378 family member includes a 7 transmembrane receptor profile and three extracellular loops. In another embodiment, a TANGO 378 family member includes a 7 transmembrane receptor profile, three extracellular loops, and three cytoplasmic loops. In yet another embodiment, a TANGO 378 family member includes a 7 transmembrane receptor profile, three extracellular loops, three cytoplasmic loops, and an extracellular N-terminal domain. In another embodiment, a TANGO 378 family member includes a 7 transmembrane receptor profile, three extracellular loops, three cytoplasmic loops, an extracellular N-terminal domain, and a C-terminal cytoplasmic domain.
  • a TANGO 378 family member includes a 7 transmembrane receptor profile, three extracellular loops, three cytoplasmic loops, an extracellular N-terminal domain, a C-terminal cytoplasmic domain, and a signal peptide.
  • a cDNA encoding INTERCEPT 340 was identified by analyzing the sequences of clones present in a human fetal spleen cDNA library.
  • Human INTERCEPT 340 that has not been post-translationally modified is predicted to have a molecular weight of 27.2 kDa.
  • Human INTERCEPT 340 includes three fibrillar collagen C-terminal (COLF) domains at amino acids 58-116 of SEQ ID NO:2 (SEQ ID NO:34); amino acids 126-151 of SEQ ID NO:2 (SEQ ID NO:35); and amino acids 186-217 of SEQ ID NO:2 (SEQ ID NO: 36).
  • Figure 3 depicts alignments of each of the COLF domains of human INTERCEPT 340 with consensus hidden Markov model COLF domains (SEQ ID NOs:31, 32, and 33).
  • INTERCEPT 340 is a secreted protein
  • INTERCEPT 340 is a membrane-associated protein.
  • N-glycosylation site is present at amino acids 105-108 of SEQ ID NO:2.
  • a glycosaminoaglycan attachment site is present at amino acids 161-164 of SEQ ID NO:2.
  • Protein kinase C phosphorylation sites are present at amino acids 57-59, 152-154, and 227-
  • a tyrosine kinase phosphorylation site is present at amino acids 81- 87 of SEQ ID NO:2.
  • Casein kinase II phosphorylation sites are present at amino acids 36- 39, 120-123 and 181-184.
  • N-myristylation sites are present at amino acids 109-114 and 164-169 of SEQ ID NO:2.
  • Figure 2 depicts a hydropathy plot of human INTERCEPT 340. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are 5 below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace.
  • INTERCEPT 340 includes three fibrillar collagen C-terminal domains. Proteins
  • INTERCEPT 340 includes fibrillar collagen C-terminal domains
  • INTERCEPT 340 polypeptides, nucleic acids, and modulators thereof can be used to treat connective tissue disorders, including a skin disorder and or a skeletal disorder (e.g., Marfan syndrome and osteogenesis imperfecta); cardiovascular disorders including hyperproliferative vascular diseases (e.g., hypertension, vascular restenosis and atherosclerosis), ischemia reperfusion injury, cardiac hypertrophy, coronary artery disease, myocardial infarction, arrhythmia, cardiomyopathies, and congestive heart failure); and/or hematopoietic disorders (e.g., myeloid disorders, lymphoid malignancies, T cell disorders).
  • a skin disorder and or a skeletal disorder e.g., Marfan syndrome and osteogenesis imperfecta
  • cardiovascular disorders including hyperproliferative vascular diseases (e.g., hypertension, vascular restenosis and atherosclerosis), ischemia reperfusion injury, cardiac hypertrophy, coronary artery disease, myocardial infarction, arrhythm
  • 340 nucleic acids, proteins, and modulators thereof can be used to modulate the function, survival, morphology, migration, proliferation and/or differentiation of cells that form the spleen, e.g., cells of the splenic connective tissue, e.g., splenic smooth muscle cells and/or endothehal cells of the splenic blood vessels.
  • INTERCEPT 340 nucleic acids, proteins, and modulators thereof can also be used to modulate the proliferation, differentiation, and/or function of cells that are processed, e.g., regenerated or phagocytized within the spleen, e.g., erythrocytes and/or B and T lymphocytes and macrophages.
  • INTERCEPT 340 nucleic acids, proteins, and modulators thereof can be used to treat spleen, e.g., the fetal spleen, associated diseases and disorders.
  • splenic diseases and disorders include e.g., splenic lymphoma and/or splenomegaly, and/or phagocytotic disorders, e.g., those inhibiting macrophage engulfment of bacteria and viruses in the bloodstream.
  • INTERCEPT 340 expression can be utilized as a marker for specific tissues (e.g., lymphoid tissues such as the spleen) and/or cells (e.g., splenic) in which INTERCEPT 340 is expressed.
  • INTERCEPT 340 nucleic acids can also be utilized for chromosomal mapping.
  • a cDNA encoding human MANGO 003 was identified by analyzing the sequences of clones present in a human thyroid cDNA library.
  • Human MANGO 003 that has not been post-translationally modified is predicted to have a molecular weight of 54.5 kDa prior to cleavage of its signal peptide (52.1 kDa after cleavage of its signal peptide).
  • the signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human MANGO 003 includes a 24 amino acid signal peptide at amino acid 1 to about amino acid 24 of SEQ ID NO:5 (SEQ ED NO: 101) preceding the mature human MANGO 003 protein which corresponds to about amino acid 25 to amino acid 504 of SEQ ID NO:5 (SEQ ID NO: 102).
  • Human MANGO 003 is a transmembrane protein having an extracellular domain which extends from about amino acid 25 to about amino acid 374 of SEQ ED NO:5 (SEQ ED NO: 103), a transmembrane domain which extends from about amino acid 375 to about amino acid 398 of SEQ ID NO:5 (SEQ ID NO: 104), and a cytoplasmic domain which extends from about amino acid 399 to amino acid 504 of SEQ ID NO:5 (SEQ ID NO: 105).
  • a human MANGO 003 protein contains an extracellular domain which extends from about amino acid 399 to amino acid 504 of SEQ ID NO: 5 (SEQ ID NO: 105), a transmembrane domain which extends from about amino acid 375 to about amino acid 398 of SEQ ED NO:5 (SEQ ID NO: 104), and a cytoplasmic domain which extends from about amino acid 25 to about amino acid 374 of SEQ ID NO:5 15 (SEQ ID NO: 103).
  • Human MANGO 003 includes three immunoglobulin domains at amino acids 44- 101 of SEQ ID NO:5 (SEQ ID NO:38); amino acids 165-223 of SEQ ID NO:5 (SEQ ID NO:39); and amino acids 261-340 of SEQ ID NO:5 (SEQ ID NO:40).
  • Figure 6 depicts alignments of each of the immunoglobulin domains of MANGO 003 with a consensus
  • Human MANGO 003 includes a neurotransmitter gated ion channel domain at amino acids 388-397 of SEQ ID NO:5 (SEQ ID NO:43).
  • Figure 7 depicts an alignment of the neurotransmitter gated ion channel domain of human MANGO 003 with a neurotransmitter gated ion channel domain derived from a hidden Markov model (SEQ ID NO:43).
  • N-glycosylation sites are present at amino acids 111-114, 231-234, 255-258, and 293-296 of SEQ ID NO:5.
  • a cAMP and cGMP-dependent protein kinase phosphorylation site is present at amino acids 202-205 of SEQ ID NO:5.
  • Protein kinase C phosphorylation sites are present at amino acids 44-48, 167-169, 207-209, 216-218, 220-222, 224-226, 233-
  • Casein kinase II phosphorylation sites are present at amino acids 192-195, 256-259, 294-297, 313-316, 422-425, and 490-493 of SEQ D NO:5.
  • Tyrosine kinase phosphorylation sites are present at amino acids 212-219 and 329-336 of SEQ ID NO:5.
  • N-myristylation sites are present at amino acids 95-100, 228-
  • 35 Clone jthYa030d03 which encodes human MANGO 003, was deposited as a composite deposit having a designation EpthLa6al with the American Type Culture Collection (ATCC ® 10801 University Boulevard, Manassas, NA 20110-2209) on March 27, 1999 and assigned Accession Number 207178.
  • This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. ⁇ 112.
  • Figure 5 depicts a hydropathy plot of human MANGO 003. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The hydropathy plot of Figure 5 indicates the presence of a hydrophobic domain within human MANGO 003, suggesting that human MANGO 003 is a transmembrane protein.
  • a cDNA encoding mouse MANGO 003 was identified by analyzing the sequences of clones present in a mouse choroid plexus cDNA library.
  • Mouse MANGO 003 that has not been post-translationally modified is predicted to have a molecular weight of 22.3 kDa.
  • Mouse MANGO 003 is a transmembrane protein having an extracellular domain which extends from about amino acid 1 to about amino acid 73 of SEQ ID NO: 8 (SEQ ID NO: 107), a transmembrane domain which extends from about amino acid 74 to about amino acid 96 of SEQ ED NO:8 (SEQ ID NO.T 08), and a cytoplasmic domain which extends from about amino acid 97 to amino acid 208 of SEQ ID NO:8 (SEQ ID NOT09).
  • N-glycosylation site is present at amino acids 190-193 of SEQ ID NO:8.
  • Protein kinase C phosphorylation sites are present at amino acids 44-46, 98-100, 119-121, and 197- 199 of SEQ ED NO: 8.
  • Casein kinase II phosphorylation sites are present at amino acids 10- 13, and 119-122 of SEQ ED NO: 8.
  • a tyrosine kinase phosphorylation site is present at amino acids 26-33 of SEQ ID NO:8.
  • N-myristylation sites are present at amino acids 14- 19, 31-36, and 79-84 of SEQ ID NO: 8.
  • Figure 9 depicts a hydropathy plot of mouse MANGO 003.
  • the global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of -1212; Myers and Miller, 1989 CABIOS 4:11-1).
  • the local alignment was performed using the L-ALIGN program version 2.0u54 July 1996 (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a score of 3241; Huang and Miller, 1991, Adv. Appl. Math. 12:373-81).
  • the global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of -488; Myers and Miller, 1989, CABIOS 4:11-1).
  • MANGO 003 includes three immunoglobulin-like domains. Proteins having such domains play a role in mediating protein-protein and protein-ligand interactions, and thus can influence a wide variety of biological processes, including cell surface recognition; transduction of an extracellular signal (e.g., by interacting with a ligand and/or a cell- surface receptor); and/or modulation of signal transduction pathways.
  • MANGO 003 further includes a neurotransmitter-gated ion channel domain.
  • Proteins having such domains play a role in modulating signal transmission at chemical synapses by, for example, influencing processes, such as the release of neurotransmitters from a cell (e.g., a neuronal cell); modulating membrane excitability and/or resting potential; and/or modulating ion flux across a membrane of a cell (e.g., a neuronal or a muscle cell).
  • a cell e.g., a neuronal cell
  • modulating membrane excitability and/or resting potential e.g., a neuronal or a muscle cell
  • MANGO 003 includes a neurotransmitter-gated ion channel domain
  • MANGO 003 polypeptides, nucleic acids, and modulators thereof can be used to treat neural disorders (e.g., a CNS disorder, including Alzheimer's disease, Pick's disease, Parkinson's and other Lewy diffuse body diseases, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease; psychiatric disorders, e.g., depression, schizophrenic disorders, Korsakoffs psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss; and neurological disorders, e.g., migraine).
  • a CNS disorder including Alzheimer's disease, Pick's disease, Parkinson's and other Lewy diffuse body diseases, multiple sclerosis, amyotrophic lateral sclerosis, progressive supra
  • MANGO 003 polypeptides, nucleic acids, and modulators thereof can be used to modulate function, survival, morphology, migration, proliferation and/or differentiation of cells in the tissues in which it is expressed (e.g. thyroid, liver, skeletal muscle, kidney, heart, lung, testis and brain).
  • MANGO 003 polypeptides, nucleic acids, and modulators thereof can be used to modulate endocrine, hepatic, skeletal muscular, renal, cardiac, reproductive and/or brain function.
  • these molecules can be used to treat a variety of disease including, but not limited to, endocrine disorders (e.g., hypothyroidism, hyperthyroidism, dwarfism, giantism, acromegaly); hepatic disorders (e.g., hepatitis, liver cirrhosis, hepatoma, liver cysts, and hepatic vein thrombosis); skeletal muscular disorders; renal disorders (e.g., renal cell carcinoma, nephritis, polycystic kidney disease); cardiovascular disorders (e.g., atherosclerosis, ischemia reperfusion injury, cardiac hypertrophy, hypertension, coronary artery disease, myocardial infarction, arrhythmia, cardiomyopathies, and congestive heart failure); and/or reproductive disorders (e.g., sterility).
  • endocrine disorders e.g., hypothyroidism, hyperthyroidism, dwarfism, giantism, acromegaly
  • MANGO 003 polypeptides, nucleic acids, or modulators thereof can be used to treat hepatic (liver) disorders, such as jaundice, hepatic failure, hereditary hyperbiliruinemias (e.g., Gilbert's syndrome, Crigler-Naijar syndromes and Dubin- Johnson and Rotor's syndromes), hepatic circulatory disorders (e.g., hepatic vein thrombosis and portal vein obstruction and thrombosis) hepatitis (e.g., chronic active hepatitis, acute viral hepatitis, and toxic and drug-induced hepatitis) cirrhosis (e.g., alcoholic cirrhosis, biliary cirrhosis, and hemochromatosis), or malignant tumors (e.g., primary carcinoma, hepatoblastoma, and angiosarcoma).
  • hepatic (liver) disorders such as jaundice, hepatic failure,
  • MANGO 003 polypeptides, nucleic acids, or modulators thereof can be used to treat disorders of skeletal muscle, such as muscular dystrophy (e.g., Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, Emery-Dreifuss Muscular Dystrophy, Limb-Girdle Muscular Dystrophy, Facioscapulohumeral Muscular Dystrophy, Myotonic Dystrophy, Oculopharyngeal Muscular Dystrophy, Distal Muscular Dystrophy, and Congenital Muscular Dystrophy), motor neuron diseases (e.g., Amyotrophic Lateral Sclerosis, Infantile Progressive Spinal Muscular Atrophy, Intermediate Spinal Muscular Atrophy, Spinal Bulbar Muscular Atrophy, and Adult Spinal Muscular Atrophy), myopathies (e.g., inflammatory myopathies (e.g., Dermatomyositis and Polymyositis), Myotonia Congenita, Para
  • MANGO 003 polypeptides, nucleic acids, or modulators thereof can be used to treat renal disorders, such as glomerular diseases (e.g., acute and chronic glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, focal proliferative glomerulonephritis, glomerular lesions associated with systemic disease, such as systemic lupus erythematosus, Goodpasture's syndrome, multiple myeloma, diabetes, neoplasia, sickle cell disease, and chronic inflammatory diseases), tubular diseases (e.g., acute tubular necrosis and acute renal failure, polycystic renal diseasemedullary sponge kidney, medullary cystic disease, nephrogenic diabetes, and renal tubular acidosis), tubulointerstitial diseases (e.g., pyelonephritis, drug and toxin induced tubulointerstitial nephritis,
  • MANGO 003 expression can be utilized as a marker for specific tissues (e.g., liver, skeletal muscle, kidney) and/or cells (e.g., hepatic, skeletal muscle, renal) in which MANGO 003 is expressed.
  • MANGO 003 nucleic acids can also be utilized for chromosomal mapping.
  • a cDNA encoding human MANGO 347 was identified by analyzing the sequences of clones present in a human brain cDNA library.
  • the signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human MANGO 347 includes a 35 amino acid signal peptide at amino acid 1 to about amino acid 35 of SEQ ED NOT 1 (SEQ ID NOT10) preceding the mature human MANGO 347 protein which corresponds to about amino acid 36 to amino acid 138 of SEQ ID NOT1 (SEQ ED NOT11).
  • Human MANGO 347 that has not been post-translationally modified is predicted to have a molecular weight of 15.4 kDa prior to cleavage of its signal peptide and a molecular weight of 11.3 kDa subsequent to cleavage of its signal peptide.
  • Human MANGO 347 includes a CUB domain at amino acids 40-136 of SEQ ID NOT 1 (SEQ ID NO:45).
  • SEQ ID NO:45 An alignment of the CUB domain of human MANGO 347 with a consensus hidden Markov model CUB domain amino acid sequence derived from a hidden Markov model (SEQ ID NO:44) is shown in Figure 12.
  • Casein kinase II phosphorylation sites are present at amino acids 67-70, and 108-111
  • N-myristylation sites are present at amino acids 19-24, 31-36, 64-69, and 113-118 of SEQ ID NOT 1.
  • Clone jlhbad295gl2 which encodes human MANGO 347, was deposited as a composite deposit having a designation EpM347 with the American Type Culture Collection (ATCC ® 10801 University Boulevard, Manassas, VA 20110-2209) on June 18, .
  • Figure 11 depicts a hydropathy plot of human MANGO 347. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The hydropathy plot of Figure 11 indicates that human MANGO 347 has a signal peptide at its amino terminus, suggesting that human MANGO
  • 30 347 is a secreted protein.
  • MANGO 347 includes a CUB domain. Proteins having such a domain play a role in mediating cell interactions during development, and thus can influence a wide variety of
  • MANGO 347 polypeptides are expressed in neural (e.g., brain cells). Because MANGO 347 includes a CUB domain and is expressed in neural cells, MANGO 347 polypeptides, nucleic acids, and modulators thereof can be used to treat disorders involving, e.g., cellular migration, proliferation, and differentiation of a cell, e.g., a neural cell (e.g., a CNS disorder, including Alzheimer's disease, Pick's disease, Parkinson's and other Lewy diffuse body diseases, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease; psychiatric disorders, e.g., depression, schizophrenic disorders, Korsakoffs psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.
  • a neural cell e.g., a CNS disorder, including Alzheimer's disease, Pick'
  • MANGO 347 expression can be utilized as a marker for specific tissues (e.g., brain) and/or cells (e.g., brain) in which MANGO 347 is expressed.
  • MANGO 347 nucleic acids can also be utilized for chromosomal mapping.
  • a cDNA encoding human TANGO 272 was identified by analyzing the sequences of clones present in a human microvascular endothehal cell library (HMVEC) cDNA library.
  • HMVEC human microvascular endothehal cell library
  • Northern blot analysis using the human clone jthda089h03 revealed strong expression of the human TANGO 272 gene in the heart. Moderate expression was detected in the placenta, lung, and liver, and lower levels of expression were detected in the brain, skeletal muscle, kidney, and pancreas.
  • the signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human TANGO 272 includes an 20 amino acid signal peptide at amino acid 1 to about amino acid 20 of SEQ ID NO: 14 (SEQ ED NO: 112) preceding the mature human TANGO 272 protein which corresponds to about amino acid 21 to amino acid 1050 of SEQ ID NOT4 (SEQ ID NOT13).
  • Human TANGO 272 that has not been post-translationally modified is predicted to have a molecular weight of 112 kDa prior to cleavage of its signal peptide and a molecular weight of 110 kDa subsequent to cleavage of its signal peptide.
  • Human TANGO 272 is a transmembrane protein having an extracellular domain which extends from about amino acid 21 to about amino acid 767 of SEQ ID NO: 14 (SEQ ED NOT 14), a transmembrane domain which extends from about amino acid 768 to about amino acid 791 of SEQ ID NOT4 (SEQ ID NOT 15), and a cytoplasmic domain which extends from about amino acid 792 to amino acid 1050 of SEQ ID NOT4 (SEQ ED NO: 116).
  • a human TANGO 272 protein contains an extracellular domain which extends from about amino acid 792 to amino acid 1050 of SEQ ID NO 4 (SEQ ED NOT 16), a transmembrane domain which extends from about amino acid 768 to about amino acid 791 of SEQ ID NO: 14 (SEQ ID NO: 115), and a cytoplasmic domain which extends from about amino acid 21 to about amino acid 767 of SEQ ID NO:14 (SEQ ID NO:114).
  • Human TANGO 272 includes fourteen EGF-like domains at amino acids 151-181 of SEQ ID NO: 14 (SEQ ID NO:49); amino acids 200-229 of SEQ ID NO: 14 (SEQ ID NO:50); amino acids 242-272 of SEQ ID NO: 14 (SEQ ID NO:51); amino acids 285-315 of SEQ ID NO: 14 (SEQ ID NO:52); amino acids 328-358 of SEQ ID NO: 14 (SEQ ED NO:53); amino acids 378-404 of SEQ ID NO: 14 (SEQ ID NO:54); amino acids 417-447 of SEQ ID NO: 14 (SEQ ID NO:55); amino acids 460-490 of SEQ ID NO: 14 (SEQ ID NO:49); amino acids 200-229 of SEQ ID NO: 14 (SEQ ID NO:50); amino acids 242-272 of SEQ ID NO: 14 (SEQ ID NO:51); amino acids 285-315 of SEQ ID NO: 14 (SEQ ID NO:52); amino acids 328-358 of SEQ ID NO
  • FIG. 15A-15C depict alignments of each of the EGF-like domains of TANGO 272 with consensus hidden Markov model EGF-like domains (SEQ ID NO: 46).
  • Human TANGO 272 further includes a delta serrate ligand domain from amino acids 518 to 576 of SEQ ID NO: 14 (SEQ ID NO:63).
  • SEQ ID NO:63 An alignment of the delta serrate ligand domain of human TANGO 272 with a consensus hidden Markov model of this domain (SEQ ID NO:47) is also depicted ( Figure 15B).
  • RGD cell attachment site is present at amino acids 177-179 of SEQ ID NO: 14.
  • N-glycosylation sites are present at amino acids 284-287, 405-408, 459-462, 489-492, 504- 507, 588-591, 639-642, 647-650, 716-719, and 873-876 of SEQ ID NOT4.
  • An a idation site is present at amino acids 628-631 of SEQ D NO.T 4.
  • Protein kinase C phosphorylation sites are present at amino acids 38-40, 70-72, 107-109, 359-361, 461-463, 594-596, 809- 811, 896-898, 940-942, 977-979, and 1022-1024 of SEQ ED NO: 14.
  • Casein kinase II phosphorylation sites are present at amino acids 30-33, 38-41, 473-476, 548-551, 579-582, 657-660, 897-900, 921-924, 940-943, and 955-958 of SEQ ID NOT4.
  • a tyrosine kinase phosphorylation site is present at amino acids 361-368 of SEQ ID NO: 14.
  • N-myristylation sites are present at amino acids 14-19, 103-108, 269-274, 302-307, 325-330, 345-350, 401- 406, 427-432, 434-439, 457-462, 520-525, 586-591, 606-611, 648-653, 707-712, 714-719, 769-774, 866-871, 926-931, and 1014-1019 of SEQ ID NOT4.
  • Clone jthda089h03 which encodes human TANGO 272
  • a description of the deposit conditions used is set forth in the section entitled "Deposit of Clones” below. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. ⁇ 112.
  • Figure 14 depicts a hydropathy plot of human TANGO 272. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The hydropathy plot of Figure 16 indicates the presence of a hydrophobic domain within human TANGO 272, suggesting that human TANGO 272 is a transmembrane protein.
  • a cDNA encoding mouse TANGO 272 was identified by analyzing the sequences of clones present in a mouse testis cDNA library.
  • Mouse TANGO 272 that has not been post-translationally modified is predicted to have a molecular weight of 53.5 kDa.
  • Mouse TANGO 272 is a transmembrane protein having an extracellular domain which extends from about amino acid 1 to about amino acid 216 of SEQ ID NO: 17 (SEQ ID NOT 18), a transmembrane domain which extends from about amino acid 217 to about amino acid 240 of SEQ ID NO: 17 (SEQ ID NOT 19), and a cytoplasmic domain which extends from about amino acid 241 to amino acid 497 of SEQ ID NOT7 (SEQ ID NOT20).
  • a mouse TANGO 272 protein contains an extracellular domain which extends from about amino acid 241 to amino acid 497 of SEQ ID NO: 17 (SEQ ID NO.T 20), a transmembrane domain which extends from about amino acid 217 to about amino acid 240 of SEQ ID NOT7 (SEQ ED NOT19), and a cytoplasmic domain which extends from about amino acid 1 to about amino acid 216 of SEQ ID NO: 17 (SEQ ID NO: 118).
  • Mouse TANGO 272 includes four EGF-like domains at about amino acids 37-67 of SEQ ID NO: 17 (SEQ ID NO:64); amino acids 80-110 of SEQ ID NO: 17 (SEQ ID NO:65); amino acids 123-153 of SEQ ID NOT7 (SEQ ED NO:66); and amino acids 166-196 of SEQ ID NO: 17 (SEQ ID NO:67).
  • Mouse TANGO 272 further includes four laminin-EGF-like domains at about amino acids 3-37 of SEQ ID NO: 17 (SEQ ID NO:68); amino acids 41-80 of SEQ ID NOT7 (SEQ ID NO:69); amino acids 83-123 of SEQ ID NOT7 (SEQ ID NO:70); and amino acids 127-172 of SEQ ED NOT7 (SEQ ID NO:71).
  • Figures 39A-39B depict alignments of each of the EGF-like- and laminin-EGF-like domains of TANGO 272 with consensus hidden Markov model EGF-like domains (SEQ ID NOs:46 and 48, respectively).
  • Mouse TANGO 272 further includes a delta serrate ligand domain from amino acids 10 to 67 of SEQ ID NOT7 (SEQ ID NO:72).
  • SEQ ID NO:72 An alignment of the delta serrate ligand domain of mouse TANGO 272 with a consensus hidden Markov model of this domain (SEQ ID NO:47) is also depicted in Figures 39A-39B.
  • EGF-like domain cysteine pattern signature are present at amino acids 13-24, 56-67, 99-110, 142-153, and 185-196 of SEQ ID NOT7.
  • N-glycosylation sites are present at amino acids 36-39, 88-91, 165-168, and 323-326 of SEQ ID NO: 17.
  • An amidation site is present at amino acids 76-79 of SEQ ID NO: 17.
  • Protein kinase C phosphorylation sites are present at amino acids 42-44, 258-260, 354-356, 388-390, 469-471, and 492-494 of SEQ ID NOT7.
  • Casein kinase II phosphorylation sites are present at amino acids 106-109, 192-195, 343-346, 388-391, and 446-449 of SEQ ID NO: 17.
  • N-myristylation sites are present at amino acids 11-16, 34-39, 47-52, 54-59, 97- 102, 120-125, 140-145, 163-168, 199-204, 218-223, 372-377, and 461-466 of SEQ ID NOTE
  • Figure 17 depicts a hydropathy plot of mouse TANGO 272. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The hydropathy plot of Figure 17 indicates the presence of a hydrophobic domain within mouse TANGO 272, suggesting that mouse TANGO 272 is a transmembrane protein.
  • a cDNA encoding rat TANGO 272 was identified by analyzing the sequences of clones present in a rat neonatal sciatic nerve cDNA library.
  • Rat TANGO 272 is a transmembrane protein having an extracellular domain which extends from about amino acid 1 to about amino acid 500 of SEQ ED NO:20 (SEQ ID- NO: 122), a transmembrane domain which extends from about amino acid 501 to about amino acid 524 of SEQ ID NO:20 (SEQ ID NO: 123), and a cytoplasmic domain which extends from about amino acid 525 to amino acid 636 of SEQ ID NO:20 (SEQ ID NO: 124).
  • a rat TANGO 272 protein contains an extracellular domain which extends from about amino acid 525 to amino acid 636 of SEQ ID NO:20 (SEQ ID NO: 124), a transmembrane domain which extends from about amino acid 501 to about amino acid 524 of SEQ ID NO:20 (SEQ ED NO: 123), and a cytoplasmic domain which extends from about amino acid 1 to about amino acid 500 of SEQ ID NO:20 (SEQ ID NO: 122).
  • Rat TANGO 272 includes eleven EGF-like domains at about amino acids 18-48 of SEQ ID NO:20 (SEQ ID NO:73); amino acids 61-91 of SEQ ID NO:20 (SEQ ID NO:74); amino acids 105-137 of SEQ ID NO:20 (SEQ ID NO:75); amino acids 150-180 of SEQ ED NO:20 (SEQ ID NO:76); amino acids 193-223 of SEQ ID NO:20 (SEQ ID NO:77); amino acids 236-266 of SEQ ID NO:20 (SEQ ID NO:78); amino acids 279-309 of SEQ ID NO:20 (SEQ ID NO:79); amino acids 322-352 of SEQ ID NO:20 (SEQ ID NO:80); amino acids 365-394 of SEQ ID NO:20 (SEQ ID NO:81); amino acids 407-437 of SEQ ID NO:20 (SEQ ID NO:82); and amino acids 450-480 of SEQ ID NO:20 (SEQ ID NO:83).
  • Figures 41 A- 41D
  • Rat TANGO 272 further includes eleven laminin/EGF-like domains at about amino acids 22-61 of SEQ ID NO:20 (SEQ ID NO:84); arnino acids 65-105 of SEQ ID NO:20 (SEQ ID NO:85); amino acids 109-150 of SEQ ID NO:20 (SEQ ID NO:86); amino acids 154-193 of SEQ ID NO:20 (SEQ ID NO:87); amino acids 197-236 of SEQ ID NO:20 (SEQ ID NO:88); amino acids 240-279 of SEQ ID NO:20 (SEQ ID NO:89); amino acids 283-322 of SEQ ID NO:20 (SEQ ID NO:90); amino acids 326-365 of SEQ ID NO:20 (SEQ ID NO:91); amino acids 368-407 of SEQ ID NO:20 (SEQ ID NO:92); amino acids 411-450; and amino acids 454-489 of SEQ ID NO:20 (SEQ ID NO:93).
  • Figures 41A-41D depict alignments of each of the
  • Rat TANGO 272 further includes a delta serrate ligand domain from amino acids
  • SEQ ID NO:95 An alignment of the delta serrate ligand domain of rat TANGO 272 with a consensus hidden Markov model of this domain (SEQ ID NO:95).
  • EGF-like domain cysteine pattern signature are present at amino acids 37-48, 80-91, 126-137, 169-180, 255-266, 298-309, 341-352, 383- 394, 426-437, and 469-480 of SEQ ID NO:20.
  • N-glycosylation sites are present at amino acids 17-20, 138-141, 192-195, 222-225, 237-240, 321-324, 372-375, 436-439, and 449-452 of SEQ ID NO:20.
  • a cAMP/cGMP-dependent protein kinase phosphorylation site is present at amino acids 618-621 of SEQ ID NO:20.
  • An amidation site is present at amino acids 361-364 of SEQ ID NO:20.
  • Protein kinase C phosphorylation sites are present at amino acids 92-94, 327-329, 542-544, and 596-598 of SEQ ID NO:20.
  • Casein kinase II phosphorylation sites are present at amino acids 104-107, 206-209, 281-284, and 390-393 of SEQ ID NO:20.
  • 10 phosphorylation site is present at amino acids 94-101 of SEQ ID NO:20.
  • N-myristylation sites are present at amino acids 2-7, 35-40, 58-63, 78-83, 134-139, 160-165, 167-172, 190- 195, 210-215, 253-258, 319-324, 339-344, 381-386, 404-409, 424-429, 447-452, 483-488, and 502-507 of SEQ ID NO:20.
  • Figure 40 depicts a hydropathy plot of rat TANGO 272. Relatively hydrophobic
  • TANGO 272 includes fourteen EGF-like domains. Proteins having such domains play a role in mediating protein-protein interactions, and thus can influence a wide variety of biological processes, including cell surface recognition; modulation of cell-cell contact; modulation of cell fate determination; and modulation of wound healing and tissue repair.
  • TANGO 272 further includes an RGD cell attachment site.
  • Proteins having such domains are typically extracellular matrix proteins such as collagens, laminin and fibronectin, among others (reviewed in Ruoslahti, 1996, Annu. Rev. Cell Dev. Biol. 12:691- 715).
  • An RGD cell attachment site typically interacts (e.g., binds to) a cell surface receptor, such as an integrin receptor, and thus mediates a variety of biological processes, including cellular adhesion, migration, among others.
  • TANGO 272 includes EGF-like domains and an RGD cell attachment site
  • TANGO 272 polypeptides, nucleic acids, and modulators thereof can be used to treat disorders involving, e.g., cellular migration, proliferation, and differentiation of a cell.
  • TANGO 272 polypeptides, nucleic acids, and modulators thereof can be used to treat neoplastic disorders, e.g., cancer, tumor metastasis.
  • TANGO 272 polypeptides, nucleic acids, and modulators thereof can be used to modulate function, survival, morphology, migration, proliferation, tissue repair and/or differentiation of cells in the tissues in which it is expressed (e.g., micro vascular endothehal cells).
  • TANGO 272 polypeptides, nucleic acids, and modulators thereof can be used to modulate cardiovascular function, and/or to promote wound healing and tissue repair (e.g., of the skin, cornea and mucosal lining).
  • these molecules can be used to treat a variety of cardiovascular diseases including, but not limited to, atherosclerosis, ischemia reperfusion injury, cardiac hypertrophy, hypertension, coronary artery disease, myocardial infarction, an-hythmia, cardiomyopathies, and congestive heart failure.
  • cardiovascular diseases including, but not limited to, atherosclerosis, ischemia reperfusion injury, cardiac hypertrophy, hypertension, coronary artery disease, myocardial infarction, an-hythmia, cardiomyopathies, and congestive heart failure.
  • TANGO 272 exhibits expression in the heart
  • TANGO 272 nucleic acids, proteins, and modulators thereof can be used to treat heart disorders, e.g., ischemic heart disease, atherosclerosis, hypertension, angina pectoris, Hypertrophic Cardiomyopathy, and congenital heart disease.
  • TANGO 272 polypeptides, nucleic acids, or modulators thereof can be used to treat placental disorders, such as toxemia of pregnancy (e.g., preeclampsia and eclampsia), placentitis, or spontaneous abortion.
  • placental disorders such as toxemia of pregnancy (e.g., preeclampsia and eclampsia), placentitis, or spontaneous abortion.
  • TANGO 272 polypeptides, nucleic acids, or modulators thereof can be used to treat pulmonary (lung) disorders, such as atelectasis, cystic fibrosis, rheumatoid lung disease, pulmonary congestion or edema, chronic obstructive airway disease (e.g., emphysema, chronic bronchitis, bronchial asthma, and bronchiectasis), diffuse interstitial diseases (e.g., sarcoidosis, pneumoconiosis, hypersensitivity pneumonitis, Goodpasture's syndrome, idiopathic pulmonary hemosiderosis, pulmonary alveolar proteinosis, desquamative interstitial pneumonitis, chronic interstitial pneumonia, fibrosing alveolitis, hamman-rich syndrome, pulmonary eosinophilia, diffuse interstitial fibrosis, Wegener' s granulomatosis, lymphomatoid granul
  • lung disorders
  • TANGO 272 polypeptides, nucleic acids, or modulators thereof can be used to treat hepatic (liver) disorders, such as jaundice, hepatic failure, hereditary hyperbiliruinemias (e.g., Gilbert's syndrome, Crigler-Naijar syndromes and Dubin- Johnson and Rotor's syndromes), hepatic circulatory disorders (e.g., hepatic vein thrombosis and portal vein obstruction and thrombosis) hepatitis (e.g., chronic active hepatitis, acute viral hepatitis, and toxic and drug-induced hepatitis) cirrhosis (e.g., alcoholic cirrhosis, biliary cirrhosis, and hemochromatosis), or malignant tumors (e.g., primary carcinoma, hepatoblastoma, and angiosarcoma).
  • hepatic (liver) disorders such as jaundice, hepatic failure, her
  • TANGO 272 polypeptides, nucleic acids, or modulators thereof can be used to treat disorders of the brain, such as cerebral edema, hydrocephalus, brain herniations, iatro genie disease (due to, e.g., infection, toxins, or drugs), inflammations (e.g., bacterial and viral meningitis, encephalitis, and cerebral toxoplasmosis), cerebrovascular diseases (e.g., hypoxia, ischemia, and infarction, intracranial hemorrhage and vascular malformations, and hypertensive encephalopathy), and tumors (e.g., neuroglial tumors, neuronal tumors, tumors of pineal cells, meningeal tumors, primary and secondary lymphomas, intracranial tumors, and medulloblastoma), and to treat injury or trauma to the brain.
  • disorders of the brain such as cerebral edema, hydrocephalus, brain herniations, iatro genie
  • TANGO 272 polypeptides, nucleic acids, or modulators thereof can be used to treat disorders of skeletal muscle, such as muscular dystrophy (e.g., Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, Emery-Dreifuss Muscular Dystrophy, Limb-Girdle Muscular Dystrophy, Facioscapulohumeral Muscular Dystrophy, Myotonic Dystrophy, Oculopharyngeal Muscular Dystrophy, Distal Muscular Dystrophy, and Congenital Muscular Dystrophy), motor neuron diseases (e.g., motor neuron diseases (e.g., Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, Emery-Dreifuss Muscular Dystrophy, Limb-Girdle Muscular Dystrophy, Facioscapulohumeral Muscular Dystrophy, Myotonic Dystrophy, Oculopharyngeal Muscular Dyst
  • myopathies e.g., inflammatory myopathies (e.g., Dermatomyositis and Polymyositis), Myotonia Congenita, Paramyotonia Congenita, Central Core Disease, Nemaline Myopathy,
  • myopathies e.g., inflammatory myopathies (e.g., Dermatomyositis and Polymyositis), Myotonia Congenita, Paramyotonia Congenita, Central Core Disease, Nemaline Myopathy,
  • Myotubular Myopathy, and Periodic Paralysis and metabolic diseases of muscle (e.g., Phosphorylase Deficiency, Acid Maltase Deficiency, Phosphofructokinase Deficiency, Debrancher Enzyme Deficiency, Mitochondrial Myopathy, Carnitine Deficiency, Carnitine Palmityl Transferase Deficiency, Phosphoglycerate Kinase Deficiency, Phosphoglycerate Mutase Deficiency, Lactate Dehydrogenase Deficiency, and Myoadenylate Deaminase Deficiency).
  • Phosphorylase Deficiency Acid Maltase Deficiency
  • Phosphofructokinase Deficiency Debrancher Enzyme Deficiency
  • Mitochondrial Myopathy Carnitine Deficiency
  • Carnitine Palmityl Transferase Deficiency Phosphoglycerate Kinase Deficiency
  • TANGO 272 polypeptides, nucleic acids, or modulators thereof can be used to treat renal disorders, such as glomerular diseases (e.g., acute and chronic glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, focal proliferative glomerulonephritis, glomerular lesions associated with systemic disease, such 0 as systemic lupus erythematosus, Goodpasture's syndrome, multiple myeloma, diabetes, neoplasia, sickle cell disease, and chronic inflammatory diseases), tubular diseases (e.g., acute tubular necrosis and acute renal failure, polycystic renal diseasemedullary sponge kidney, medullary cystic disease, nephrogenic diabetes, and renal tubular acidosis), tubulointerstitial diseases (e.g., pyelonephritis, drug and toxin induced tubulointerstitial 5 nephriti
  • TANGO 272 polypeptides, nucleic acids, or modulators thereof can be used to treat pancreatic disorders, such as pancreatitis (e.g., acute hemorrhagic pancreatitis and chronic pancreatitis), pancreatic cysts (e.g., congenital cysts, pseudocysts, and benign or malignant neoplastic cysts), pancreatic tumors (e.g., pancreatic carcinoma and adenoma), diabetes mellitus (e.g., insulin- and non-insulin-dependent types, impaired 5 glucose tolerance, and gestational diabetes), or islet cell tumors (e.g., insulinomas, adenomas, Zollinger-Ellison syndrome, glucagonomas, and somatostatinoma).
  • pancreatitis e.g., acute hemorrhagic pancreatitis and chronic pancreatitis
  • pancreatic cysts e.g., congenital cysts, pseudo
  • TANGO 272 expression can be utilized as a marker for specific tissues (e.g., cardiovascular) and/or cells (e.g., cardiac) in which TANGO 272 is expressed.
  • TANGO 272 nucleic acids can also be utilized for chromosomal mapping.
  • a cDNA encoding human TANGO 295 was identified by analyzing the sequences of clones present in a human mammary epithelium cDNA library.
  • the signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human TANGO 295 includes a 28 amino acid signal peptide at amino acid 1 to about amino acid 28 of SEQ ID NO:23 (SEQ ID NOT25) preceding the mature human TANGO 295 protein which corresponds to about amino acid 29 to amino acid 156 of SEQ ID NO:23 (SEQ ID NO: 126).
  • Human TANGO 295 that has not been post-translationally modified is predicted to have a molecular weight of 17.5 kDa prior to cleavage of its signal peptide and a molecular weight of 14.6 kDa subsequent to cleavage of its signal peptide.
  • Secretion assays reveal that human TANGO 295 protein is secreted as a 17 kDa protein.
  • the secretion assays were performed as follows: 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/streptomycin) at 37 °C, 5% CO 2 overnight. 293T cells were transfected with 2 ⁇ g of full-length MANGO 245 inserted in the pMET7 vector/well and 10 ⁇ g LipofectAMINE (GIBCO/BRL Cat. # 18324-012) /well according to the protocol for
  • GIBCO/BRL LipofectAMINE The transfectant was removed 5 hours later and fresh growth medium was added to allow the cells to recover overnight. The medium was removed and each well was gently washed twice with DMEM without methionine and cysteine (ICN Cat. # 16-424-54). 1 ml DMEM without methionine and cysteine with 50 ⁇ Ci Trans- 35 S (ICN Cat. # 51006) was added to each well and the cells were incubated at
  • Human TANGO 295 includes a pancreatic ribonuclease domain at amino acids 32- 156 of SEQ ID NO:23 (SEQ ID NO:97).
  • Figure 20 depicts an alignment of pancreatic ribonuclease domain of human TANGO 295 with a consensus hidden Markov model pancreatic ribonuclease domain (SEQ ID NO:96).
  • N-glycosylation site is present at amino acids 127-130 of SEQ ID NO:23.
  • a cAMP/cGMP dependent protein kinase site is present at amino acids 139-142 of SEQ ED NO:23.
  • Protein kinase C phosphorylation sites are present at amino acids 27-29, 62-64, 85- 87, and 113-115 of SEQ ID NO:23.
  • N-myristylation sites are present at amino acids 18-23, and 32-37 of SEQ ID NO:23.
  • Figure 19 depicts a hydropathy plot of human TANGO 295. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The hydropathy plot of Figure 19 indicates that human TANGO 295
  • TANGO 295 is a secreted protein.
  • TANGO 295 includes a pancreatic ribonuclease domain. Proteins having such domains have pyrimidine-specific endonuclease activity, and are present at elevated levels in the pancreas of various mammals and few reptiles.
  • TANGO 295 shows some structural similarities to Ribonuclease k6 (RNase k6). RNase k6 is expressed in human monocytes and monophils (but not in eosinophils), suggesting a role for this ribonuclease in regulating host defense. Based on the structural similarities between TANGO 295 and RNase k6,
  • TANGO 295 may play a role in regulating host defense.
  • TANGO 295 polypeptides, nucleic acids, and modulators thereof can be used to modulate the function, morphology, proliferation and/or differentiation of cells in the tissues in which it is expressed (e.g., mammary epithelium). Accordingly, TANGO 295 polypeptides, nucleic acids, and modulators thereof can be used to treat epithelial disorders, e.g., mammary epithelial disorders (e.g., breast cancer).
  • epithelial disorders e.g., mammary epithelial disorders (e.g., breast cancer).
  • TANGO 295 expression can be utilized as a marker for specific tissues (e.g., breast) and/or cells (e.g., mammary) in which TANGO 295 is expressed.
  • TANGO 295 nucleic acids can also be utilized for chromosomal mapping.
  • a cDNA encoding human TANGO 354 was identified by analyzing the sequences of clones present in a Mixed Lymphocyte Reaction (MLR) cDNA library.
  • MLR Mixed Lymphocyte Reaction
  • Human TANGO 354 that has not been post-translationally modified is predicted to have a molecular weight of 33.8 kDa prior to cleavage of its signal peptide (31.6 kDa after cleavage of its signal peptide).
  • human TANGO 354 includes a 19 amino acid signal peptide at amino acid 1 to about amino acid 19 of SEQ ID NO:26 (SEQ ID NO: 127) preceding the mature human TANGO 354 protein which corresponds to about amino acid
  • Human TANGO 354 is a transmembrane protein having an extracellular domain which extends from about amino acid 20 to about amino acid 169 of SEQ ID NO:26 (SEQ ID NO: 129), a transmembrane domain which extends from about amino acid 170 to about amino acid 193 of SEQ ID NO:26 (SEQ ID NOT30), and a cytoplasmic domain which extends from about amino acid 194 to amino acid 305 of SEQ ID NO:26 (SEQ ID NOT31).
  • a human TANGO 354 protein contains an extracellular domain which extends from about amino acid 194 to amino acid 305 of SEQ ID NO:26 (SEQ ID NO: 131), a transmembrane domain which extends from about amino acid 170 to about amino acid 193 of SEQ ID NO:26 (SEQ ID NOT30), and a cytoplasmic domain which extends from about amino acid 20 to about amino acid 169 of SEQ ID NO:26 (SEQ ID NO:129).
  • Human TANGO 354 includes an immunoglobulin domain at amino acids 33-110 of SEQ ID NO:26 (SEQ ID NO:41).
  • Figure 23 depicts alignments of the immunoglobulin domains of TANGO 354 with consensus hidden Markov model immunoglobulin domains (SEQ ID NO:37).
  • N-glycosylation site is present at amino acids 88-91 of SEQ ID NO:26.
  • a cAMP and cGMP-dependent protein kinase phosphorylation site is present at amino acids 233-236 of SEQ ID NO:26.
  • Protein kinase C phosphorylation sites are present at amino acids 81-83, 231-233, and 236-238 of SEQ ID NO:26.
  • Casein kinase II phosphorylation sites are present at amino acids 44-47, 69-72, 81-84, 94-97, 101-104, 113-116, and 146-149 of SEQ ID NO:26.
  • a tyrosine kinase phosphorylation site is present at amino acids 291- 299 of SEQ ID NO:26.
  • N-myristylation sites are present at amino acids 30-35, and 109-114 of SEQ ID NO:26.
  • Clone jthLa042a04 which encodes human TANGO 354, was deposited as EpT354 with the American Type Culture Collection (ATCC ® 10801 University Boulevard, Manassas, VA 20110-2209) on June 18, 1999 and assigned Accession Number PTA-249. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. ⁇ 112.
  • Figure 22 depicts a hydropathy plot of human TANGO 354. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The hydropathy plot of Figure 22 indicates the presence of a hydrophobic domain within human TANGO 354, suggesting that human TANGO 354 is a transmembrane protein. Use of TANGO 354 Nucleic Acids. Polypeptides. and Modulators Thereof
  • TANGO 354 includes an immuno globulin-like domain. Proteins having such domains play a role in mediating protein-protein and protein-ligand interactions, and thus can influence a wide variety of biological processes, including modulation of cell surface recognition; modulation of cellular motility, e.g., chemotaxis and chemokinesis; transduction of an extracellular signal (e.g., by interacting with a ligand and or a cell- surface receptor); and/or modulation of a signal transduction pathways.
  • TANGO 354 polypeptides, nucleic acids, and modulators thereof can be used to modulate function, survival, morphology, migration, proliferation and/or differentiation of cells in the tissues in which it is expressed (e.g., hematopoietic tissues).
  • TANGO 354 in hematopoietic cells can be used to modulate (e.g., increase or decrease) hematopoietic function, thereby influencing one or more of: (1) regulation of hematopoiesis; (2) modulation of haemostasis; (3) modulation of an inflammatory response; (4) modulation of neoplastic growth, e.g., inhibition of tumor growth; and/or (5) regulation of thrombolysis.
  • TANGO 354 polypeptides, nucleic acids, and modulators thereof can be used to treat a variety of hematopoietic diseases including, but not limited to, myeloid disorders and/or lymphoid malignancies.
  • myeloid diseases that can be treated include acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Naickus, 1991, Crit Rev. in
  • lymphoid malignancies that can be treated using these molecules include acute lymphoblastic leukemia (ALL) which includes B-lineage
  • ALL and T-lineage ALL ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia
  • PLL hairy cell leukemia
  • HLL hairy cell leukemia
  • WM Waldenstrom's macroglobulinemia
  • malignant lymphomas include non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T- cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF) and Hodgkin's disease.
  • ATL adult T cell leukemia/lymphoma
  • CCL cutaneous T- cell lymphoma
  • LGF large granular lymphocytic leukemia
  • Hodgkin's disease Hodgkin's disease.
  • TANGO 354 polypeptides, nucleic acids, and modulators thereof can be used to treat a variety of neoplastic diseases, including malignancies of the various organ systems, such as affecting lung, breast, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • malignancies of the various organ systems such as affecting lung, breast, lymphoid, gastrointestinal, and genito-urinary tract
  • adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
  • Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • carcinosarcomas e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • An "adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • sarcoma is art recognized and refers to malignant tumors of mesenchymal derivation.
  • TANGO 354 polypeptides, nucleic acids, and modulators thereof can also be used to treat a variety of non-cancerous diseases or conditions involving, for example, aberrant T cell activity (e.g., aberrant T cell proliferation and/or secretion).
  • aberrant T cell activity e.g., aberrant T cell proliferation and/or secretion.
  • T cell diseases or conditions include inflammation; allergy, for example, atopic allergy; organ rejection after transplantation (e.g., skin graft, cardiac graft, islet graft); graft- versus-host disease; autoimmune diseases (including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, diabetes, myasthenia gravis, systemic lupus erythematosus, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sj ⁇ gren's Syndrome, including keratoconjunctivitis sicca secondary to Sj ⁇ gren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, ulceris, conjunctivitis
  • TANGO 345 expression can be utilized as a marker for specific tissues (e.g., lymphoid tissues such as the thymus and spleen) and/or cells (e.g., lymphocytes) in which TANGO 345 is expressed.
  • tissue e.g., lymphoid tissues such as the thymus and spleen
  • cells e.g., lymphocytes
  • TANGO 345 nucleic acids can also be utilized for chromosomal mapping.
  • a cDNA encoding human TANGO 378 was identified by analyzing the sequences of clones present in a human natural killer cell cDNA library. This analysis led to the identification of a clone, jthta028f04, encoding full-length human TANGO 378.
  • the cDNA of this clone is 3258 nucleotides long ( Figures 24A-24C; SEQ ID NO:28).
  • the signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human TANGO 378 includes a 21 amino acid signal peptide at amino acid 1 to about amino acid 21 of SEQ ID NO:29 (SEQ ID NO: 132) preceding the mature human MANGO 347 protein which corresponds to about amino acid 22 to amino acid 528 of SEQ ID NO:29 (SEQ ID NO: 133).
  • Human TANGO 378 that has not been post-translationally modified is predicted to have a molecular weight of 59.0 kDa prior to cleavage of its signal peptide and a molecular weight of 56.7 kDa subsequent to cleavage of its signal peptide.
  • Human TANGO 378 is a seven transmembrane G-protein coupled receptor (GPCR) protein having an N-terminal extracellular domain which extends from about amino acid 22 to about amino acid 244 of SEQ ID NO:29 (SEQ ID NO: 134); seven transmembrane domains which extend from about amino acids 245 to about amino acid 269 of SEQ ID NO:29 (SEQ ID NO: 135), about amino acids 287 to about amino acid 306 of SEQ ID NO:29 (SEQ ID NOT36), about amino acids 323 to about amino acid 343 of SEQ ID NO:29 (SEQ ID NOT37), about amino acids 358 to about amino acid 376 of SEQ ID NO:29 (SEQ ID NO: 138), about amino acids 414 to about amino acid 438 of SEQ ID NO:29 (SEQ ID NO: 139), about amino acids 457 to about amino acid 477 of SEQ ID NO:29 (SEQ ID NO: 140), and about amino acids 485 to about amino acid 504 of SEQ ID NO:29 (SEQ
  • a human TANGO 378 protein contains an N- terminal extracellular domain which extends from about amino acid 505 to amino acid 528 of SEQ ID NO:29 (SEQ ID NO: 142); seven transmembrane domains which extend from about amino acids 245 to about amino acid 269 of SEQ ID NO:29 (SEQ ID NOT35), about amino acids 287 to about amino acid 306 of SEQ ID NO:29 (SEQ ID NO: 136), about amino acids 323 to about amino acid 343 of SEQ ID NO:29 (SEQ ID NOT37), about amino acids 358 to about amino acid 376 of SEQ ID NO:29 (SEQ ID NOT38), about amino acids 414 to about amino acid 438 of SEQ ED NO:29 (SEQ ID NO: 139), about amino acids 457 to about amino acid 477 of SEQ ID NO:29 (SEQ ID NO: 140), and about amino acids 485 to about amino acid 504 of SEQ ED NO:29 (SEQ ID NO: 141); and a C-
  • Human TANGO 378 includes three extracellular loops which extend from about amino acid 307 to about amino acid 322 of SEQ ID NO:29 (SEQ ID NO: 143), about amino acid 377 to about amino acid 413 of SEQ ID NO:29 (SEQ ID NO: 144), and about amino acid 478 to about amino acid 484 of SEQ ID NO:29 (SEQ ID NO.T 45).
  • Human TANGO 378 includes three intracellular loops which extend from about amino acid 270 to about amino acid 286 of SEQ ID NO:29 (SEQ ID NO: 146), about amino acid 344 to about amino acid 357 of SEQ ID NO:29 (SEQ ID NO: 147), and about amino acid 439 to about amino acid 456 of SEQ ID NO:29 (SEQ ID NO: 148).
  • N-glycosylation sites are present at amino acids 18-21, 58-61, 65-68, 146-149, 173- 176, 179-182, 394-397, and 400-403 of SEQ ID NO:29.
  • a cAMP and cGMP-dependent protein kinase phosphorylation site is present at amino acids 274-277 of SEQ ID NO:29.
  • Protein kinase C phosphorylation sites are present at amino acids 45-47, 93-95, 375-377, 437-439, 449-451, and 505-507 of SEQ ID NO:29.
  • Casein kinase II phosphorylation sites are present at amino acids 23-26, 29-32, and 510-513 of SEQ ID NO:29.
  • N-myristylation sites are present at amino acids 86-91, 101-106, 157-162, 255-260, 311-316, 420-425, and 467-472 of SEQ ID NO:29.
  • a thiol (cysteine) protease histidine site is present at amino acid 410-420 of SEQ ID NO:29.
  • Figure 25 depicts a hydropathy plot of human TANGO 378. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace.
  • the hydropathy plot of Figure 25 indicates that human TANGO 378 has a signal peptide at its amino terminus and seven hydrophobic domains within human TANGO 378, suggesting that human TANGO 378 is a transmembrane protein.
  • TANGO 378 includes a seven transmembrane domain which is typically found in G-protein coupled receptors. Proteins having such a domain play a role in transducing an extracellular signal, e.g., by interacting with a ligand and/or a cell-surface receptor, followed by mobilization of intracellular molecules that participate in signal transduction pathways (e.g., adenylate cyclase, or phosphatidylinositol 4,5-bisphosphate (PIP 2 ), inositol 1,4,5-triphosphate (EP 3 )).
  • signal transduction pathways e.g., adenylate cyclase, or phosphatidylinositol 4,5-bisphosphate (PIP 2 ), inositol 1,4,5-triphosphate (EP 3 )
  • TANGO 378 polypeptides, nucleic acids, and modulators thereof can be used to modulate function, survival, morphology, migration, proliferation and/or differentiation of cells in the tissues in which it is expressed (e.g., natural killer cells).
  • TANGO 354 polypeptides, nucleic acids, and modulators thereof can be used to modulate an immune response in a subject by, for example, (1) modulating immune cytotoxic responses against pathogenic organisms, e.g., viruses, bacteria, and parasites; (2) by modulating organ rejection after transplantation (e.g., skin graft, cardiac graft, islet graft); (3) by modulating immune recognition and lysis of normal and malignant cells; (4) by modulating T cell diseases; and (5) by controlling neoplastic growth, e.g., inhibition of tumor growth.
  • pathogenic organisms e.g., viruses, bacteria, and parasites
  • organ rejection after transplantation e.g., skin graft, cardiac graft, islet graf
  • TANGO 378 polypeptides, nucleic acids, and modulators thereof can be used to treat a variety of diseases involving aberrant immune responses, for example, aberrant T cell activity (e.g., aberrant T cell proliferation and/or secretion).
  • aberrant T cell activity e.g., aberrant T cell proliferation and/or secretion.
  • a non-limiting list of diseases involving aberrant T cell activity is provided in the section entitled "TANGO 354" above.
  • TANGO 378 polypeptides, nucleic acids, and modulators thereof can be used to treat a variety of neoplastic diseases, including hematopoietic malignancies and including, but not limited to, myeloid disorders, lymphoid malignancies, and/or malignancies of the various organ systems. ).
  • neoplastic diseases including hematopoietic malignancies and including, but not limited to, myeloid disorders, lymphoid malignancies, and/or malignancies of the various organ systems.
  • TANGO 354 A non-limiting list of such neoplastic diseases is provided in the section entitled "TANGO 354" above.
  • TANGO 378 expression can be utilized as a marker for specific tissues (e.g., lymphoid tissues such as the thymus and spleen) and/or cells (e.g., Natural Killer cells) in which
  • TANGO 345 is expressed.
  • TANGO 345 nucleic acids can also be utilized for chromosomal mapping.
  • Tables 1 and 2 below provide summaries of INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, and TANGO 378 sequence information.
  • nucleic acid molecules that encode a polypeptide of the invention or a biologically active portion thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single- stranded or double-stranded, but preferably is double-stranded DNA.
  • An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • an "isolated" nucleic acid molecule is free of sequences (preferably protein encoding sequences) which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the "isolated" nucleic acid is free of intron sequences.
  • the isolated nucleic acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the nucleic acid molecules of the invention comprise a contiguous open reading frame encoding a polypeptide of the invention.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOsT, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, or a complement thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequences of SEQ ID NOs.T, 3, 4, 6, 1, 9, 10, 12, 13, 15, 16, 18,
  • nucleic acid molecules of the invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, 2nd ed.,1989, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold
  • a nucleic acid molecule of the invention can be amplified using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard
  • nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to all or a portion of a nucleic acid molecule of the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence of
  • a nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence thereby forming a stable duplex.
  • a nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence encoding a full length polypeptide of the invention for example, a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a polypeptide of the invention.
  • the nucleotide sequence determined from the cloning one gene allows for the generation of probes and primers designed for use in identifying and/or cloning homologues in other cell types, e.g., from other tissues, as well as homologues from other mammals.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide
  • Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts or genomic sequences encoding the same protein molecule encoded by a selected nucleic acid molecule.
  • the probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • a label group attached thereto e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which
  • mis-express the protein such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted.
  • a nucleic acid fragment encoding a biologically active portion of a polypeptide of the invention can be prepared by isolating a portion of any of SEQ ID NOs: 1 , 3, 4, 6, 7, 9,
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of SEQ ID NOsT, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24,
  • DNA sequence polymorphisms that lead to changes in the amino acid sequence may exist within a population (e.g., the human population). Such genetic polymorphisms may exist among individuals within a population due to natural allehc variation.
  • An allele is one of a group of genes which occur alternatively at a given genetic locus.
  • allelic variant refers to a nucleotide sequence which occurs at a given locus or to a polypeptide encoded by the nucleotide sequence.
  • gene and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention.
  • allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given 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 or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention.
  • nucleic acid molecules encoding proteins of the invention from other species which have a nucleotide sequence which differs from that of the human protein described herein are intended to be within the scope of the invention.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of a cDNA of the invention can be isolated based on their identity to the human nucleic acid molecule disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • a cDNA encoding a soluble form of a membrane-bound protein of the invention isolated based on its hybridization to a nucleic acid molecule encoding all or part of the membrane-bound form.
  • a cDNA encoding a membrane-bound form can be isolated based on its hybridization to a nucleic acid molecule encoding all or part of the soluble form.
  • an isolated nucleic acid molecule of the invention is at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900,
  • nucleic acid molecule comprising the nucleotide sequence, preferably the coding sequence, of SEQ ID NOsT, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, or a complement thereof.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 10%, 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, 1989, John Wiley & Sons, NY, sections
  • 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.
  • SSC sodium chloride/sodium citrate
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NOsT, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, or a complement thereof corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • allelic variants of a nucleic acid molecule of the invention sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation thereby leading to changes in the amino acid sequence of the encoded protein, without altering the biological activity of the protein. For example, one can make nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues.
  • a "non-essential” amino acid residue is a residue that can be altered from the wild-type sequence without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are not conserved or only semi-conserved among homologues of various species may be non-essential for activity and thus would be likely targets for alteration.
  • amino acid residues that are conserved among the homologues of various species e.g., murine and human
  • amino acid residues that are conserved among the homologues of various species may be essential for activity and thus would not be likely targets for alteration.
  • nucleic acid molecules encoding a polypeptide of the invention that contain changes in amino acid residues that are not essential for activity.
  • polypeptides differ in amino acid sequence from SEQ ID NO: 1
  • 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 NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29.
  • An isolated nucleic acid molecule encoding a variant protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, 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. Briefly, PCR primers are designed that delete the trinucleotide codon of the amino acid to be changed and replace it with the trinucleotide codon of the amino acid to be included. This primer is used in the PCR amplification of DNA encoding the protein of interest. This fragment is then isolated and inserted into the full length cDNA encoding the protein of interest and expressed recombinantly. The resulting protein now includes the amino acid replacement.
  • conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
  • a mutant polypeptide that is a variant of a polypeptide of the invention can be assayed for: (1) the ability to form protein-protein interactions with proteins in a signaling pathway of the polypeptide of the invention; (2) the ability to bind a ligand of the polypeptide of the invention; or (3) the ability to bind to an intracellular target protein of the polypeptide of the invention.
  • the mutant polypeptide can be assayed for the ability to modulate cellular proliferation, cellular migration or chemotaxis, or cellular differentiation.
  • the present invention encompasses antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid encoding a polypeptide of the invention, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame).
  • An antisense nucleic acid molecule can be antisense to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding a polypeptide of the invention.
  • the non-coding regions (“5' and 3' untranslated regions") are the 5' and 3' sequences which flank the coding region and are not translated into amino acids.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40,
  • 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, ⁇ -D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a selected polypeptide of the invention to thereby inhibit expression, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • An antisense nucleic acid molecule of the invention can be an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al., 1987, Nucleic Acids Res. 15:6625-41).
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al.,1987, Nucleic Acids Res. 15:6131-48) or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-30).
  • 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- 91
  • a ribozyme having specificity for a nucleic acid molecule encoding a polypeptide of the invention can be designed based upon the nucleotide sequence of a cDNA disclosed herein.
  • a derivative of a Tetrahymena L-l 9 INS R ⁇ A can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742.
  • an mRNA encoding a polypeptide of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak, 1993, Science 261:1411-8.
  • the invention also encompasses nucleic acid molecules which form triple helical structures.
  • expression of a polypeptide of the invention can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g., the promoter and/or enhancer) to form triple helical structures that prevent transcription of the gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide e.g., the promoter and/or enhancer
  • 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 93:14670-5.
  • PNAs can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
  • PNAs can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup, 1996, supra); or as probes or primers for DNA sequence and hybridization (Hyrup, 1996, supra; Perry-O'Keefe et al., 1996, Proc. Natl Acad. Sci. USA 93:14670-675).
  • PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras can be generated which may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup, 1996, supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996, supra) and Finn et al. (1996, Nucleic Acids Res. 24(17):3357-63).
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6; Lemaitre et al, 1987, Proc. Natl. Acad. Sci. USA 84:648-52; PCT Publication No. W0 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W0 89/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6; Lemaitre et al, 1987, Proc. Natl. Acad. Sci.
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988, Bio/Techniques 6:958-16) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-49).
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • One aspect of the invention pertains to isolated proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise antibodies directed against a polypeptide of the invention.
  • the native polypeptide can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • polypeptides of the invention are produced by recombinant DNA techniques.
  • a polypeptide of the invention can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein").
  • the protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.
  • pure or “isolated” as used herein preferably has the same numerical limits as “purified” or "isolated” immediately above.
  • isolated and purified do not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure (e.g., lacking contaminating proteins, or chromatography reagents such as denaturing agents and polymers, e.g., acrylamide or agarose) substances or solutions.
  • purified or isolated preparations will lack any contaminating proteins from the same animal from which the protein is normally produced, as can be accomplished by recombinant expression of, for example, a human protein in a non-human cell.
  • Biologically active portions of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the protein (e.g., the amino acid sequence shown in any of SEQ ID NOs:2, 5, 8, 11, 14, or 17), which include fewer amino acids than the full length protein, and exhibit at least one activity of the corresponding full-length protein.
  • biologically active portions comprise a domain or motif with at least one activity of the corresponding protein.
  • a biologically active portion of a protein of the invention can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the invention.
  • Preferred polypeptides have the amino acid sequence of SEQ ID Nos:2, 5, 8, 11, 14,
  • 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 determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-8), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-7).
  • Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389- 402).
  • PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • Another preferred, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988, CABIOS 4:11-7). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
  • a "chimeric protein” or “fusion protein” comprises all or part (preferably biologically active) of a polypeptide of the invention operably linked to a heterologous polypeptide (i.e., a polypeptide other than the same polypeptide of the invention).
  • a heterologous polypeptide i.e., a polypeptide other than the same polypeptide of the invention.
  • the tenn "operably linked” is intended to indicate that the polypeptide of the invention and the heterologous polypeptide are fused in-frame to each other.
  • the heterologous polypeptide can be fused to the N-terminus or C-terminus of the polypeptide of the invention.
  • fusion protein is a GST fusion protein in which the polypeptide of the invention is fused to the C-terminus of GST sequences.
  • Such fusion proteins can facilitate the purification of a recombinant polypeptide of the invention.
  • the fusion protein contains a heterologous signal peptide at its N-terminus.
  • the native signal peptide of a polypeptide of the invention can be removed and replaced with a signal peptide from another protein.
  • the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal peptide (Current Protocols in Molecular Biology, 1992, Ausubel et al., eds., John Wiley & Sons).
  • eukaryotic heterologous signal peptides include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California).
  • useful prokaryotic heterologous signal peptides include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).
  • the fusion protein is an immunoglobulin fusion protein in which all or part of a polypeptide of the invention is fused to sequences derived from a member of the immunoglobulin protein family.
  • the immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand (soluble or membrane-bound) and a protein on the surface of a cell (receptor), to thereby suppress signal transduction in vivo.
  • the immunoglobulin fusion protein can be used to affect the bioavailability of a cognate ligand of a polypeptide of the invention.
  • Inhibition of ligand receptor interaction may be useful therapeutically, both for treating proliferative and differentiative disorders and for modulating (e.g., promoting or inhibiting) cell survival.
  • the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies directed against a polypeptide of the invention in a subject, to purify ligands and in screening assays to identify molecules which inhibit the interaction of receptors with ligands.
  • Chimeric and fusion proteins of the invention can be produced by standard recombinant DNA techniques.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel et al., supra).
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide of the invention.
  • a signal peptide of a polypeptide of the invention can be used to facilitate secretion and isolation of the secreted protein or other proteins of interest.
  • Signal peptides are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events.
  • Such signal peptides contain processing sites that allow cleavage of the signal peptide from the mature proteins as they pass through the secretory pathway.
  • a nucleic acid sequence encoding a signal peptide of the invention can be operably linked in an expression vector to a protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate.
  • the signal peptide directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal peptide is subsequently or concurrently cleaved.
  • the protein can then be readily purified from the extracellular medium by art recognized methods.
  • the signal peptide can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.
  • the signal peptides of the present invention can be used to identify regulatory sequences, e.g., promoters, enhancers, repressors. Since signal peptides are the most amino-terminal sequences of a peptide, it is expected that the nucleic acids which flank the signal peptide on its amino-terminal side will be regulatory sequences which affect transcription. Thus, a nucleotide sequence which encodes all or a portion of a signal peptide can be used as a probe to identify and isolate signal peptides and their flanking regions, and these flanking regions can be studied to identify regulatory elements therein. 0 The present i •nventi •on also pertai •ns to vari ⁇ ants of the polypeptides of the invention.
  • Such variants have an altered amino acid sequence which can function as either agonists
  • Nariants can be generated by mutagenesis, e.g., discrete point mutation or truncation.
  • An agonist can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the protein.
  • An antagonist of a 5 protei ⁇ n can i ⁇ nhi ⁇ bit one or more of the activities of the naturally occurring form of the protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest.
  • specific biological effects can be elicited by treatment with a variant of limited function.
  • Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring 0 form of the protein can have fewer side effects in a subject relative to treatment with the naturally occurring form of the protein.
  • Modification of the structure of the subject polypeptides can be for such purposes as enhancing therapeutic or prophylactic efficacy, stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo), or post-translational modifications (e.g., to
  • modified peptides when designed to retain at least one activity of the naturally-occurring form of the protein, or to produce specific antagonists thereof, are considered functional equivalents of the polypeptides described in more detail herein.
  • modified peptides can be produced, for instance, by amino acid substitution, deletion, or addition.
  • Whether a change in the amino acid sequence of a peptide results in a functional homolog can be readily determined by assessing the ability of the variant peptide to produce a response in cells in a fashion similar to the wild-type protein, or competitively inhibit such a response.
  • Polypeptides in which more than one replacement has taken place can readily be tested in the same manner.
  • Nariants of a protein of the invention which function as either agonists (mimetics) or as antagonists can be identified by screening combinatorial libraries of mutants, e.g. , truncation mutants, of the protein of the invention for agonist or antagonist activity.
  • a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).
  • a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).
  • libraries of fragments of the coding sequence of a polypeptide of the invention can be used to generate a variegated population of polypeptides for screening and subsequent selection of variants.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded D ⁇ A, renaturing the D ⁇ A to form double stranded D ⁇ A which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest.
  • REM Recursive ensemble mutagenesis
  • An isolated polypeptide of the invention, or a fragment thereof, can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length polypeptide or protein can be used or, alternatively, the invention provides antigenic peptide fragments for use as immunogens.
  • the antigenic peptide of a protein of the invention comprises at least 8 (preferably 10, 15, 20, or 30) amino acid residues of the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23,
  • Preferred epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, e.g., hydrophilic regions. Hydropathy plots or similar analyses can be used to identify hydrophilic regions.
  • An immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal).
  • a suitable subject e.g., rabbit, goat, mouse or other mammal.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed or chemically synthesized polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention, e.g., an epitope of a polypeptide of the invention.
  • a molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies.
  • the term "monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope.
  • Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide of the invention as an immunogen.
  • Preferred polyclonal antibody compositions are ones that have been selected for antibodies directed against a polypeptide or polypeptides of the invention.
  • Particularly preferred polyclonal antibody preparations are ones that contain only antibodies directed against a polypeptide or polypeptides of the invention.
  • Particularly preferred immunogen compositions are those that contain no other human proteins such as, for example, immunogen compositions made using a non-human host cell for recombinant expression of a polypeptide of the invention. In such a manner, the only human epitope or epitopes recognized by the resulting antibody compositions raised against this immunogen will be present as part of a polypeptide or polypeptides of the invention.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibodies specific for a protein or polypeptide of the invention can be selected for (e.g., partially purified) or purified by, e.g., affinity chromatography.
  • a recombinantly expressed and purified (or partially purified) protein of the invention is produced as described herein, and covalently or non-covalently coupled to a solid support such as, for example, a chromatography column.
  • the column can then be used to affinity purify antibodies specific for the proteins of the invention from a sample containing antibodies directed against a large number of different epitopes, thereby generating a substantially purified antibody composition, i.e., one that is substantially free of contaminating antibodies.
  • a substantially purified antibody composition is meant, in this context, that the antibody sample contains at most only 30% (by dry weight) of contaminating antibodies directed against epitopes other than those on the desired protein or polypeptide of the invention, and preferably at most 20%>, yet more preferably at most 10%, and most preferably at most 5% (by dry weight) of the sample is contaminating antibodies.
  • a purified antibody composition means that at least 99% of the antibodies in the composition are directed against the desired protein or polypeptide of the invention.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique (Kohler and Milstein, 1975, Nature 256:495-1), 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., pgs. 77-96) or trioma techniques.
  • standard techniques such as the hybridoma technique (Kohler and Milstein, 1975, Nature 256:495-1), 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., pgs. 77-96) or trioma techniques.
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.
  • a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide of interest.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAPJ Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No.
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; and Boss et al., U.S. Patent No.
  • Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • 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.
  • Fully human antibodies are particularly desirable for therapeutic treatment of human patients.
  • Such antibodies can be produced , for example, using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
  • Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA and IgE antibodies.
  • Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g., a murine antibody
  • a completely human antibody recognizing the same epitope is used to guide the selection of a completely human antibody recognizing the same epitope.
  • an antibody may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxcrubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (I) (IDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincri
  • the drug moiety can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example , a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, ⁇ -interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2
  • IL-2 interleukin-6
  • GM- 1 granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent
  • An antibody directed against a polypeptide of the invention can be used to isolate the polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, such an antibody can be used to detect the protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide.
  • the antibodies can also be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, 8-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 1, 131 1, 35 S or 3 H.
  • an antibody can be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorabicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents
  • antimetabolites e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine
  • alkylating agents e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine
  • cyclothosphamide busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin
  • anthracyclines e.g., daunorubicin (formerly daunomycin) and doxorabicin
  • antibiotics e.g., dactinomycin
  • anti- mitotic agents e.g., vincristine and vinblastine.
  • the drug moiety can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, ⁇ - interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
  • the invention provides substantially purified antibodies or fragment thereof, and human or non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of any one of SEQ
  • amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4; and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of any one of SEQ ID NOsT, 3, 4, 6, 7, 9, 10, 12, 13, 15,
  • the substantially purified antibodies of the invention, or fragments thereof can be human, non- human, chimeric and/or humanized antibodies.
  • the invention provides human or non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, or an amino acid sequence encoded by the cDNA of a clone deposited as ATCC ® Accession Number 207178, ATCC ® Accession Number PTA-249, or ATCC ® Accession Number PTA-250; a fragment of at least 15 amino acid residues of the amino acid sequence of any one of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, an amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 2, 5, 8, 11,
  • non-human antibodies can be goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies.
  • the non-human antibodies of the invention can be chimeric and/or humanized antibodies.
  • the human or non-human antibodies of the invention can be polyclonal antibodies or monoclonal antibodies.
  • the invention provides monoclonal antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, or an amino acid sequence encoded by the cDNA of a clone deposited as ATCC ® Accession Number 207178, ATCC ®
  • amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4; and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of any one of SEQ ID NOs: 1, 3, 4, 6, 7,
  • the monoclonal antibodies can be human, humanized, chimeric and/or non-human antibodies.
  • the substantially purified antibodies or fragments thereof specifically bind to a signal peptide, a secreted sequence, an extracellular domain, a transmembrane or a cytoplasmic domain cytoplasmic membrane of a polypeptide of the invention.
  • the substantially purified antibodies or fragments thereof, the human or non-human antibodies or fragments thereof, and/or the monoclonal antibodies or fragments thereof, of the invention specifically bind to a secreted sequence or an extracellular domain of the amino acid sequence of SEQ ID NOs:103, 107, 114, 118, 122, 129, or 134.
  • the secreted sequence or extracellular domain to which the secreted sequence or extracellular domain specifically bind to a signal peptide, a secreted sequence, an extracellular domain, a transmembrane or a cytoplasmic domain cytoplasmic membrane of a polypeptide of the invention.
  • a antibody, or fragment thereof, binds comprises from about amino acids 25-374 of SEQ ID NO:5 (SEQ ID NO: 103), from amino acids 1-73 of SEQ ID NO:8 (SEQ ID NO: 107), from amino acids 21-767 of SEQ ID NOT4 (SEQ ID NOT 14), from amino acids 1-216 of SEQ ID NOT7 (SEQ ID NOT 18), from amino acids 1-500 of SEQ ID NO:20 (SEQ ID NO: 122) from amino acids 20-169 of SEQ ID NO:26 (SEQ ID NO: 129), and from amino acids 22- 15 244 of SEQ ID NO:29 (SEQ ID NO:134).
  • any of the antibodies of the invention can be conjugated to a therapeutic moiety or to a detectable substance.
  • detectable substances that can be conjugated to the antibodies of the invention are an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, and a radioactive 0 material.
  • the invention also provides a kit containing an antibody of the invention conjugated to a detectable substance, and instructions for use.
  • Still another aspect of the invention is a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical 5 composition contains an antibody of the invention, a therapeutic moiety, and a pharmaceutically acceptable carrier.
  • Still another aspect of the invention is a method of making an antibody that specifically recognizes INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272,
  • TANGO 295, TANGO 354, and TANGO 378 the method comprising immunizing a 0 mammal with a polypeptide.
  • the polypeptide used as an immunogen comprises an amino acid sequence selected from the group consisting of: the amino acid sequence of any one of
  • a sample is collected from the mammal that contains an antibody that specifically recognizes GPVI.
  • the polypeptide is recombinantly produced using a non-human host cell.
  • the antibodies can be further purified from the sample using techniques well known to those of skill in the art.
  • the method can further comprise producing a monoclonal antibody-producing cell from the cells of the mammal.
  • antibodies are collected from the antibody-producing cell.
  • vectors preferably expression vectors, containing a nucleic acid encoding a polypeptide of the invention (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • 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 retrovirases, 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.
  • 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, 1990, Academic Press, San Diego, CA. Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
  • the recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells (using baculovirus expression vectors), yeast cells or mammalian cells). Suitable host cells are discussed further in Goeddel, supra.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three 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,
  • GST glutathione S-transferase
  • suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al.,1988, Gene 69:301-15) and pET lid (Studier et al., Gene Expression Technology: Methods in Enzymology, 1990, Academic Press, San Diego, CA pgs. 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET 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.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, Gene Expression Technology: Methods in Enzymology, 1990, Academic Press, San Diego, CA pgs. 119-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., 1992, Nucleic Acids Res. 20:2111-8). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerivisae include pYepSecl (Baldari et al., 1987, EMBOJ. 6:229-34), pMFa (Kurjan and Herskowitz, 1982, Cell 30:933-43), pJRY88 20 (Schultz et al, 1987, Gene 54:113-23), pYES2 (Invitrogen Corporation, San Diego, CA), and pPicZ (Invitrogen Corp, San Diego, CA).
  • the expression vector is a baculoviras expression vector.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al., 1983, Mol. Cell Biol. 3:2156-65) and the pVL series (Lucklow 25 and Summers, 1989, Virology 170:31-9).
  • 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 (Kaufinan et al., 1987, EMBO J. 6:187-95).
  • pCDM8 Seed, 1987, Nature 329:840
  • pMT2PC Kaufinan et al., 1987, EMBO J. 6:187-95.
  • control functions are often provided by viral regulatory elements.
  • promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian
  • Virus 40 for other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al., supra.
  • 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. 1:268-77), lymphoid-specific promoters (Calame and Eaton, 1988, Adv. Immunol. 43:235- 75), in particular promoters of T cell receptors (Winoto and Baltimore, 1989, EMBO J.
  • promoters are also encompassed, for example the murine hox promoters (Kessel and Grass, 1990, Science 249:314-9) and the ⁇ - fetoprotein promoter (Campes and Tilghman, 1989, Genes Dev. 3:537-46).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to the mRNA encoding a polypeptide of the invention.
  • Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • host cell and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell (e.g., insect cells, yeast or mammalian cells).
  • prokaryotic e.g., E. coli
  • eukaryotic cell e.g., insect cells, yeast or mammalian cells.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drags, such as G418, hygromycin and methotrexate.
  • Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • an endogenous e.g., INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, and TANGO 3708 nucleic acid within a cell, cell line or microorganism
  • a DNA regulatory element heterologous to the endogenous gene of interest into the genome of a cell, stable cell line or cloned microorganism such that the inserted regulatory element is operatively linked with the endogenous gene (e.g., INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, and TANGO 378) and controls, modulates or activates the endogenous gene.
  • INTERCEPT 340 endogenous INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, and TANGO 378 which are normally "transcriptionally silent", i.e., INTERCEPT 340,
  • MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, andTANGO 378 genes which are normally not expressed, or are expressed only at very low levels in a cell line or microorganism, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell line or microorganism.
  • a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell line or microorganism.
  • MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, andTANGO 378 genes may be activated by insertion of a promiscuous regulatory element that works across cell types.
  • a heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with and activates expression of endogenous INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295,
  • TANGO 354, and TANGO 378 genes using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, and described e.g., in Chappel, U.S. Patent No. 5,272,071 ; PCT publication No. WO 91/06667, published May 16, 1991.
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce a polypeptide of the invention. Accordingly, the invention further provides methods for producing a polypeptide of the invention using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell.
  • the host cells of the invention can also be used to produce nonhuman transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a sequences encoding a polypeptide of the invention have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous sequences encoding a polypeptide of the invention have been introduced into their genome or homologous recombinant animals in which endogenous encoding a polypeptide of the invention sequences have been altered.
  • Such animals are useful for studying the function and/or activity of the polypeptide and for identifying and/or evaluating modulators of polypeptide activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • an "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing nucleic acid encoding a polypeptide of the invention (or a homologue thereof) into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the polypeptide of the invention to particular cells.
  • transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of mRNA encoding the transgene in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying the transgene can further be bred to other transgenic animals carrying other transgenes.
  • a vector which contains at least a portion of a gene encoding a polypeptide of the invention into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the gene.
  • the vector is designed such that, upon homologous recombination, the endogenous gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous protein).
  • the altered portion of the gene is flanked at its 5' and 3' ends by additional nucleic acid of the gene to allow for homologous recombination to occur between the exogenous gene carried by the vector and an endogenous gene in an embryonic stem cell.
  • the additional flanking nucleic acid sequences are of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5' and 3' ends
  • flanking DNA both at the 5' and 3' ends
  • the vector is introduced into an embryonic stem cell line (e.g., by elecfroporation) and cells in which the introduced gene has homologously recombined with the endogenous gene are selected (see, e.g., Li et al., 1992, Cell 69:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see, e.g., Bradley in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, 1987, . Robertson, ed., IRL, Oxford pgs. 113-52).
  • 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.
  • FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al., 1991, Science 251:1351-5).
  • 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 385:810-3 and PCT Publication NOs. WO 97/07668 and WO 97/07669.
  • compositions suitable for administration 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.
  • the invention includes methods for preparing pharmaceutical compositions for modulating the expression or activity of a polypeptide or nucleic acid of the invention. Such methods comprise formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of the invention. Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of the invention and one or more additional active compounds.
  • the agent which modulates expression or activity may, for example, be a small molecule.
  • such small molecules include peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • organic or inorganic compounds i.e., including heteroorganic and organometallic compounds
  • doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
  • Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g. about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram.
  • appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drag combination, and the degree of expression or activity to be modulated.
  • 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
  • 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 ampules, 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 ELJ (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 polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, 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.
  • compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or saccharin
  • the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza 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 preferred dosage is 0.1 mg/kg to 100 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. (1997, J Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).
  • a therapeutically effective amount of protein or polypeptide ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Patent 5,328,470) or by stereotactic injection (see, e.g., Chen et al., 1994, Proc. Natl. Acad. Sci. USA 91:3054-7).
  • 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.
  • methods of treatment e.g., therapeutic and prophylactic.
  • the INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295 e.g., the INTERCEPT 340
  • TANGO 354, and TANGO 378 polypeptides of the invention can to used to modulate cellular function, survival, morphology, proliferation, and/or differentiation of the cells in which they are expressed.
  • the polypeptides of the invention can be used to treat diseases such as neoplastic disorders (e.g., cancer, tumors), hematopoietic disorders
  • the isolated nucleic acid molecules of the invention can be used to express proteins (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect mRNA (e.g., in a biological sample) or a genetic lesion, and to modulate activity of a polypeptide of the invention.
  • the polypeptides of the invention can be used to screen drags or compounds which modulate activity or expression of a polypeptide of the invention as well as to treat disorders characterized by insufficient or excessive production of a protein of the invention or production of a form of a protein of the invention which has decreased or aberrant activity compared to the wild type protein.
  • the antibodies of the invention can be used to detect and isolate a protein of the invention and modulate activity of a protein of the invention.
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drags) which bind to polypeptide of the invention or have a stimulatory or inhibitory effect on, for example, expression or activity of a polypeptide of the invention.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drags) which bind to polypeptide of the invention or have a stimulatory or inhibitory effect on, for example, expression or activity of a polypeptide of the invention.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a polypeptide of the invention or biologically active portion thereof.
  • the test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non- peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12:145).
  • Libraries of compounds may be presented in solution (e.g., Houghten, 1992, Bio/Techniques 13:412-21), or on beads (Lam, 1991, Nature 354:82-4), chips (Fodor, 1993, Nature 364:555-6), bacteria (U.S. Patent No. 5,223,409), spores (U.S. Patent NOs. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al, 1992, Proc. Natl. Acad.
  • an assay is a cell-based assay in which a cell which expresses a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to the polypeptide determined.
  • the cell for example, can be a yeast cell or a cell of mammalian origin. Determining the ability of the test compound to bind to the polypeptide can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the polypeptide or biologically active portion thereof can be determined by detecting the labeled compound in a complex.
  • test compounds can be labeled with 125 1, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay comprises contacting a cell which expresses a membrane- bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or a biologically active portion thereof as compared to the known compound.
  • the assay involves assessment of an activity characteristic of the polypeptide, wherein binding of the test compound with the polypeptide or a biologically active portion thereof alters (e.g., increases or decreases) the activity of the polypeptide.
  • an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the polypeptide or biologically active portion thereof.
  • Detennining the ability of the test compound to modulate the activity of the polypeptide or a biologically active portion thereof can be accomplished, for example, by determining the ability of the polypeptide protein to bind to or interact with a target molecule or to transport molecules across the cytoplasmic membrane.
  • a target molecule is a molecule with which a selected polypeptide (e.g., a polypeptide of the invention binds or interacts with in nature, for example, a molecule on the surface of a cell which expresses the selected protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.
  • a target molecule can be a polypeptide of the invention or some other polypeptide or protein.
  • a target molecule can be a component of a signal transduction pathway which facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a compound to a polypeptide of the invention) through the cell membrane and into the cell or a second intercellular protein which has catalytic activity or a protein which facilitates the association of downstream signaling molecules with a polypeptide of the invention. Determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule.
  • an extracellular signal e.g., a signal generated by binding of a compound to a polypeptide of the invention
  • the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca 2+ , diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g. luciferase), or detecting a cellular response, for example, cellular differentiation, or cell proliferation.
  • a reporter gene e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g. luciferase
  • detecting a cellular response for example, cellular differentiation, or cell proliferation.
  • an assay of the present invention is a cell-free assay comprising contacting a polypeptide of the invention or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to the polypeptide or biologically active portion thereof. Binding of the test compound to the polypeptide can be determined either directly or indirectly as described above.
  • the assay includes contacting the polypeptide of the invention or biologically active portion thereof with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or biologically active portion thereof as compared to the known compound.
  • an assay is a cell-free assay comprising contacting a polypeptide of the invention or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the polypeptide or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished, for example, by determining the ability of the polypeptide to bind to a target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished by determining the ability of the polypeptide of the invention to further modulate the target molecule.
  • the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as previously described.
  • the cell-free assay comprises contacting a polypeptide of the invention or biologically active portion thereof with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the polypeptide to preferentially bind to or modulate the activity of a target molecule.
  • the cell-free assays of the present invention are amenable to use of both a soluble form or the membrane-bound form of a polypeptide of the invention.
  • non-ionic detergents such as n-octylglucoside, n-d
  • binding of a test compound to the polypeptide, or interaction of the polypeptide with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or A polypeptide of the invention, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above.
  • glutathione sepharose beads Sigma Chemical; St. Louis, MO
  • glutathione derivatized microtiter plates which are then combined with the test compound or the test compound and either the non-adsorbed target protein or A polypeptide of the invention, and the mixture incubated under conditions conducive to complex formation (e
  • the complexes can be dissociated from the matrix, and the level of binding or activity of the polypeptide of the invention can be determined using standard techniques.
  • Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention.
  • either the polypeptide of the invention or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated polypeptide of the invention or target molecules can be prepared from biotin- NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with the polypeptide of the invention or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the invention trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the polypeptide of the invention or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the polypeptide of the invention or target molecule.
  • modulators of expression of a polypeptide of the invention are identified in a method in which a cell is contacted with a candidate compound and the expression of the selected mRNA or protein (i.e., the mRNA or protein corresponding to a polypeptide or nucleic acid of the invention) in the cell is determined.
  • the level of expression of the selected mRNA or protein in the presence of the candidate compound is compared to the level of expression of the selected mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of expression of the polypeptide of the invention based on this comparison. For example, when expression of the selected mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of the selected mRNA or protein expression.
  • the candidate compound when expression of the selected mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of the selected mRNA or protein expression.
  • the level of the selected mRNA or protein expression in the cells can be determined by methods described herein.
  • polypeptide of the inventions can be used as
  • bait proteins in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No.
  • binding proteins are also likely to be involved in the propagation of signals by the polypeptide of the inventions as, for example, upstream or downstream elements of a signaling pathway involving the polypeptide of the invention.
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.
  • sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. Accordingly, nucleic acid molecules described herein or fragments thereof, can be used to map the location of the corresponding genes on a chromosome. The mapping of the sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
  • genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the sequence of a gene of the invention.
  • Computer analysis of the sequence of a gene of the invention can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process.
  • These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the gene sequences will yield an amplified fragment.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the nucleic acid sequences of the invention to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a gene to its chromosome include in situ hybridization (described in Fan et al., 1990, Proc. Natl. Acad. Sci. USA 87:6223-7), pre-screening with labeled flow-sorted chromosomes (CITE), and pre-selection by hybridization to chromosome specific cDNA libraries.
  • in situ hybridization described in Fan et al., 1990, Proc. Natl. Acad. Sci. USA 87:6223-7
  • CITE labeled flow-sorted chromosomes
  • Fluorescence in situ hybridization of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
  • FISH Fluorescence in situ hybridization
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping 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 a gene of the invention can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • nucleic acid sequences disclosed herein can be used to perform searches against "mapping databases", e.g., BLAST-type search, such that the chromosome position of the gene is identified by sequence homology or identity with known sequence fragments which have been mapped to chromosomes.
  • mapping databases e.g., BLAST-type search
  • a polypeptide and fragments and sequences thereof and antibodies specific thereto can be used to map the location of the gene encoding the polypeptide on a chromosome. This mapping can be carried out by specifically detecting the presence of the polypeptide in members of a panel of somatic cell hybrids between cells of a first species of animal from which the protein originates and cells from a second species of animal and then determining which somatic cell hybrid(s) expresses the polypeptide and noting the chromosome(s) from the first species of animal that it contains. For examples of this technique, see Pajunen et al, 1988, Cytogenet. Cell Genet. 47:37-41 and Van Keuren et al., 1986, Hum. Genet. 74:34-40.
  • the presence of the polypeptide in the somatic cell hybrids can be determined by assaying an activity or property of the polypeptide, for example, enzymatic activity, as described in Bordelon-Riser et al., 1979, Somatic Cell Genetics 5:597-613 and Owerbach et al., 1978, Proc. Natl. Acad. Sci. USA 75:5640-5644.
  • the nucleic acid sequences of the present invention can also be used to identify individuals from minute biological samples.
  • the United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymorphism
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the c sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the nucleic acid sequences described herein can 0 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 5 be used to obtain such identification sequences from individuals and from tissue.
  • the nucleic acid sequences of the invention uniquely represent portions of the human genome.
  • allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
  • Each of the 0 sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals.
  • 22, 25, and 28 can comfortably provide positive individual identification with a panel of 5 perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases.
  • a panel of reagents from the nucleic acid sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • positive identification of the individual, living or dead can be made from extremely small tissue samples.
  • DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a 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 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 nucleic acid sequences of the invention or portions thereof, e.g., fragments derived from noncoding regions having a length of at least 20 or 30 bases.
  • nucleic acid sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such probes can be used to identify tissue by species and/or by organ type.
  • polynucleotide reagents e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such probes can be used to identify tissue by species and/or by organ type.
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically.
  • one aspect of the present invention relates to diagnostic assays for determining INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 protein and/or nucleic acid expression as well as INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 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 or unwanted INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, in
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 protein or nucleic acid expression or activity. For example, mutations in a 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 protein or nucleic acid expression or activity.
  • determinations may be based on the normalized expression levels of these genes.
  • Expression levels are normalized by correcting the absolute expression level of a INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 gene by comparing its expression to the expression of a gene that is not a INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378, e.g., a housekeeping gene that is constitutively expressed.
  • Suitable genes for normalization include housekeeping genes such as the actin gene. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, e.g., a non-disease sample, or between samples from different sources.
  • the expression level can be provided as a relative expression level.
  • the level of expression of the gene is determined for 10 or more samples of different cell isolates, preferably 50 or more samples, prior to the determination of the expression level for the sample in question.
  • the mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the gene(s) in question.
  • the expression level of the gene determined for the test sample is then divided by the mean expression value obtained for that gene. This provides a relative expression level and aids in identifying extreme cases of disease.
  • the samples used in the baseline determination will be from diseased or from non-diseased cells of tissue.
  • the choice of the cell source is dependent on the use of the relative expression level. Using expression found in normal tissues as a mean expression score aids in validating whether the INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 gene assayed is diseased cell-type specific (versus normal cells). Such a use is particularly important in identifying whether a INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 gene can serve as a target gene.
  • the mean expression value can be revised, providing improved relative expression values based on accumulated data. Expression data from cells provide a means for grading the severity of the disease state.
  • Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 genes in clinical trials.
  • agents e.g., drugs, compounds
  • An exemplary method for detecting the presence or absence of a polypeptide or nucleic acid of the invention in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of the invention such that the presence of a polypeptide or nucleic acid of the invention is detected in the biological sample.
  • a preferred agent for detecting mRNA or genomic DNA encoding a polypeptide of the invention is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA encoding a polypeptide of the invention.
  • the nucleic acid probe can be, for example, a full-length cDNA, such as the nucleic acid of SEQ ID NOs , 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a polypeptide of the invention.
  • a full-length cDNA such as the nucleic acid of SEQ ID NOs , 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a polypeptide of the invention.
  • Other suitable probes for use in the diagnostic assays of the invention are described
  • a preferred agent for detecting a polypeptide of the invention is an antibody capable of binding to a polypeptide of the invention, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g. , Fab or F(ab') 2 ) can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of a polypeptide of the invention include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of a polypeptide of the invention include introducing into a subject a labeled antibody directed against the polypeptide.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting a polypeptide of the invention or mRNA or genomic DNA encoding a polypeptide of the invention, such that the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide is detected in the biological sample, and comparing the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide in the control sample with the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide in the test sample.
  • kits for detecting the presence of a polypeptide or nucleic acid of the invention in a biological sample can be used to determine if a subject is suffering from or is at increased risk of developing a disorder associated with aberrant expression of a polypeptide of the invention (e.g., a proliferative disorder, e.g., psoriasis or cancer).
  • a disorder associated with aberrant expression of a polypeptide of the invention e.g., a proliferative disorder, e.g., psoriasis or cancer.
  • the kit can comprise a labeled compound or agent capable of detecting the polypeptide or mRNA encoding the polypeptide in a biological sample and means for determining the amount of the polypeptide or mRNA in the sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide).
  • Kits can also include instractions for observing that the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of the polypeptide if the amount of the polypeptide or mRNA encoding the polypeptide is above or below a normal level.
  • the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.
  • a first antibody e.g., attached to a solid support
  • a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.
  • the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule encoding a polypeptide of the invention.
  • the kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent.
  • the kit can also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate).
  • the kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained.
  • Each component of the kit is usually enclosed within an individual container and all of the various containers are within a single package along with instractions for observing whether the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of the polypeptide.
  • the methods described herein can furthermore be utilized as diagnostic or prognostic assays to identify subjects having or at risk of developing a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention.
  • the assays described herein such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with aberrant expression or activity of a polypeptide of the invention.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing such a disease or disorder.
  • the present invention provides a method in which a test sample is obtained from a subject and a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of the invention is detected, wherein the presence of the polypeptide or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant expression or activity of the polypeptide.
  • a 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.
  • a biological fluid e.g., serum
  • cell sample e.g., cell sample
  • tissue e.g., tissue
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • such methods can be used to determine whether a subject can be effectively treated with a specific agent or class of agents (e.g., agents of a type which decrease activity of the
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant expression or activity of a polypeptide of the invention in which a test sample is obtained and the polypeptide or nucleic acid encoding the polypeptide is detected (e.g., wherein the presence of the polypeptide or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant expression or activity of the polypeptide).
  • the methods of the invention can also be used to detect genetic lesions or mutations in a gene of the invention, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized aberrant expression or activity of a polypeptide of the invention.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion or mutation characterized by at least one of an alteration affecting the integrity of a gene encoding the polypeptide of the invention, or the mis-expression of the gene encoding the polypeptide of the invention.
  • such genetic lesions or mutations can be detected by ascertaining the existence of at least one of: 1) a deletion of one or more nucleotides from the gene; 2) an addition of one or more nucleotides to the gene; 3) a substitution of one or more nucleotides of the gene; 4) a chromosomal rearrangement of the gene; 5) an alteration in the level of a messenger RNA transcript of the gene; 6) an aberrant modification of the gene, such as of the methylation pattern of the genomic DNA; 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of the gene; 8) a non- wild type level of a the protein encoded by the gene; 9) an allelic loss of the gene; and 10) an inappropriate post-translational modification of the protein encoded by the gene.
  • assay techniques known in the art which can be used for detecting lesions in a gene.
  • detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent NOs. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., 1988, Science 241:1077-80; and Nakazawa et al., 1994, Proc. Natl. Acad.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to the selected gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • nucleic acid e.g., genomic, mRNA or both
  • Alternative amplification methods include: self sustained sequence replication (Guatelli et al, 1990, Proc. Natl. Acad. Sci. USA 87:1874-78), transcriptional amplification system (Kwoh, et al, 1989, Proc. Natl. Acad. Sci. USA 86:1173-7), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6:1191), 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 selected gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes 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 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 7:244-55;
  • genetic mutations can be identified in two-dimensional arrays containing light-generated DNA probes as described in
  • a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
  • Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the selected gene and detect mutations by comparing the sequence of the sample nucleic acids with the corresponding wild-type (control) sequence.
  • sequencing reactions include those based on techniques developed by Maxim and Gilbert (1977, Proc. Natl. Acad. Sci. USA 74:560) or Sanger (1977, Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays developed by Naeve et al. (1995, Bio/Techniques 19:448-53), including sequencing by mass spectrometry (see, e.g., PCT Publication No. WO 94/16101; Cohen et al., 1996, Adv. Chromatogr. 36:127-62; and Griffin et al., 1993, Appl. Biochem. Biotechnol. 38:147-59).
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in a selected gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al., 1985, Science 230:1242).
  • the technique of mismatch cleavage entails providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • 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 217:286-95.
  • the control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called DNA mismatch repair enzymes) in defined systems for detecting and mapping point mutations in cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes proteins that recognize mismatched base pairs in double-stranded DNA
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al., 1994, Carcinogen esis 15:1657-62).
  • a probe based on a selected sequence is hybridized to a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in genes.
  • SSCP single strand conformation polymorphism
  • SSCP single strand conformation polymorphism
  • DNA fragments of sample and control nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, and the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al.,
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely 1 c denature, for example by adding a "GC clamp of approximately 40 bp of high-melting GC- rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA
  • 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
  • oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • PCR amplification may be used in conjunction with the instant invention. Oligonucleotides
  • 30 used as p ⁇ mers 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,
  • 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 aperfect 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 prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a gene encoding a polypeptide of the invention.
  • any cell type or tissue, preferably peripheral blood leukocytes, in which the polypeptide of the invention is expressed may be utilized in the prognostic assays described herein.
  • Agents, or modulators which have a stimulatory or inhibitory effect on activity or expression of a polypeptide of the invention as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders associated with aberrant activity of the polypeptide.
  • the pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drag
  • 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 drag.
  • the pharmacogenomics of the individual permits the selection of effective agents (e.g., drags) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of a polypeptide of the invention, expression of a nucleic acid of the invention, or mutation content of a gene of the invention in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drags due to altered drag disposition and abnormal action in affected persons. See, e.g., Linder, 1997, Clin. Chem. 43(2):254-66. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drags act on the body are referred to as "altered drug action.”
  • G6PD glucose-6-phosphate dehydrogenase deficiency
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • drag metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, a PM will show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • the activity of a polypeptide of the invention, expression of a nucleic acid encoding the polypeptide, or mutation content of a gene encoding the polypeptide in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • pharmacogenetic studies can be used to apply genotyping of polymo ⁇ hic alleles encoding drag-metabolizing enzymes to the identification of an individual's drag responsiveness phenotype. This knowledge, when applied to dosing or drag selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a modulator of activity or expression of the polypeptide, such as a modulator identified by one of the exemplary screening assays described herein.
  • Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of a polypeptide of the invention can be applied not only in basic drug screening, but also in clinical trials.
  • agents e.g., drugs, compounds
  • the effectiveness of an agent, as determined by a screening assay as described herein, to increase gene expression, protein levels or protein activity can be monitored in clinical trials of subjects exhibiting decreased gene expression, protein levels, or protein activity.
  • the effectiveness of an agent, as determined by a screening assay, to decrease gene expression, protein levels or protein activity can be monitored in clinical trials of subjects exhibiting increased gene expression, protein levels, or protein activity.
  • expression or activity of a polypeptide of the invention and preferably, that of other polypeptide that have been implicated in for example, a cellular proliferation disorder can be used as a marker of the immune responsiveness of a particular cell.
  • genes including those of the invention, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates activity or expression of a polypeptide of the invention (e.g., as identified in a screening assay described herein) can be identified.
  • an agent e.g., compound, drug or small molecule
  • a polypeptide of the invention e.g., as identified in a screening assay described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of a gene of the invention and other genes implicated in the disorder.
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of a gene of the invention or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of the polypeptide or nucleic acid of the invention in the preadministration sample;
  • 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
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant expression or activity of a polypeptide of the invention, e.g., cardiac infection (e.g., myocarditis or dilated cardiomyopathy), central nervous system infection (e.g., nonspecific febrile illness or meningoencephalitis), pancreatic infection (e.g., acute pancreatitis), respiratory infection (pneumonia), gastrointestinal infection, type I diabetes, cancer, familia hypercholesterolemia, treat hemophilia B, Marfan syndrome, protein S deficiency, allergy, inflammation, and gastroduodenal ulcer.
  • the polypeptides of the invention can be used to modulate cellular function, survival, mo ⁇ hology, proliferation
  • the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant expression or activity of a polypeptide of the
  • Subjects at risk for a disease which is caused or contributed to by aberrant expression or activity of a polypeptide of the invention can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms
  • an agonist or antagonist agent can be used for treating the subject.
  • the modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of the polypeptide.
  • An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of the
  • the agent stimulates one or more of the biological activities of the polypeptide.
  • stimulatory agents include the active polypeptide of the invention and a nucleic acid molecule encoding the polypeptide of the invention that has been introduced into the cell.
  • the agent inhibits one or more of the biological activities of the
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a polypeptide of the invention.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) expression or activity.
  • the method involves administering a polypeptide of the invention or a nucleic acid molecule of the invention as therapy to compensate for reduced or aberrant expression or activity of the polypeptide.
  • Stimulation of activity is desirable in situations in which activity or expression is abnormally low or downregulated and/or in which increased activity is likely to have a beneficial effect. Conversely, inhibition of activity is desirable in situations in which activity or expression is abnormally high or upregulated and/or in which decreased activity is likely to have a beneficial effect.
  • Clones containing cDNA molecules encoding human MANGO 003 were deposited with the American Type Culture Collection (ATCC ® 10801 University Boulevard, Manassas, VA 20110-2209) on March 30, 1999 as Accession Number 207178, as part of a composite deposit representing a mixture of three strains, each carrying one recombinant plasmid harboring a particular cDNA clone.
  • ATCC ® 10801 University Boulevard, Manassas, VA 20110-2209 American Type Culture Collection
  • an aliquot of the mixture can be streaked out to single colonies on nutrient medium (e.g., LB plates) supplemented with 100 g/ml ampicillin, single colonies grown, and then plasmid DNA extracted using a standard minipreparation procedure.
  • a sample of the DNA minipreparation can be digested with a combination of the restriction enzymes Sal I and Not I, and the resultant products resolved on a 0.8% agarose gel using standard DNA electrophoresis conditions. The digest liberates fragments as follows:
  • the identity of the strains can be inferred from the fragments liberated.
  • Clones containing cDNA molecules encoding human INTERCEPT 340, MANGO 347, and TANGO 272 were deposited with the American Type Culture Collection (ATCC ® 10801 University Boulevard, Manassas, NA 20110-2209) on June 18, 1999 as Accession Number PTA-250, as part of a composite deposit representing a mixture of three strains, each carrying one recombinant plasmid harboring a particular cDNA clone.
  • ATCC ® 10801 University Boulevard, Manassas, NA 20110-2209 American Type Culture Collection
  • an aliquot of the mixture can be streaked out to single colonies on nutrient medium (e.g., LB plates) supplemented with 100 g/ml ampicillin, single colonies grown, and then plasmid DNA extracted using a standard minipreparation procedure.
  • a sample of the DNA minipreparation can be digested with a combination of the restriction enzymes Sal I and Not I, and the resultant products resolved on a 0.8% agarose gel using standard DNA electrophoresis conditions. The digest liberates fragments as follows:
  • human INTERCEPT 340 (clone EpI340): 3.3 kB human MANGO 347 (clone EpM347): 1.4 kB human TANGO 272 (clone EpT272): 5.0 kB
  • the identity of the strains can be inferred from the fragments liberated.
  • Clones containing cDNA molecules encoding human TANGO 295, TANGO 354, and TANGO 378 were deposited with the American Type Culture Collection (ATCC ® 10801 University Boulevard, Manassas, VA 20110-2209) on June 18, 1999 as Accession Number PTA-249, as part of a composite deposit representing a mixture of three strains, each carrying one recombinant plasmid harboring a particular cDNA clone.
  • ATCC ® 10801 University Boulevard, Manassas, VA 20110-2209 American Type Culture Collection
  • an aliquot of the mixture can be streaked out to single colonies on nutrient medium (e.g., LB plates) supplemented with 100 g/ml ampicillin, single colonies grown, and then plasmid DNA extracted using a standard minipreparation procedure.
  • a sample of the DNA minipreparation can be digested with a combination of the restriction enzymes Sal I and Not I, and the resultant products resolved on a 0.8% agarose gel using standard DNA electrophoresis conditions. The digest liberates fragments as follows:
  • human TANGO 295 (clone EpT295) 1.5 kB human TANGO 354 (clone EpT354) 1.8 kB human TANGO 378 (clone EpT378) 3.3 kB

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Abstract

The invention provides isolated nucleic acid molecules, designated INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, and TANGO 378 which encode wholly secreted or membrane-associated proteins. The invention also provides antisense nucleic acid molecules, expression vectors containing the nucleic acid molecules of the invention, host cells into which the expression vectors have been introduced, and non-human transgenic animals in which a nucleic acid molecule of the invention has been introduced or disrupted. The invention still further provides isolated polypeptides, fusion polypeptides, antigenic peptides and antibodies. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

Description

MEMBRANE-ASSOCIATED AND SECRETED PROTEINS AND USES THEREOF
This application claims priority to co-pending U.S. Application No. 09/345,464, filed June 30, 1999, the entire contents of which are incorporated herein by reference in its entirety.
Background of the Invention
Many secreted proteins, for example, cytokines, play a vital role in the regulation of cell growth, cell differentiation, and a variety of specific cellular responses. A number of medically useful proteins, including erythropoietin, granulocyte-macrophage colony stimulating factor, human growth hormone, and various interleukins, are secreted proteins.
Many membrane-associated proteins are receptors which bind a ligand and transduce an intracellular signal, leading to a variety of cellular responses. The identification and characterization of such a receptor enables one to identify both the ligands which bind to the receptor and the intracellular molecules and signal transduction pathways associated with the receptor, permitting one to identify or design modulators of receptor activity, e.g., receptor agonists or antagonists and modulators of signal transduction.
Thus, an important goal in the design and development of new therapies is the identification and characterization of membrane-associated and secreted proteins and the genes which encode them.
Summary of the Invention
The present invention is based, at least in part, on the discovery of cDNA molecules encoding INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, and TANGO 378 all of which are either wholly secreted or transmembrane proteins. These proteins, fragments, derivatives, and variants thereof are collectively referred to as "polypeptides of the invention" or "proteins of the invention." Nucleic acid molecules encoding the polypeptides or proteins of the invention are collectively referred to as "nucleic acids of the invention."
The nucleic acids and polypeptides of the present invention are useful as modulating agents in regulating a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding a polypeptide of the invention or a biologically active portion thereof. The present invention also provides nucleic acid molecules which are suitable for use as primers or hybridization probes for the detection of nucleic acids encoding a polypeptide of the invention. The invention features nucleic acid molecules which are at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequence of SEQ ID NOs:l, 3, 4, 6, 1, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, the nucleotide sequence of the cDNA insert of a clone deposited with ATCC® as Accession Number 207178 (the "cDNA of ATCC® Accession Number 207178"), the nucleotide sequence of the cDNA insert of a clone deposited with ATCC® as Accession Number PTA-249 (the "cDNA of ATCC® Accession Number PTA-249"), or the nucleotide sequence of the cDNA insert of a clone deposited with ATCC® as Accession Number PTA-250 (the "cDNA of ATCC® Accession Number PTA-250"), or a complement thereof.
The invention features nucleic acid molecules which include a fragment of at least
10 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, or 4000) nucleotides of the nucleotide sequence of SEQ ID NOs:l, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, the nucleotide sequence of the cDNA of ATCC® Accession Number 207178, the nucleotide sequence of the cDNA of ATCC® Accession Number PTA-249, or
15 the nucleotide sequence of the cDNA of ATCC® Accession Number PTA-250, or a complement thereof.
The invention also features nucleic acid molecules which include a nucleotide sequence encoding a protein having an amino acid sequence that is at least 45 % (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NOs:2, 5, 8,
20 11, 14, 17, 20, 23, 26, or 29, the amino acid sequence encoded by the cDNA of ATCC® Accession Number 207178, the amino acid sequence encoded by the cDNA of ATCC® Accession Number PTA-249, or the amino acid sequence encoded by the cDNA of ATCC® Accession Number PTA-250.
In preferred embodiments, the nucleic acid molecules have the nucleotide sequence
25 of SEQ ID NOs:l, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, the nucleotide sequence of the cDNA of ATCC® Accession Number 207178, the nucleotide sequence of the cDNA of ATCC® Accession Number PTA-249, or the nucleotide sequence of the cDNA of ATCC® Accession Number PTA-250, or a complement thereof.
Also within the invention are nucleic acid molecules which encode a fragment of a
30 polypeptide having the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, or a fragment including at least 15 (25, 30, 50, 100, 150, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, or 1400) contiguous amino acids of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, the amino acid sequence encoded by the cDNA of ATCC® Accession Number 207178, the amino acid sequence encoded by the cDNA of ATCC®
35 Accession Number PTA-249, or the amino acid sequence encoded by the cDNA of ATCC® Accession Number PTA-250. The invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, the amino acid sequence encoded by the cDNA of ATCC® Accession Number 207178, the amino acid sequence encoded by the cDNA of ATCC® Accession Number PTA-249, or the amino acid sequence encoded by the cDNA of ATCC® Accession Number PTA-250, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule consisting of a nucleic acid sequence encoding SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, the nucleotide sequence of the cDNA of ATCC® Accession Number 207178, the nucleotide sequence of the cDNA of ATCC® Accession Number PTA-249, or the nucleotide sequence of the cDNA of ATCC® Accession Number PTA-250, or a complement thereof under stringent conditions.
Also within the invention are isolated polypeptides or proteins having an amino acid sequence that is at least about 60%, preferably 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, the amino acid sequence encoded by the cDNA of ATCC® Accession Number 207178, the amino acid sequence encoded by the cDNA of ATCC® Accession Number PTA-249, or the amino acid sequence encoded by the cDNA of ATCC® Accession Number PTA-250.
Also within the invention are isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 60%o, preferably
65%), 15%, 85%o, or 95%> identical the nucleic acid sequence encoding SEQ ID NOs:2, 5, 8,
20 11, 14, 17, 20, 23, 26, or 29, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ
ID NOs:l, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, or complement thereof, the non-coding strand of the cDNA of ATCC® Accession Number
25 207178, the non-coding strand of the cDNA of ATCC® Accession Number PTA-249, or the non-coding strand of the cDNA of ATCC® Accession Number PTA-250.
Also within the invention are polypeptides which are naturally occurring allelic variants of a polypeptide that includes the amino acid sequence of SEQ ID NOs:2, 5, 8, 11,
14, 17, 20, 23, 26, or 29, the amino acid sequence encoded by the cDNA of ATCC®
Accession Number 207178, the amino acid sequence encoded by the cDNA of ATCC®
Accession Number PTA-249, or the amino acid sequence encoded by the cDNA of ATCC®
Accession Number PTA-250, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule having the sequence of SEQ ID
NOs.l, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, or a complement
35 thereof, under stringent conditions. Such allelic variant differ at 1%, 2%, 3%, 4%, or 5% of the amino acid residues. The invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NOs: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, the cDNA of ATCC® Accession Number 207178, the cDNA of ATCC® Accession Number PTA-249, or the cDNA of ATCC® Accession Number PTA-250, or a complement thereof. In other embodiments, the nucleic acid molecules are at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, or 4200) nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NOs.T, 3, 4, 6, 1, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, the cDNA of ATCC® Accession Number 207178, the cDNA of ATCC® Accession Number PTA-249, or the cDNA of ATCC® Accession Number PTA-250, or a complement thereof.
In other embodiments, the isolated nucleic acid molecules encode an extracellular, transmembrane, or cytoplasmic domain of a polypeptide of the invention. h another embodiment, the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a nucleic acid of the invention.
Another aspect of the invention provides vectors, e.g., recombinant expression vectors, comprising a nucleic acid molecule of the invention. In another embodiment, the invention provides host cells containing such a vector or a nucleic acid molecule of the invention. The invention also provides methods for producing a polypeptide of the invention by culturing, in a suitable medium, a host cell of the invention containing a recombinant expression vector such that a polypeptide is produced.
Another aspect of this invention features isolated or recombinant proteins and polypeptides of the invention. Preferred proteins and polypeptides possess at least one biological activity possessed by the corresponding naturally-occurring human polypeptide.
An activity, a biological activity, or a functional activity of a polypeptide or nucleic acid of the invention refers to an activity exerted by a protein, polypeptide or nucleic acid molecule of the invention on a responsive cell as determined in vivo, or in vitro, according to standard techniques. Such activities can be a direct activity, such as an association with or an enzymatic activity on a second protein or an indirect activity, such as a cellular signaling activity mediated by interaction of the protein with a second protein.
In one embodiment, the isolated polypeptide of the invention lacks both a transmembrane and a cytoplasmic domain. In another embodiment, the polypeptide lacks both a transmembrane domain and a cytoplasmic domain and is soluble under physiological conditions. For INTERCEPT 340, biological activities include, e.g., (1) the ability to form protein-protein interactions with proteins in the signaling pathway of the naturally- occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; (3) the ability to interact with an INTERCEPT 340 receptor, e.g., a cell surface receptor (e.g., an integrin); (4) the ability to modulate the activity of an intracellular molecule that participates in a signal transduction pathway, e.g., an intracellular molecule in the integrin signalling (e.g., a cdk2 inhibitor); (5) the ability to assemble into fibrils; (6) the ability to strengthen and organize the extracellular matrix; (7) the ability to modulate the shape of tissues and cells; (8) the ability to interact with (e.g., bind to) components of the extracellular matrix; and (9) the ability to modulate cell migration. Other activities include the ability to modulate function, survival, morphology, migration, proliferation and/or differentiation of cells of tissues in which it is expressed (e.g., splenic cells). For example, additional biological activities of INTERCEPT 340 include: (1) the ability to modulate splenic cell activity; (2) the ability to modulate skeletal morphogenesis; and/or (3) the ability to modulate smooth muscle cell proliferation and differentiation.
For MANGO 003, biological activities include, e.g., (1) the ability to form protein- protein (e.g., protein-ligand) interactions with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to interact with (e.g., bind to) a ligand of the naturally-occurring polypeptide; (3) the ability to interact with a MANGO 003 receptor, e.g., a cell surface receptor; (4) the ability to modulate cell surface recognition; (5) the ability to transduce an extracellular signal (e.g., by interacting with a ligand and/or a cell- surface receptor); (6) the ability to modulate a signal transduction pathway; and (7) the ability to modulate signal transmission at a chemical synapse. Other activities include the ability to modulate function, survival, morphology, proliferation and/or differentiation of cells of tissues in which it is expressed (e.g., thyroid, liver, skeletal muscle, kidney, heart, lung, testis and brain). For example, the activities of MANGO 003 can include modulation of endocrine, hepatic, skeletal muscular, renal, cardiovascular, reproductive and/or brain function.
For MANGO 347, biological activities include, e.g., (1) the ability to form protein- protein interactions with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to interact with a ligand of the naturally-occurring polypeptide;
(3) the ability to interact with a MANGO 347 receptor; and (4) the ability to modulate a developmental process, e.g., morphogenesis, cellular migration, adhesion, proliferation, differentiation, and/or survival. Other activities include the ability to modulate function, survival, morphology, proliferation and/or differentiation of cells of tissues in which it is expressed (e.g., brain cells). For example, the activities of MANGO 347 can include modulation of neural (e.g., CNS) function.
For TANGO 272, biological activities include, e.g., (1) the ability to form protein- protein interactions with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; (3) the ability to interact with a TANGO 272 receptor, e.g., a cell surface receptor (e.g., an integrin); (4) the ability to modulate cell-cell contact; (5) the ability to modulate cell attachment; (6) the ability to modulate cell fate; and (7) the ability to modulate tissue repair and/or wound healing. Other activities include the ability to modulate function, survival, morphology, proliferation and/or differentiation of cells of tissues in which it is expressed (e.g., microvascular endothelial cells). For example, the activities of MANGO 347 can include modulation of cardiovascular function.
For TANGO 295, biological activities include, e.g., (1) the ability to form protein- protein interactions with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; (3) the ability to interact with a TANGO 295 receptor; (4) the ability to interact with (e.g., bind to) a nucleic acid; and (5) the ability to elicit pyrimidine-specific endonuclease activity. Other activities include the ability to modulate function, survival, morphology, proliferation and/or differentiation of cells of tissues in which it is expressed (e.g., mammary epithelium).
For TANGO 354, biological activities include, e.g., (1) the ability to fomi protein- protein interactions with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; (3) the ability to interact with (e.g., bind to) a TANGO 354 receptor, e.g., a cell surface receptor; (4) the ability to modulate cell surface recognition; (5) the ability to modulate cellular motility, e.g., chemotaxis and/or chemokinesis; (6) the ability to transduce an extracellular signal (e.g., by interacting with a ligand and/or a cell-surface receptor); and (7) the ability to modulate a signal transduction pathway. Other activities include the ability to modulate function, survival, morphology, proliferation and/or differentiation of cells of tissues in which it is expressed (e.g., hematopoietic tissues). For example, TANGO 354 biological activities can further include: (1) regulation of hematopoiesis; (2) modulation (e.g., increasing or decreasing) of haemostasis; (3) modulation of an inflammatory response; (4) modulation of neoplastic growth, e.g., inhibition of tumor growth; and (5) modulation of thrombolysis.
For TANGO 378, biological activities include, e.g., (1) the ability to form protein- protein interactions with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; (3) the ability to interact with a TANGO 378 receptor; (4) the ability to transduce an extracellular signal; and (5) the ability to modulate a signal transduction pathway (e.g., adenylate cyclase, or phosphatidylinositol 4,5-bisphosphate (PrP2), inositol 1,4,5-triphosphate (IP3)). Other activities include the ability to modulate function, survival, morphology, proliferation and/or differentiation of cells of tissues in which it is expressed (e.g., natural killer cells). For example, TANGO 378 biological activities can further include the ability to modulate an immune response in a subject, for example, (1) by modulating immune cytotoxic responses against pathogenic organisms, e.g., viruses, bacteria, and parasites; (2) by modulating organ rejection after transplantation; and (3) by modulating immune recognition and lysis of normal and malignant cells.
In one embodiment, a polypeptide of the invention has an amino acid sequence sufficiently identical to an identified domain of a polypeptide of the invention. As used herein, the term "sufficiently identical" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity. For example, amino acid or nucleotide sequences which contain a common structural domain having about 60% identity, preferably 65%> identity, more preferably 75%, 85%, 95%>, 98% or more identity are defined herein as sufficiently identical.
In one embodiment, a MANGO 003, MANGO 347, TANGO 272, TANGO 295,
TANGO 354, or TANGO 378 polypeptide of the invention includes a signal peptide.
In another embodiment, a nucleic acid molecule of the invention encodes a
MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, orTANGO 378 polypeptide which includes a signal peptide.
In another embodiment, a MANGO 003, TANGO 272, TANGO 354, or TANGO
378 polypeptide of the invention includes one or more of the following domains: (1) a signal peptide; (2) an N-terminal extracellular domain; (3) a C-terminal transmembrane domain; and (4) a cytoplasmic domain.
The polypeptides of the present invention, or biologically active portions thereof, can be operably linked to a heterologous amino acid sequence to form fusion proteins. In one embodiment, the fusion protein consists of a chimeric protein assembled from portions of the protein from different species.
In one embodiment, the isolated polypeptide of the invention lacks both a transmembrane and a cytoplasmic domain. In another embodiment, the polypeptide lacks both a transmembrane domain and a cytoplasmic domain and is soluble under physiological conditions.
The invention further features antibodies that specifically bind a polypeptide of the invention such as monoclonal or polyclonal antibodies. In addition, the polypeptides of the invention or biologically active portions thereof, or antibodies of the invention, can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
In another aspect, the present invention provides methods for detecting the presence of the activity or expression of a polypeptide of the invention in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of activity such that the presence of activity is detected in the biological sample.
In another aspect, the invention provides methods for modulating activity of a polypeptide of the invention comprising contacting a cell with an agent that modulates (inhibits or stimulates) the activity or expression of a polypeptide of the invention such that activity or expression in the cell is modulated. In one embodiment, the agent is an antibody that specifically binds to a polypeptide of the invention.
In another embodiment, the agent modulates expression of a polypeptide of the invention by modulating transcription, splicing, or translation of an mRNA encoding a polypeptide of the invention. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an mRNA encoding a polypeptide of the invention.
The present invention also provides methods to treat a subject having a disorder characterized by aberrant activity of a polypeptide of the invention or aberrant expression of a nucleic acid of the invention by administering an agent which is a modulator of the activity of a polypeptide of the invention or a modulator of the expression of a nucleic acid of the invention to the subject. In one embodiment, the modulator is a protein of the invention. In another embodiment, the modulator is a nucleic acid of the invention. In other embodiments, the modulator is a peptide, peptidomimetic, or other small organic molecule. The present invention also provides diagnostic assays for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of: (i) aberrant modification or mutation of a gene encoding a polypeptide of the invention, (ii) mis- regulation of a gene encoding a polypeptide of the invention, and (iii) aberrant post- translational modification of the invention wherein a wild-type form of the gene encodes a protein having the activity of the polypeptide of the invention.
In another aspect, the invention provides a method for identifying a compound that binds to or modulates the activity of a polypeptide of the invention. In general, such methods entail measuring a biological activity of the polypeptide in the presence and absence of a test compound and identifying those compounds which alter the activity of the polypeptide.
The invention also features methods for identifying a compound which modulates the expression of a polypeptide or nucleic acid of the invention by measuring the expression of the polypeptide or nucleic acid in the presence and absence of the compound. In yet a further aspect, the invention provides substantially purified antibodies or fragments thereof including human and non-human antibodies or fragments thereof which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number 207178, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-249, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-250; a fragment of at least 15 amino acid residues of the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29; an amino acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4; and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of SEQ ID NOs:l, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C. In various embodiments, the substantially purified antibodies of the invention, or fragments thereof can be human, non-human, chimeric and/or humanized antibodies.
Any of the antibodies of the invention can be conjugated to a therapeutic moiety or to a detectable substance. Non-limiting examples of detectable substances that can be conjugated to the antibodies of the invention are an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, and a radioactive material.
The invention also provides a kit containing an antibody of the invention conjugated to a detectable substance, and instructions for use. Still another aspect of the invention is a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier. In preferred embodiments, the pharmaceutical composition contains an antibody of the invention, a therapeutic moiety, and a pharmaceutically acceptable carrier.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
Brief Description of the Drawings
Figures 1A-1B depict the cDNA sequence of human INTERCEPT 340 (SEQ ID NO:l) and the predicted amino acid sequence of INTERCEPT 340 (SEQ ID NO:2). The open reading frame of SEQ ID NO.T extends from nucleotide 1222 to nucleotide 1944 of SEQ ID NO.T (SEQ ID NO:3).
Figure 2 depicts a hydropathy plot of human INTERCEPT 340. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) and potential N- glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of INTERCEPT 340 are indicated. The amino acid sequence of each of the fibrillar collagen C-terminal domains are indicated by underlining and the abbreviation "COLF".
Figure 3 depicts an alignment of each of the fibrillar collagen C-terminal domains (also referred to herein as "COLF domains") of human INTERCEPT 340 with consensus hidden Markov model COLF domains. For each alignment, the upper sequence is the consensus amino acid sequence (SEQ ID NOs:31, 32, and 33), while the lower sequence amino acid sequence corresponds to amino acid 58 to amino acid 116 of SEQ ID NO:2 (SEQ ID NO:34), amino acid 126 to amino acid 151 of SEQ ID NO:2 (SEQ ID NO:35), and amino acid 186 to amino acid 217 of SEQ ID NO:2 (SEQ ID NO:36).
Figures 4A-4C depict the cDNA sequence of human MANGO 003 (SEQ ID NO:4) and the predicted amino acid sequence of MANGO 003 (SEQ ID NO:5). The open reading frame of SEQ ID NO:4 extends from nucleotide 57 to nucleotide 1568 of SEQ ID NO:4 (SEQ ED NO:6).
Figure 5 depicts a hydropathy plot of human MANGO 003. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) and potential N-glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of MANGO 003 are indicated. The amino acid sequence of each of the immunoglobulin domains, and the neurotransmitter gated ion channel domain are indicated by underlining and the abbreviations "ig" and "neur chan", respectively.
Figure 6 depicts an alignment of each of the immunoglobulin domains (also referred to herein as "Ig domains") of human MANGO 003 with the consensus hidden Markov model immunoglobulin domains. For each alignment, the upper sequence is the consensus sequence (SEQ ID NO:37), while the lower sequence corresponds to amino acid 44 to amino acid 101 of SEQ ID NO:5 (SEQ ID NO:38), amino acid 165 to amino acid 223 of SEQ ID NO:5 (SEQ ID NO:39), and amino acid 261 to amino acid 340 of SEQ ID NO:5 (SEQ ID NO:40).
Figure 7 depicts an alignment of the neurotransmitter gated ion channel domain of human MANGO 003 with the consensus hidden Markov model neurotransmitter gated ion channel domain. The upper sequence is the consensus sequence (SEQ ID NO:42), while the lower sequence corresponds to amino acid 388 amino acid 397 of SEQ ID NO: 5 (SEQ ID NO:43).
Figure 8 depicts the cDNA sequence of mouse MANGO 003 (SEQ ID NO:7) and the predicted amino acid sequence of MANGO 003 (SEQ ID NO:8). The open reading frame of SEQ ID NO:7 extends from nucleotide 1 to nucleotide 626 of SEQ ID NO:4 (SEQ ID NO:9).
Figure 9 depicts a hydropathy plot of mouse MANGO 003. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) and potential N-glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of mouse MANGO 003 are indicated.
Figure 10 depicts the cDNA sequence of human MANGO 347 (SEQ ID NO: 10) and the predicted amino acid sequence of MANGO 347 (SEQ ID NOT 1). The open reading frame of SEQ ID NO:10 extends from nucleotide 31 to nucleotide 444 of SEQ ID NO:10 (SEQ ID NO: 12).
Figure 11 depicts a hydropathy plot of human MANGO 347. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of MANGO 347 are indicated. The amino acid sequence of the CUB domain is indicated by underlining and the abbreviation "CUB".
Figure 12 depicts an alignment of the CUB domain of human MANGO 347 with a consensus hidden Markov model CUB domain. The upper sequence is the consensus amino acid sequence (SEQ ID NO:44), while the lower sequence corresponds to amino acid 40 to amino acid 136 of SEQ ID NO: 11 (SEQ ID NO:45).
Figures 13A-13D depict the cDNA sequence of human TANGO 272 (SEQ ID NO:13) and the predicted amino acid sequence of TANGO 272 (SEQ ID NO:14). The open reading frame of SEQ ID NO:13 extends from nucleotide 230 to nucleotide 3379 of SEQ ID NO:13 (SEQ ID O:15).
Figure 14 depicts a hydropathy plot of human TANGO 272. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) and potential N- glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of TANGO 272 are indicated. The amino acid sequence of each of the fourteen EGF-like domains and the delta serrate ligand domain is indicated by underlining and the abbreviation "EGF-like" and "DSL", respectively.
Figures 15A-15C depict an alignment of each of the EGF-like domains of human TANGO 272 with consensus hidden Markov model EGF-like domains. The upper sequence is the consensus amino acid sequence (SEQ ID NO:46), while the lower sequence corresponds to amino acid 151 to amino acid 181 of SEQ ID NO: 14 (SEQ ID NO:49); amino acid 200 to amino acid 229 of SEQ ID NO: 14 (SEQ ID NO:50); amino acid 242 to amino acid 272 of SEQ ID NO: 14 (SEQ ID NO:51); amino acid 285 to amino acid 315 of SEQ ID NO: 14 (SEQ ID NO:52); amino acid 328 to amino acid 358 of SEQ ID NO: 14
10 (SEQ ID NO:53); amino acid 378 to amino acid 404 of SEQ ID NO: 14 (SEQ ID NO:54); amino acid 417 to amino acid 447 of SEQ ID NO:14 (SEQ ID NO:55); amino acid 460 to amino acid 490 of SEQ ID NO: 14 (SEQ ID NO:56); amino acid 503 to amino acid 533 of SEQ ID NO:14 (SEQ ID NO:57); amino acid 546 to amino acid 576 of SEQ ID NO:14 (SEQ ID NO:58); amino acid 589 to amino acid 619 of SEQ ID NO: 14 (SEQ ID NO:59);
15 amino acid 632 to amino acid 661 of SEQ ID NO: 14 (SEQ ID NO:60); amino acid 674 to amino acid 704 of SEQ ID NO:14 (SEQ ID NO:61); and amino acid 717 amino acid 747 of SEQ ID NO.T4 (SEQ ID NO:62). For alignment of the delta serrate ligand domain, the upper sequence is the consensus hidden Markov model (SEQ ID NO:47), while the lower sequence corresponds to amino acid 518 to amino acid 576 of SEQ ID NO: 14 (SEQ ID
20 NO:63).
Figures 16A-16B depict the cDNA sequence of mouse TANGO 272 (SEQ ID NO:16) and the predicted amino acid sequence of TANGO 272 (SEQ ID NO:17). The open reading frame of SEQ ID NO: 16 extends from nucleotide 1 to nucleotide 1492 of SEQ ID NO:16 (SEQ ID NO:18).
25 Figure 17 depicts a hydropathy plot of mouse TANGO 272. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) and potential N-glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of mouse TANGO
30 272 are indicated.
Figure 18 depicts the cDNA sequence of human TANGO 295 (SEQ ID NO:22) and the predicted amino acid sequence of TANGO 295 (SEQ ID NO:23). The open reading frame of SEQ ID NO:22 extends from nucleotide 217 to nucleotide 684 of SEQ ID NO:28 (SEQ ID NO:24).
35 Figure 19 depicts a hydropathy plot of human TANGO 295. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) and potential N- glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of human TANGO 295 are indicated. The amino acid sequence of the pancreatic ribonuclease domain is indicated by underlining and the abbreviation "RNase A".
Figure 20 depicts an alignment of the pancreatic ribonuclease domain of human TANGO 295 with a consensus hidden Markov model pancreatic ribonuclease domain. The upper sequence is the consensus amino acid sequence (SEQ ID NO:96), while the lower sequence corresponds to amino acid 32 to amino acid 156 of SEQ ID NO:23 (SEQ ID NO:97).
Figures 21A-21B depict the cDNA sequence of human TANGO 354 (SEQ ID NO:25) and the predicted amino acid sequence of TANGO 354 (SEQ ID NO:26). The open reading frame of SEQ ID NO:25 extends from nucleotide 62 to nucleotide 976 of SEQ ID NO:25 (SEQ ID NO:27).
Figure 22 depicts a hydropathy plot of human TANGO 354. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) and potential N- glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of human TANGO 354 are indicated. The amino acid sequence of the immunoglobulin domain is indicated by underlining and the abbreviation "ig".
Figure 23 depicts an alignment of the immunoglobulin domain of human TANGO 354 with a consensus hidden Markov model immunoglobulin domains. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 37), while the lower sequence corresponds to amino acid 33 to amino acid 110 of SEQ ID NO:26 (SEQ ID NO:41).
Figures 24A-24C depict the cDNA sequence of human TANGO 378 (SEQ ID NO:28) and the predicted amino acid sequence of TANGO 378 (SEQ ID NO:29). The open reading frame of SEQ ID NO:28 extends from nucleotide 42 to nucleotide 1625 of SEQ ID NO:28 (SEQ ID NO:30).
Figure 25 depicts a hydropathy plot of human TANGO 378. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) and potential N- glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of human TANGO 378 are indicated. The amino acid sequence of the seven transmembrane domain is indicated by underlining and the abbreviation "7tm". Figure 26 depicts an alignment of the seven transmembrane receptor domain of human TANGO 378 with a consensus hidden Markov model of this domain. The upper sequence is the consensus amino acid sequence (SEQ ID NO:98), while the lower sequence corresponds to amino acid 187 to amino acid 515 of SEQ ID NO:29 (SEQ ID NO:99).
Figures 27A-27C depict a global alignment between the nucleotide sequence of the open reading frame (ORF) of human MANGO 003 (SEQ ID NO:6) and the nucleotide sequence of the open reading frame of mouse MANGO 003 (SEQ ID NO:9). The upper sequence is the human MANGO 003 ORF nucleotide sequence, while the lower sequence is the mouse MANGO 003 ORF nucleotide sequence. These nucleotides sequences share a 31.1% identity. The global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of -1212; Myers and Miller, 1989, CABIOS 4:11-1).
Figures 28A-28B depict a local alignment between the nucleotide sequence of human MANGO 003 (SEQ ID NO:4) and the nucleotide sequence of mouse MANGO 003 (SEQ ID NO:7). The upper sequence is the human MANGO 003 nucleotide sequence, while the lower sequence is the mouse MANGO 003 nucleotide sequence. These nucleotides sequences share a 62.8 % identity over nucleotide 970 to nucleotide 2080 of the human MANGO 003 sequence (nucleotide 10 to nucleotide 1070 of mouse MANGO 003). The local alignment was performed using the L- ALIGN program version 2.0u54 July 1996 (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a score of 3241; Huang and Miller, 1991, Adv. Appl. Math. 12:373-381).
Figure 29 depicts a global alignment between the amino acid sequence of human MANGO 003 (SEQ ID NO:5) and the amino acid sequence of mouse MANGO 003 (SEQ ID NO:8). The upper sequence is the human MANGO 003 amino acid sequence, while the lower sequence is the mouse MANGO 003 amino acid sequence. These amino acid sequences share a 30.1% identity. The global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of -488; Myers and Miller, 1989, C4R/OS4.T 1-7).
Figures 30A-30E depict a global alignment between the nucleotide sequence of the open reading frame (ΟRF) of human TANGO 272 (SEQ ID NO: 15) and the nucleotide sequence of the open reading frame of mouse TANGO 272 (SEQ ID NO:18). The upper sequence is the mouse TANGO 272 ORF nucleotide sequence, while the lower sequence is the human TANGO 272 ORF nucleotide sequence. These nucleotides sequences share a 39.1% identity. The global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of -79; Myers and Miller, 1989, CABIOS 4:11-1). Figures 31A-31D depict a local alignment between the nucleotide sequence of human TANGO 272 (SEQ ID NO: 13) and the nucleotide sequence of mouse TANGO 272 (SEQ ID NO: 16). The upper sequence is the human TANGO 272 nucleotide sequence, while the lower sequence is the mouse TANGO 272 nucleotide sequence. These nucleotides sequences share a 67.6 % identity over nucleotide 1890 to nucleotide 4610 of the human TANGO 272 sequence (nucleotide 10 to nucleotide 2560 of mouse TANGO 272). The local alignment was performed using the L-ALIGN program version 2.0u54 July 1996 (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a score of 8462; Huang and Miller, 1991, Adv. Appl. Math. 12:373-381).
Figures 32A-32B depict a global alignment between the amino acid sequence of human TANGO 272 (SEQ ID NO: 14) and the amino acid sequence of mouse TANGO 272 (SEQ ID NO: 17). The upper sequence is the human TANGO 272 amino acid sequence, while the lower sequence is the mouse TANGO 272 amino acid sequence. These amino acid sequences share a 38.2% identity. The global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of -19; Myers and Miller, 1989, CABIOS 4:11-7).
Figures 33A-33D depict the cDNA sequence of rat TANGO 272 (SEQ ID NO: 19) and the predicted amino acid sequence of TANGO 272 (SEQ ID NO:20). The open reading frame of SEQ ID NO:19 extends from nucleotide 925 to nucleotide 2832 of SEQ ID NO:19
(SEQ ID NO:21).
Figures 34A-34H depict a global alignment between the nucleotide sequence of human TANGO 272 (SEQ ID NO:13) and the nucleotide sequence of rat TANGO 272
(SEQ ID NO:19). The upper sequence is the human TANGO 272 nucleotide sequence, while the lower sequence is the rat TANGO 272 nucleotide sequence. These nucleotides sequences share a 55.7% identity. The global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of 8635; Myers and Miller, 1989, G4R/OS 4:l l-7).
Figures 35A-35F depict a global alignment between the nucleotide sequence of mouse TANGO 272 (SEQ ID NO:16) and the nucleotide sequence of rat TANGO 272
(SEQ ID NO: 19). The upper sequence is the mouse TANGO 272 nucleotide sequence, while the lower sequence is the rat TANGO 272 nucleotide sequence. These nucleotides sequences share a 43.1% identity. The global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of 2827; Myers and Miller, 1989, CABIOS 4:11-7).
Figure 36 depicts a global alignment of the human TANGO 295 and GenPept
AF037081 amino acid sequences. The upper sequence is the human TANGO 295 sequence
(SEQ ID NO:23), while the lower sequence is the GenPept AF037081 sequence (SEQ ID NO-.100). GenPept AF037081 encodes a ribonuclease k6 protein. The global alignment revealed a 53.2% identity between these two sequences (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of 405; Myers and Miller, 1989, CABIOS 4:11-7).
Figures 37A-37C depict a global alignment of the human TANGO 295 (SEQ ID NO:22) and GenPept AF037081 (SEQ ID NO: 100) nucleotide sequences. The upper sequence is the human TANGO 295 sequence, while the lower sequence is the GenPept AF037081 sequence. The global alignment revealed a 22.6% identity between these two sequences (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of -2718; Myers and Miller, 1989, CABIOS 4:11-1).
10 Figures 38A-38B depict a local alignment of the human TANGO 295 (SEQ ID NO:22) and GenPept AF037081 (SEQ ID NO: 100) nucleotide sequences. The upper sequence is the human TANGO 295 sequence, while the lower sequence is the GenPept AF037081 sequence. The local alignment revealed a 62.7% identity between nucleotide 235 to nucleotide 687 of human TANGO 295, and nucleotide 3 to nucleotide 453 of
15 AF037081; 43.4% identity between nucleotide 410 to nucleotide 850 of human TANGO ■ 295, and nucleotide 3 to nucleotide 450 of AF037081; and 46.5% identity between nucleotide 432 to nucleotide 700 of human TANGO 295, and nucleotide 5 to nucleotide 251 of AF037081 (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of 1214; Huang and Miller, 1991, Adv. Appl. Math. 12:373-381).
20 Figures 39A-39B depict an alignment of each of the EGF-like domains and laminin- EGF-like domains of mouse TANGO 272 with consensus hidden Markov model EGF-like domains. For alignments of the EGF-like domains, the upper sequence is the consensus amino acid sequence (SEQ ID NO:46), while the lower sequence corresponds to amino acids 37-67 of SEQ ID NO:17 (SEQ ID NO:64); amino acid 80 to amino acid 110 of SEQ
25 ID NO.T 7 (SEQ ID NO:65); amino acid 123 to amino acid 153 of SEQ ID NO.T 7 (SEQ ID NO:66); and amino acid 166 to amino acid 196 of SEQ ID NO: 17 (SEQ ID NO:67). For alignments of the laminin/EGF-like domains, the upper sequence is the consensus hidden Markov model domain (SEQ ID NO:48), while the lower sequence corresponds to amino acid 3 to amino acid 37 of SEQ ID NO: 17 (SEQ ID NO:68); amino acid 41 to amino acid
30 80 of SEQ ID NO: 17 (SEQ ID NO:69); amino acid 83 to amino acid 123 of SEQ ID NO: 17 (SEQ ID NO:70); and amino acid 127 to amino acid 172 of SEQ ID NO:17 (SEQ ID NO:71). For alignment of the delta serrate ligand domain, the upper sequence is the consensus hidden Markov model domain (SEQ ID NO:47), while the lower sequence corresponds to amino acid 10 to amino acid 67 of SEQ ID NO: 17 (SEQ ID NO:72).
35 Figure 40 depicts a hydropathy plot of rat TANGO 272. Relatively hydrophobic residues are above the dashed horizontal line, and relatively hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) and potential N-glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. Below the hydropathy plot, the numbers corresponding to the amino acid sequence of rat TANGO 272 are indicated.
Figures 41A-41D depict an alignment of each of the EGF-like domains and laminin- EGF-like domains of rat TANGO 272 with consensus hidden Markov model of EGF-like domains. For alignments of the EGF-like domains, the upper sequence is the consensus amino acid sequence (SEQ ID NO:46), while the lower sequence corresponds to amino acid 18 to amino acid 48 of SEQ ID NO:20 (SEQ ED NO:73); amino acid 61 to amino acid 91 of SEQ ID NO:20 (SEQ ID NO:74); amino acids 105-137 of SEQ ID NO:20 (SEQ ID
10 NO:75); amino acids 150-180 of SEQ ID NO:20 (SEQ ID NO:76); amino acids 193-223 of SEQ ID NO:20 (SEQ ID NO:77); amino acids 236-266 of SEQ ID NO:20 (SEQ ID NO:78); amino acids 279-309 of SEQ ID NO:20 (SEQ ID NO:79); amino acids 322-352 of SEQ ID NO:20 (SEQ ID NO:80); amino acids 365-394 of SEQ ID NO:20 (SEQ ID NO:81); amino acids 407-437 of SEQ ID NO:20 (SEQ ID NO:82); and amino acids 450-
15 480 of SEQ ID NO:20 (SEQ ED NO:83). For alignments of the laminin/EGF-like domains, the upper sequence is the consensus hidden Markov model domain (SEQ ID NO:48), while the lower sequence corresponds to amino acids 22-61 of SEQ ID NO:20 (SEQ ID NO:84); amino acids 65-105 of SEQ ID NO:20 (SEQ ID NO:85); amino acids 109-150 of SEQ ID NO:20 (SEQ ID NO:86); amino acids 154-193 of SEQ ID NO:20 (SEQ ID NO:87); amino 0 acids 197-236 of SEQ ID NO:20 (SEQ ID NO:88); amino acids 240-279 of SEQ ID NO:20 (SEQ ID NO:89); amino acids 283-322 of SEQ ID NO:20 (SEQ ID NO:90); amino acids 326-365 of SEQ ID NO:20 (SEQ ID NO:91); amino acids 368-407 of SEQ ID NO:20 (SEQ ID NO:92); amino acids 411-450 of SEQ ID NO:20 (SEQ ID NO:93); and amino acids 454- 489 of SEQ ID NO:20 (SEQ ID NO:94). For alignment of the delta serrate ligand domain, 5 the upper sequence is the consensus hidden Markov model domain (SEQ ID NO:47), while the lower sequence corresponds to amino acids 246-309 of SEQ ID NO:20 (SEQ ID NO:95).
Detailed Description of the Invention 0 The present invention is based, at least in part, on the discovery of cDNA molecules encoding INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, and TANGO 378, all of which are either wholly secreted or transmembrane proteins.
The proteins and nucleic acid molecules of the present invention comprise a family 5 of molecules having certain conserved structural and functional features. As used herein, the term "family" is intended to mean two or more proteins or nucleic acid molecules having a common structural domain and having sufficient amino acid or nucleotide sequence identity as defined herein. Family members can be from either the same or different species. For example, a family can comprise two or more proteins of human origin, or can comprise one or more proteins of human origin and one or more of non- human origin. Members of the same family may also have common structural domains.
For example, INTERCEPT 340 family members can include at least one, preferably two, and more preferably three fibrillar collagen C-terminal domains (also referred to herein as "COLF domains"). As used herein, a "fibrillar collagen C-terminal domain" refers to an amino acid sequence of about 15 to 65, preferably about 20-60, more preferably about 25, 31-58 amino acids in length. Consensus hidden Markov model COLF domains contain the sequence of SEQ ID NOs:31, 32, and 33 (Figure 3). The more conserved residues in the consensus sequence are indicated by uppercase letters and the less conserved residues in the consensus sequence are indicated by lowercase letters. A comparison of the C-terminal sequences of fibrillar collagens, collagens X, NIII, and the collagen Clq revealed a conserved cluster of amino acid residues having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) that exhibited marked similarities in hydrophilicity profiles between the different collagens, despite a low level of sequence similarity. These similarities in hydrophilicity profiles within their C-termini suggest that these proteins may adopt a common tertiary structure and that the conserved cluster of aromatic residues in this domain may be involved in C-terminal trimerization. The COLF domains of INTERCEPT 340 extend from about amino acids 58 to 116, 126 to 151, and 186 to 217 of SEQ ID NO:2 (SEQ ID NOs:34, 35, and 36, respectively) (Figure 3). By alignment of the amino acid sequence of the consensus hidden Markov model COLF amino acid sequence with the amino acid sequence of the COLF domains of INTERCEPT 340, conserved amino acid residues having aromatic side chains can be found. For example, conserved tyrosine, tryptophan and phenylalanine residues can be found at amino acid 87, 88 and 133 of SEQ
ID NO:2.
MANGO 003 and TANGO 354 family members can include at least one, preferably two, and more preferably three immunoglobulin domains. As used herein, an
"immunoglobulin domain" (also referred to herein as "Ig") refers to an amino acid sequence of about 45 to 85, preferably about 55-80, more preferably about 57, 58, or 78, 79 amino acids in length. Preferably, the immunoglobulin domains have a bit score for the alignment of the sequence to the Ig family Hidden Markov Model (HMM) of at least 10, preferably
20-30, more preferably 22-40, more preferably 40-50, 50-75, 75-100, 100-200 or greater.
The Ig family HMM has been assigned the PFAM Accession PF00047. Consensus hidden
Markov model immunoglobulin domains are shown Figures 6 and 23 (SEQ ID NO:37).
The more conserved residues in the consensus sequence are indicated by uppercase letters and the less conserved residues in the consensus sequence are indicated by lowercase letters. Immunoglobulin domains are present in a variety of proteins (including secreted and membrane-associated proteins). Membrane-associated proteins may be involved in protein-protein, and protein-ligand interaction at the cell surface, and thus may influence diverse activities including cell surface recognition and or signal transduction. The immunoglobulin domains of MANGO 003 extend from about amino acids 44 to 101, 165 to 223, and 261 to 240 of SEQ ED NO:5 (SEQ ID NOs:38, 39, and 40, respectively) (Figure 6). The immunoglobulin domain of TANGO 354 extend from about amino acids 33 to 110 of SEQ ID NO:26 (SEQ ED NO:41) (Figure 23).
MANGO 003 family member can include a neurotransmitter-gated ion channel domain. As used herein, a "neurotransmitter-gated ion channel domain" refers to an amino acid sequence of about 5 to 20, preferably about 7 to 12, more preferably about 9 to 10 amino acids in length. The neurotransmitter-gated ion channel domain HMM has been assigned the PFAM Accession PF00065. A consensus hidden Markov model neurotransmitter-gated ion chaimel domain contain the sequence of SEQ ED NO:42 shown in Figure 7. The more conserved residues in the consensus sequence are indicated by uppercase letters and the less conserved residues in the consensus sequence are indicated by lowercase letters. The neurotransmitter-gated ion channel domains of MANGO 003 extend from about amino acids 388 to 397 of SEQ ID NO:5 (SEQ ID NO:43).
TANGO 272 family members can include at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, preferably thirteen, and more preferably fourteen
EGF-like domains. Preferably, the EGF-like domains are found in the extracellular domain of a TANGO 272 protein. As used herein, an "EGF-like domain" refers to an amino acid sequence of about 25 to 50, preferably about 30 to 45, and more preferably 30 to 40 amino acid residues in length. An EGF domain further contains at least about 2 to 10, preferably,
3 to 9, 4 to 8, or 6 to 7 conserved cysteine residues. A consensus hidden Markov model
EGF-like domain sequence includes six cysteines, all of which are thought to be involved in disulfide bonds having the following amino acid sequence: Cys-Xaa(5, 7)-Cys-Xaa(4, 5,
12)-Cys-Xaa(l, 5, 6)-Cys-Xaa(l)-Cys-Xaa(l)- Cys-Xaa(8)-Cys (SEQ ID NO:46), where
Xaa is any amino acid. The region between the fifth and the sixth cysteine typically contains two conserved glycines of which at least one is present in most EGF-like domains.
In one embodiment, TANGO 272 includes at least one EGF-like domain having the sequences selected from the group consisting of: amino acids 151-181 of SEQ ID NO: 14
(SEQ ID NO:49); amino acids 200-229 of SEQ ID NO: 14 (SEQ ID NO:50); amino acids
242-272 of SEQ ID NO:14 (SEQ ID NO:51); amino acids 285-315 of SEQ ID NO:14 (SEQ ID NO:52); amino acids 328-358 of SEQ ID NO.T4 (SEQ ID NO:53); amino acids 378-404 of SEQ ID NO: 14 (SEQ ID NO:54); amino acids 417-447 of SEQ ID NO: 14 (SEQ ID NO:55); amino acids 460-490 of SEQ ID NO:14 (SEQ ED.NO:56); amino acids 503-533 of SEQ ID NO: 14 (SEQ ID NO:57); amino acids 546-576 of SEQ ID NO: 14 (SEQ ID NO:58); amino acids 589-619 of SEQ ID NO:14 (SEQ ID NO:59); amino acids 632-661 of SEQ ID NO: 14 (SEQ ID NO:60); amino acids 674-704 of SEQ ID NO: 14 (SEQ ID NO:61); and amino acids 717-747 of SEQ ED NO: 14 (SEQ ID NO:62).
In another embodiment, TANGO 272 includes at least one EGF-like domain having the sequences selected from the group consisting of: 37-67 of SEQ ID NO: 17 (SEQ ID NO:64); amino acids 80-110 of SEQ ID NO:17 (SEQ ID NO:65); amino acids 123-153 of SEQ ID NO: 17 (SEQ ID NO:66); and amino acids 166-196 of SEQ ED NO: 17 (SEQ ID NO:67).
In yet another embodiment, TANGO 272 includes at least one EGF-like domain having the sequences selected from the group consisting of: amino acids 18-48 of SEQ ID NO:20 (SEQ ID NO:73); amino acids 61-91 of SEQ ID NO:20 (SEQ ID NO:74); amino acids 105-137 of SEQ ID NO:20 (SEQ ID NO:75); amino acids 150-180 of SEQ ID NO.20 (SEQ ID NO:76); amino acids 193-223 of SEQ ID NO:20 (SEQ ID NO:77); amino acids 15 236-266 of SEQ ID NO:20 (SEQ ID NO:78); amino acids 279-309 of SEQ ID NO:20 (SEQ ID NO:79); amino acids 322-352 of SEQ ID NO:20 (SEQ ID NO:80); amino acids 365-394 of SEQ ID NO:20 (SEQ ID NO:81); amino acids 407-437 of SEQ ID NO:20 (SEQ ID NO:82); and amino acids 450-480 of SEQ ID NO:20 (SEQ ID NO:83).
An alignment of the consensus hidden Markov model EGF-like domains with the 20 EGF-like domains of human TANGO 272 is shown in Figures 15A-15C. The more conserved residues in the consensus sequence are indicated by uppercase letters and the less conserved residues in the consensus sequence are indicated by lowercase letters. By alignment of the amino acid sequence of the consensus hidden Markov model EGF-like domain with the amino acid sequence of the EGF-like domains of TANGO 272, conserved
25 cysteine residues can be found. For example, conserved cysteine residues can be found at amino acid 151, 159, 164, 167, 200, 206, 211, 218, 220, 229, 242, 249, 263, 264, 272, 285,
291, 297, 304, 306, 315, 328, 334, 340, 347, 349, 358, 378, 386, 393, 395, 404, 417, 423,
429, 436, 438, 447, 460, 466, 472, 479, 481, 490, 503, 509, 515, 522, 524, 533, 546, 552,
558, 565, 567, 576, 589, 595, 601, 608, 610, 619, 632, 637, 643, 650, 652, 661, 674, 680,
30 686, 693, 695, 717, 723, 729, 736, 738 and 747 of SEQ ID NO:14.
TANGO 272 family members can include at least one delta serrate ligand domain.
As used herein, a "delta serrate ligand domain" (also referred to herein as a "DSL domain") refers to an amino acid sequence of about 30-70, more preferably 45-60, and most preferably 58 amino acids in length typically found in transmembrane signaling molecules
35 that regulate differentiation in metazoans (Lissemore et al., 1999, Mol. Phylogenet. Evol.
11(2):308-19). In one embodiment, human TANGO 272 includes a delta serrate ligand domain from about amino acids 518 to 576 of SEQ ID NO: 14 (SEQ ID NO:63); and about amino acids 246 to 309 of SEQ ID NO:20 (SEQ ID NO:95). Figure 15B depicts an alignment of the consensus hidden Markov model delta serrate ligand domain (SEQ ID NO:47) with this domain in human TANGO 272 at amino acids 518 to 576 of SEQ ED NO: 14 (SEQ ED NO:63). Figures 39A-39B depict an alignment of the consensus hidden Markov model delta serrate ligand domain (SEQ ID NO:47) with this domain in mouse TANGO 272 at amino acids 10 to 67 of SEQ ID NO:17 (SEQ ID NO:72). Figures 41A- 41B depict an alignment of the consensus hidden Markov model delta serrate ligand domain (SEQ ID NO:47) with this domain in rat TANGO 272 at amino acids 246 to 309 of SEQ ID
NO:20 (SEQ ID NO:95).
TANGO 272 family members can include at least one RGD cell attachment site. As used herein, the term "RGD cell attachment site" refers to a cell adhesion sequence consisting of amino acids Arg-Gly-Asp typically found in extracellular matrix proteins such as collagens, laminin and fibronectin, among others (reviewed in Ruoslahti, 1996, Annu.
Rev. Cell Dev. Biol. 12:697-715). Preferably, the RGD cell attachment site is located in the extracellular domain of a TANGO 272 protein and interacts (e.g. , binds to) a cell surface receptor, such as an integrin receptor. As used herein, the term "integrin" refers to a family of receptors comprising α/β heterodimers that mediate cell attachment to extracellular matrices and cell-cell adhesion events. The subunits vary in size between 120 and 180 kDa and are each noncovalently associated with a β subunit (90-110 kDa) (reviewed by
Hynes, 1992, Cell 69:11-25). Most integrins are expressed in a wide variety of cells, and most cells express several integrins. There are at least 8 known subunits and 14 known β subunits. The majority of the integrin ligands are extracellular matrix proteins involved in substratum cell adhesion such as collagens, laminin, fibronectin among others. The RGD cell attachment site is located at about amino acid residues 177-179 of SEQ ID NO: 14.
MANGO 347 family members can include a CUB domain sequence. As used herein, the term "CUB domain" includes an amino acid sequence having at least about 80-
150, preferably 90-130, more preferably 96-120, and most preferably about 110 amino acids in length. Preferably, a CUB domain further includes at least one, preferably two, three, and most preferably four conserved cysteine residues. Preferably, the conserved cysteine residues form at least one, and preferably two disulfide bridges (e.g., Cysl-Cys2, and Cys3-
Cys4) resulting in a β-barrel configuration. The CUB domain of MANGO 347 extends from about amino acid 40 to amino acid 136 of SEQ ID NO.T 1 (SEQ ID NO:45). Figure 12 depicts an alignment of the consensus hidden Markov model CUB domain (SEQ ID NO:44) with this domain in human MANGO 347 at amino acids 40 to 136 of SEQ ED NO: 11 (SEQ ID NO:45). TANGO 295 family members can include a pancreatic ribonuclease domain sequence. As used herein, the term "pancreatic ribonuclease domain" includes an amino acid sequence having at least about 100 to 150, preferably 110-140, more preferably 120- 130, and most preferably 124 amino acids in length. Preferably, a pancreatic ribonuclease domain further includes at least one, preferably two, three, four and most preferably five conserved cysteine residues and an amino acid residue, e.g., a lysine, which is involved in catalytic activity. Preferably, at least one cysteine residue is involved in a disulfide bond, a lysine residue is involved in catalytic activity, and three other residues involved in substrate binding. Proteins having the pancreatic ribonuclease domain are pyrimidine-specific endonucleases present in high quantities in the pancreas of a number of mammalian taxa and of a few reptiles. The pancreatic ribonuclease domain of TANGO 295 extends from about amino acid 32 to amino acid 156 of SEQ ID NO:23 (SEQ ID NO:97). Figure 20 depicts an alignment of the consensus hidden Markov model pancreatic ribonuclease domain (SEQ ID NO:96) with this domain in human TANGO 295 at amino acids 32 to 156 of SEQ ID NO:23 (SEQ ID NO:97).
Based on structural similarities, TANGO 378 family members can be classified as members of the superfamily of G-protein coupled receptor. As used herein, the term "G protein-coupled receptor" or "GPCR" refers to a family of proteins that preferably comprise an N-terminal extracellular domain, seven transmembrane domains (also referred to as membrane-spanning domains), three extracellular domains (also referred to as extracellular
20 loops), three cytoplasmic domains (also referred to as cytoplasmic loops), and a C-terminal cytoplasmic domain (also referred to as a cytoplasmic tail). Members of the GPCR family also share certain conserved amino acid residues, some of which have been determined to be critical to receptor function and/or G protein signaling. An alignment of the transmembrane domains of 44 representative GPCRs can be found at
25 http://mgdkkl.nidll.nih.gov:8000/extended.html.
Accordingly, in one embodiment, TANGO 378 family members can include at least one, two, three, four, five, six, or preferably, seven transmembrane domains, and thus has a
"7 transmembrane receptor profile". As used herein, the term "7 transmembrane receptor profile" includes an amino acid sequence having at least about 10-300, preferably about 15-
30 200, more preferably about 20-100 amino acid residues, or at least about 22-100 amino acids in length and having a bit score for the alignment of the sequence to the 7tm_l family
Hidden Markov Model (HMM) of at least 10, preferably 20-30, more preferably 22-40, more preferably 40-50, 50-75, 75-100, 100-200 or greater. The 7tm_l family HMM has been assigned the PFAM Accession PF00001
35 (http://genome.wustl.edu/Pfam/V WWdata/7tm_l.html). In one embodiment, the seven transmembrane domains of TANGO 378 extend from about amino acids 245 to about amino acid 269 of SEQ ID NO:29 (SEQ ID NO: 135), about amino acids 287 to about amino acid 306 of SEQ ED NO:29 (SEQ ID NO: 136), about amino acids 323 to about amino acid 343 of SEQ ID NO:29 (SEQ ID NO: 137), about amino acids 358 to about amino acid 376 of SEQ ID NO:29 (SEQ ID NO.138), about amino acids 414 to about amino acid 438 of SEQ ID NO:29 (SEQ ID NOT39), about amino acids 457 to about amino acid 477 of SEQ ID NO:29 (SEQ ED NO: 140), and about amino acids 485 to about amino acid 504 of SEQ ID NO:29 (SEQ ID NO: 141); and a C-terminal cytoplasmic domain which extends from about amino acid 505 to amino acid 528 of SEQ ID NO:29 (SEQ ID NO:142). Figure 26 depicts an alignment of each of the transmembrane domains of TANGO 378 with the consensus hidden Markov model seven transmembrane receptor domain (SEQ ID NO:98).
To identify the presence of a 7 transmembrane receptor profile in a TANGO 378, the amino acid sequence of the protein is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters
(http://www.sanger.ac.uk/Software/Tfam/HMM_search). For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for PF00001 and score of 15 is the default threshold score for determining a hit. Alternatively, the seven transmembrane domain can be predicted based on stretches of hydrophobic amino acids forming α-helices (SOUSI server). Accordingly, proteins having at least 50-60%> identity, preferably about 60-70%, more preferably about 10-80%, or about 80-90% identity with the 7 transmembrane receptor profile of human TANGO 378 are within the scope of the invention.
TANGO 378 family members can include at least one, preferably two, and most preferably three extracellular loops. As defined herein, the term "loop" includes an amino acid sequence having a length of at least about 4, preferably about 5-10, preferably about 10-20, and more preferably about 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, or 100-150 amino acid residues, and has an amino acid sequence that connects two transmembrane domains within a protein or polypeptide. Accordingly, the N-terminal amino acid of a loop is adjacent to a C-terminal amino acid of a transmembrane domain in a naturally-occurring TANGO 378 or TANGO 378-like molecule, and the C-terminal amino acid of a loop is adjacent to an N-terminal amino acid of a transmembrane domain in a naturally-occurring TANGO 378 or TANGO 378-like molecule. As used herein, an "extracellular loop" includes an amino acid sequence located outside of a cell, or extracellularly. For example, an extracellular loop can be found at about amino acids 307- 322, 377-413, and 478-484 of SEQ ID NO:29.
TANGO 378 family members can include at least one, preferably two, and most preferably three cytoplasmic loops. As used herein, a "cytoplasmic loop" includes an amino acid sequence located within a cell or within the cytoplasm of a cell. For example, a cytoplasmic loop is found at about amino acids 270-286, 344-357, and 439-456 of SEQ ID NO:29.
In one embodiment, a MANGO 003, a TANGO 272, a TANGO 354 or a TANGO 378 family member can include one or more of the following domains: (1) an N-terminal extracellular domain, (2) a transmembrane domain, or (3) a C-terminal cytoplasmic domain.
MANGO 003, a TANGO 272, a TANGO 354 or a TANGO 378 family member can include an extracellular domain. When located at the N-terminal domain the extracellular domain is referred to herein as an "N-terminal extracellular domain" or an "extracellular domain". As used herein, an "N-terminal extracellular domain" includes an amino acid sequence having about 1-800, preferably about 1-746, more preferably about 1-650, more preferably about 1-550, more preferably about 1-369, about 150 amino acid residues in length and is located outside of a cell or extracellularly. The C-terminal amino acid residue of a "N-terminal extracellular domain" is adjacent to an N-terminal amino acid residue of a transmembrane domain in a naturally-occurring MANGO 003, TANGO 272, TANGO 354 or TANGO 378 protein. Preferably, the N-terminal extracellular domain is capable of interacting (e.g., binding to) with an extracellular signal, for example, a ligand (e.g., a glycoprotein hormone) or a cell surface receptor (e.g., an integrin receptor). Most preferably, the N-terminal extracellular domain mediates a variety of biological processes, for example, protein-protein interactions, signal transduction and/or cell adhesion. In one embodiment, an N-terminal cytoplasmic domain is located at about amino acids 25-374 of
SEQ ID NO:5 (SEQ ID NO: 103); about amino acids 1-73 of SEQ ID NO:8 (SEQ ID
NO: 107); at about amino acids 21-767 of SEQ ID NO: 14 (SEQ ID NO: 114); at about amino acids 1-216 of SEQ ID NO: 17 (SEQ ID NOT 18); at about amino acids 1-500 of SEQ ID
NO:20 (SEQ ID NO: 122); at about amino acids 20-169 of SEQ ID NO:26 (SEQ ID NO.T29); and at about amino acids 22-244 of SEQ ID NO:29 (SEQ ID NO.T34).
In another embodiment, a MANGO 003, a TANGO 272, a TANGO 354 or a
TANGO 378 family member can include a transmembrane domain. As used herein, the term "transmembrane domain" includes an amino acid sequence of about 15 amino acid residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 20, 25, 30, 35, 40, or 45 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an -helical structure. In a preferred embodiment, at least 50%o, 60%, 70%>,
80%), 90%), 95%> or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, http://pfam. wustl.edu/cgi-bi_y etdesc?name=7tm-l and Zagotta et al, 1996, Annual Rev. Neuronsci. 19: 235-63, the contents of which are incorporated herein by reference. Amino acid residues 375-398 of SEQ ED NO:5 (SEQ ID NO: 104), 74- 96 of SEQ ID NO:8 (SEQ ID NO: 108), 768-791 of SEQ ID NOT4 (SEQ ID NO: 115), 217- 240 of SEQ ID NO:17 (SEQ ID NOT19), 501-524 of SEQ ID NO:20 (SEQ ID NO: 123); 170-193 of SEQ ID NO:26 (SEQ ID NOT30), and 245-269, 287-306, 323-343, 358-376, 414-438, 457-477 and 485-504 of SEQ ED NO:29 (SEQ ID NOs: 135-141) include transmembrane domains.
A MANGO 003, TANGO 272, TANGO 354 or TANGO 378 family member can include a C-terminal cytoplasmic domain. As used herein, a "C-terminal cytoplasmic domain" includes an amino acid sequence having a length of at least about 10, preferably about 10-25, more preferably about 25-50, more preferably about 50-75, even more preferably about 75-100, 100-133, 133-150, 150-200, 200-250, 250-300, 300-400, 400-500, or 500-600 amino acid residues and is located within a cell or within the cytoplasm of a cell. Accordingly, the N-terminal amino acid residue of a "C-terminal cytoplasmic domain" is adjacent to a C-terminal amino acid residue of a transmembrane domain in a naturally- occurring MANGO 003, TANGO 272, TANGO 354 or TANGO 378 protein. For example, a C-terminal cytoplasmic domain is found at about amino acid residues 399-504 of SEQ ID NO:5, 97-208 of SEQ ID NO:8, 792-1050 of SEQ ID NOT4, 241-497 of SEQ ID NO:17, 525-636 of SEQ ID NO:20; 194-305 of SEQ ID NO:26, and 505-528 of SEQ ID NO:29.
MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, orTANGO
378 family members can include a signal peptide. As used herein, a "signal peptide" includes a peptide of at least about 15 amino acid residues in length which occurs at the N- terminus of secretory and membrane-bound proteins and which contains at least about 70% hydrophobic amino acid residues such as alanine, leucine, isoleucine, phenylalanine, proline, tyrosine, tryptophan, or valine. The sequence can contain about 15 to 45 amino acid residues or about 17-22 amino acid residues, and has at least about 60-80%, 65-75%, or about 70% hydrophobic residues. A signal peptide serves to direct a protein containing such a sequence to a lipid bilayer. Thus, in one embodiment, a MANGO 003 protein contains a signal peptide of about amino acids 1-22, 1-23, 1-24, 1-25, or 1-26 of SEQ ID
NO:5 (SEQ ED NOT01). In one embodiment, a MANGO 347 protein contains a signal peptide of about amino acids 1-33, 1-34, 1-35, 1-36, or 1-37 of SEQ ID NO:l l (SEQ ID
NOT 10). In one embodiment, a TANGO 272 protein contains a signal peptide of amino acids 1-18, 1-19, 1-20, 1-21, or 1-22 of SEQ ID NO: 14 (SEQ ID NOT 12). In yet another embodiment, a TANGO 295 protein contains a signal peptide of amino acids 1-26, 1-27, 1-
28, 1-29, or 1-30 of SEQ ID NO:23 (SEQ ID NO: 125). In another embodiment, a TANGO
354 protein contains a signal peptide of amino acids 1-17, 1-18, 1-19, 1-20, or 1-21 of SEQ ID NO:26 (SEQ ID NO: 127). In another embodiment, a TANGO 378 protein contains a signal peptide of amino acids 1-19, 1-20, 1-21, 1-22, or 1-23 of SEQ ID NO:29 (SEQ ID NO: 132). The signal peptide is cleaved during processing of the mature protein. The amino acid sequence of the mature MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 protein starts at the next amino acid after the signal peptide is cleaved. For example, the amino acid sequence of MANGO 003 may start at amino acids 23, 24, 25, 26, or 27 depending on the exact location of the cleavage of the signal peptide.
The signal peptide is cleaved during processing of the mature protein. Sometimes the initial methionine residue is also cleaved from the protein during signal peptide processing. Thus, in one embodiment, a MANGO 003 protein does not contain a signal peptide or an initial methionine residue and begins from residue 2 of SEQ ID NO: 102. In one embodiment, a MANGO 347 protein does not contain a signal peptide or an initial methionine residue and begins from residue 2 of SEQ ID NO 11. In one embodiment, a TANGO 272 protein does not contain a signal peptide or an initial methionine residue and begins from residue 2 of SEQ ID NOT 13. Thus, in one embodiment, a TANGO 295 protein does not contain a signal peptide or an initial methionine residue an begins from residue 2 of SEQ ID NOT26. Thus, in one embodiment, a TANGO 354 protein does not contain a signal peptide or an initial methionine residue an begins from residue 2 of SEQ ED NO: 128. Thus, in one embodiment, a TANGO 378 protein does not contain a signal peptide or an initial methionine residue an begins from residue 2 of SEQ ED NO: 133.
In one embodiment, a MANGO 003 family member includes three immunoglobulin domains and a neurotransmitter-gated ion channel domain. In another embodiment, a
MANGO 003 family member includes three immunoglobulin domains, a neurotransmitter- gated ion channel domain and a transmembrane domain. In yet another embodiment, a MANGO 003 family member includes three immunoglobulin domains, a neurotransmitter- gated ion channel domain, a transmembrane domain and an N-terminal extracellular domain. In another embodiment, a MANGO 003 family member includes three immunoglobulin domains, a neurotransmitter-gated ion channel domain, a transmembrane domain, an N-terminal extracellular domain and a C-terminal cytoplasmic domain. In yet another embodiment, a MANGO 003 family member includes three immunoglobulin domains, a neurotransmitter-gated ion channel domain, a transmembrane domain, an N- terminal extracellular domain, a C-terminal cytoplasmic domain, and a signal peptide.
In one embodiment, a MANGO 354 family member includes at least one immunoglobulin domain and a transmembrane domain. In another embodiment, a
MANGO 354 family member includes at least one immunoglobulin domain, a transmembrane domain and a signal peptide.
In one embodiment, a TANGO 272 family member includes fourteen EGF-like domains and a delta serrate ligand domain. In another embodiment, a TANGO 272 family member includes fourteen EGF-like domains, a delta serrate ligand domain and an RGD cell attachment site. In yet another embodiment, a TANGO 272 family member includes fourteen EGF-like domains, a delta serrate ligand domain, an RGD cell attachment site, and a transmembrane domain. In another embodiment, a TANGO 272 family member includes fourteen EGF-like domains, a delta serrate ligand domain, an RGD cell attachment site, a transmembrane domain, and an extracellular N-terminal domain. In another embodiment, a TANGO 272 family member includes fourteen EGF-like domains, a delta serrate ligand domain, an RGD cell attachment site, a transmembrane domain, an extracellular N-terminal domain and a C-terminal cytoplasmic domain. In another embodiment, a TANGO 272 family member includes fourteen EGF-like domains, a delta serrate ligand domain, an RGD cell attachment site, a transmembrane domain, an extracellular N-terminal domain, a C- terminal cytoplasmic domain, and a signal peptide.
In one embodiment, a TANGO 378 family member includes a 7 transmembrane receptor profile and three extracellular loops. In another embodiment, a TANGO 378 family member includes a 7 transmembrane receptor profile, three extracellular loops, and three cytoplasmic loops. In yet another embodiment, a TANGO 378 family member includes a 7 transmembrane receptor profile, three extracellular loops, three cytoplasmic loops, and an extracellular N-terminal domain. In another embodiment, a TANGO 378 family member includes a 7 transmembrane receptor profile, three extracellular loops, three cytoplasmic loops, an extracellular N-terminal domain, and a C-terminal cytoplasmic domain. In another embodiment, a TANGO 378 family member includes a 7 transmembrane receptor profile, three extracellular loops, three cytoplasmic loops, an extracellular N-terminal domain, a C-terminal cytoplasmic domain, and a signal peptide.
Various features of INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, and TANGO 378 are summarized below.
INTERCEPT 340
A cDNA encoding INTERCEPT 340 was identified by analyzing the sequences of clones present in a human fetal spleen cDNA library.
This analysis led to the identification of a clone, jthsal 02b 12, encoding full-length human INTERCEPT 340. The cDNA of this clone is 3284 nucleotides long (Figures 1A- 1B; SEQ ID NOT). The 723 nucleotide open reading frame of this cDNA, nucleotides 1222-1944 of SEQ ID NOT (SEQ ID NO:3), encodes a 241 amino acid protein (Figures 1A-1B; SEQ ID N0:2).
Human INTERCEPT 340 that has not been post-translationally modified is predicted to have a molecular weight of 27.2 kDa. Human INTERCEPT 340 includes three fibrillar collagen C-terminal (COLF) domains at amino acids 58-116 of SEQ ID NO:2 (SEQ ID NO:34); amino acids 126-151 of SEQ ID NO:2 (SEQ ID NO:35); and amino acids 186-217 of SEQ ID NO:2 (SEQ ID NO: 36). Figure 3 depicts alignments of each of the COLF domains of human INTERCEPT 340 with consensus hidden Markov model COLF domains (SEQ ID NOs:31, 32, and 33). In one embodiment, INTERCEPT 340 is a secreted protein, hi another embodiment, INTERCEPT 340 is a membrane-associated protein.
An N-glycosylation site is present at amino acids 105-108 of SEQ ID NO:2. A glycosaminoaglycan attachment site is present at amino acids 161-164 of SEQ ID NO:2. Protein kinase C phosphorylation sites are present at amino acids 57-59, 152-154, and 227-
10 229 of SEQ ID NO:2. A tyrosine kinase phosphorylation site is present at amino acids 81- 87 of SEQ ID NO:2. Casein kinase II phosphorylation sites are present at amino acids 36- 39, 120-123 and 181-184. N-myristylation sites are present at amino acids 109-114 and 164-169 of SEQ ID NO:2.
Clone jthsal02bl2, which encodes human INTERCEPT 340, was deposited as a
15 composite deposit having a designation EpI340 with the American Type Culture Collection (ATCC® 10801 University Boulevard, Manassas, VA 20110-2209) on June 18, 1999 and assigned Accession Number PTA-250. A description of the deposit conditions is set forth in the section entitled "Deposit of Clones" below. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of 0 Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
Figure 2 depicts a hydropathy plot of human INTERCEPT 340. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are 5 below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace.
Use of INTERCEPT 340 Nucleic Acids. Polypeptides, and Modulators Thereof
INTERCEPT 340 includes three fibrillar collagen C-terminal domains. Proteins
30 having such domains play a role in modulating connective tissue formation and/or maintenance, and thus can influence a wide variety of biological processes, including assembly into fibrils; strengthening and organization of the extracellular matrix; shaping of tissues and cells; modulation of cell migration; and/or modulation of signal transduction pathways. Because INTERCEPT 340 includes fibrillar collagen C-terminal domains,
35 INTERCEPT 340 polypeptides, nucleic acids, and modulators thereof can be used to treat connective tissue disorders, including a skin disorder and or a skeletal disorder (e.g., Marfan syndrome and osteogenesis imperfecta); cardiovascular disorders including hyperproliferative vascular diseases (e.g., hypertension, vascular restenosis and atherosclerosis), ischemia reperfusion injury, cardiac hypertrophy, coronary artery disease, myocardial infarction, arrhythmia, cardiomyopathies, and congestive heart failure); and/or hematopoietic disorders (e.g., myeloid disorders, lymphoid malignancies, T cell disorders). As INTERCEPT 340 was originally found in a fetal spleen library, INTERCEPT
340 nucleic acids, proteins, and modulators thereof can be used to modulate the function, survival, morphology, migration, proliferation and/or differentiation of cells that form the spleen, e.g., cells of the splenic connective tissue, e.g., splenic smooth muscle cells and/or endothehal cells of the splenic blood vessels. INTERCEPT 340 nucleic acids, proteins, and modulators thereof can also be used to modulate the proliferation, differentiation, and/or function of cells that are processed, e.g., regenerated or phagocytized within the spleen, e.g., erythrocytes and/or B and T lymphocytes and macrophages. Thus INTERCEPT 340 nucleic acids, proteins, and modulators thereof can be used to treat spleen, e.g., the fetal spleen, associated diseases and disorders. Examples of splenic diseases and disorders include e.g., splenic lymphoma and/or splenomegaly, and/or phagocytotic disorders, e.g., those inhibiting macrophage engulfment of bacteria and viruses in the bloodstream.
Further, in light of INTERCEPT 340's presence in a human fetal spleen cDNA library, INTERCEPT 340 expression can be utilized as a marker for specific tissues (e.g., lymphoid tissues such as the spleen) and/or cells (e.g., splenic) in which INTERCEPT 340 is expressed. INTERCEPT 340 nucleic acids can also be utilized for chromosomal mapping.
MANGO 003
A cDNA encoding human MANGO 003 was identified by analyzing the sequences of clones present in a human thyroid cDNA library.
This analysis led to the identification of a clone, jthYa030d03, encoding full-length human MANGO 003. The cDNA of this clone is 3169 nucleotides long (Figures 4A-4B; SEQ ED NO:4). The 1512 nucleotide open reading frame of this cDNA, nucleotide 57 to nucleotide 1568 of SEQ ID NO:4 (SEQ ID NO:6), encodes a 504 amino acid protein (Figures 4A-4B; SEQ ID NO:5).
Human MANGO 003 that has not been post-translationally modified is predicted to have a molecular weight of 54.5 kDa prior to cleavage of its signal peptide (52.1 kDa after cleavage of its signal peptide). The signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human MANGO 003 includes a 24 amino acid signal peptide at amino acid 1 to about amino acid 24 of SEQ ID NO:5 (SEQ ED NO: 101) preceding the mature human MANGO 003 protein which corresponds to about amino acid 25 to amino acid 504 of SEQ ID NO:5 (SEQ ID NO: 102).
Human MANGO 003 is a transmembrane protein having an extracellular domain which extends from about amino acid 25 to about amino acid 374 of SEQ ED NO:5 (SEQ ED NO: 103), a transmembrane domain which extends from about amino acid 375 to about amino acid 398 of SEQ ID NO:5 (SEQ ID NO: 104), and a cytoplasmic domain which extends from about amino acid 399 to amino acid 504 of SEQ ID NO:5 (SEQ ID NO: 105).
Alternatively, in another embodiment, a human MANGO 003 protein contains an extracellular domain which extends from about amino acid 399 to amino acid 504 of SEQ ID NO: 5 (SEQ ID NO: 105), a transmembrane domain which extends from about amino acid 375 to about amino acid 398 of SEQ ED NO:5 (SEQ ID NO: 104), and a cytoplasmic domain which extends from about amino acid 25 to about amino acid 374 of SEQ ID NO:5 15 (SEQ ID NO: 103).
Human MANGO 003 includes three immunoglobulin domains at amino acids 44- 101 of SEQ ID NO:5 (SEQ ID NO:38); amino acids 165-223 of SEQ ID NO:5 (SEQ ID NO:39); and amino acids 261-340 of SEQ ID NO:5 (SEQ ID NO:40). Figure 6 depicts alignments of each of the immunoglobulin domains of MANGO 003 with a consensus
20 hidden Markov model immunoglobulin domain (SEQ ID NO:37).
Human MANGO 003 includes a neurotransmitter gated ion channel domain at amino acids 388-397 of SEQ ID NO:5 (SEQ ID NO:43). Figure 7 depicts an alignment of the neurotransmitter gated ion channel domain of human MANGO 003 with a neurotransmitter gated ion channel domain derived from a hidden Markov model (SEQ ID
25 NO:42).
N-glycosylation sites are present at amino acids 111-114, 231-234, 255-258, and 293-296 of SEQ ID NO:5. A cAMP and cGMP-dependent protein kinase phosphorylation site is present at amino acids 202-205 of SEQ ID NO:5. Protein kinase C phosphorylation sites are present at amino acids 44-48, 167-169, 207-209, 216-218, 220-222, 224-226, 233-
30 235, 347-349, and 422-424 of SEQ ID NO:5. Casein kinase II phosphorylation sites are present at amino acids 192-195, 256-259, 294-297, 313-316, 422-425, and 490-493 of SEQ D NO:5. Tyrosine kinase phosphorylation sites are present at amino acids 212-219 and 329-336 of SEQ ID NO:5. N-myristylation sites are present at amino acids 95-100, 228-
233, 261-266, 317-322, 334-339, 382-387, and 443-448 of SEQ ID NO:5.
35 Clone jthYa030d03, which encodes human MANGO 003, was deposited as a composite deposit having a designation EpthLa6al with the American Type Culture Collection (ATCC® 10801 University Boulevard, Manassas, NA 20110-2209) on March 27, 1999 and assigned Accession Number 207178. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
Figure 5 depicts a hydropathy plot of human MANGO 003. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The hydropathy plot of Figure 5 indicates the presence of a hydrophobic domain within human MANGO 003, suggesting that human MANGO 003 is a transmembrane protein.
A cDNA encoding mouse MANGO 003 was identified by analyzing the sequences of clones present in a mouse choroid plexus cDNA library.
This analysis led to the identification of a clone, jfmjf004cl 1 , encoding partial mouse MANGO 003. The cDNA of this clone is 504 nucleotides long (Figures 8A-8B; SEQ ID NO:7). The 626 nucleotide open reading frame of this cDNA, nucleotides 1-626 of SEQ ID NO:7 (SEQ ID NO:9), encodes a 208 amino acid protein (Figures 8A-8B; SEQ ID NO:8).
Northern blot analysis using the mouse clone jfinjf004cl 1 revealed strong expression of the mouse MANGO 003 gene in the mouse liver, skeletal muscle and kidney. Moderate expression was detected in the heart, lung and testis, and lower levels of expression were detected in the mouse brain. No expression was detected in the spleen.
Mouse MANGO 003 that has not been post-translationally modified is predicted to have a molecular weight of 22.3 kDa.
Mouse MANGO 003 is a transmembrane protein having an extracellular domain which extends from about amino acid 1 to about amino acid 73 of SEQ ID NO: 8 (SEQ ID NO: 107), a transmembrane domain which extends from about amino acid 74 to about amino acid 96 of SEQ ED NO:8 (SEQ ID NO.T 08), and a cytoplasmic domain which extends from about amino acid 97 to amino acid 208 of SEQ ID NO:8 (SEQ ID NOT09).
An N-glycosylation site is present at amino acids 190-193 of SEQ ID NO:8. Protein kinase C phosphorylation sites are present at amino acids 44-46, 98-100, 119-121, and 197- 199 of SEQ ED NO: 8. Casein kinase II phosphorylation sites are present at amino acids 10- 13, and 119-122 of SEQ ED NO: 8. A tyrosine kinase phosphorylation site is present at amino acids 26-33 of SEQ ID NO:8. N-myristylation sites are present at amino acids 14- 19, 31-36, and 79-84 of SEQ ID NO: 8. Figure 9 depicts a hydropathy plot of mouse MANGO 003. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The hydropathy plot of Figure 9 indicates the presence of a hydrophobic domain within human MANGO 003, suggesting that human MANGO 003 is a transmembrane protein.
A global alignment between the nucleotide sequence of the open reading frame (ORF) of human MANGO 003 (SEQ ID NO:6) and the nucleotide sequence of the open reading frame of mouse MANGO 003 (SEQ ID NO:9) revealed a 31.1% identity (Figures 27A-27C). The global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of -1212; Myers and Miller, 1989 CABIOS 4:11-1).
A local alignment between the nucleotide sequence of human MANGO 003 (SEQ ID NO:4) and the nucleotide sequence of mouse MANGO 003 (SEQ ID NO:7) revealed a 62.8 % identity over nucleotides 970-2080 of the human MANGO 003 sequence (nucleotides 10-1070 of mouse MANGO 003) (Figures 28A-28B). The local alignment was performed using the L-ALIGN program version 2.0u54 July 1996 (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a score of 3241; Huang and Miller, 1991, Adv. Appl. Math. 12:373-81).
A global alignment between the amino acid sequence of human MANGO 003 (SEQ ID NO:5) and the amino acid sequence of mouse MANGO 003 (SEQ ID NO:8) revealed a 30.1% identity (Figure 29). The global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of -488; Myers and Miller, 1989, CABIOS 4:11-1).
Use of MANGO 003 Nucleic Acids, Polypeptides. and Modulators Thereof
MANGO 003 includes three immunoglobulin-like domains. Proteins having such domains play a role in mediating protein-protein and protein-ligand interactions, and thus can influence a wide variety of biological processes, including cell surface recognition; transduction of an extracellular signal (e.g., by interacting with a ligand and/or a cell- surface receptor); and/or modulation of signal transduction pathways.
MANGO 003 further includes a neurotransmitter-gated ion channel domain.
Proteins having such domains play a role in modulating signal transmission at chemical synapses by, for example, influencing processes, such as the release of neurotransmitters from a cell (e.g., a neuronal cell); modulating membrane excitability and/or resting potential; and/or modulating ion flux across a membrane of a cell (e.g., a neuronal or a muscle cell). Because MANGO 003 includes a neurotransmitter-gated ion channel domain, MANGO 003 polypeptides, nucleic acids, and modulators thereof can be used to treat neural disorders (e.g., a CNS disorder, including Alzheimer's disease, Pick's disease, Parkinson's and other Lewy diffuse body diseases, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease; psychiatric disorders, e.g., depression, schizophrenic disorders, Korsakoffs psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss; and neurological disorders, e.g., migraine).
MANGO 003 polypeptides, nucleic acids, and modulators thereof can be used to modulate function, survival, morphology, migration, proliferation and/or differentiation of cells in the tissues in which it is expressed (e.g. thyroid, liver, skeletal muscle, kidney, heart, lung, testis and brain). For example, MANGO 003 polypeptides, nucleic acids, and modulators thereof can be used to modulate endocrine, hepatic, skeletal muscular, renal, cardiac, reproductive and/or brain function. Accordingly, these molecules can be used to treat a variety of disease including, but not limited to, endocrine disorders (e.g., hypothyroidism, hyperthyroidism, dwarfism, giantism, acromegaly); hepatic disorders (e.g., hepatitis, liver cirrhosis, hepatoma, liver cysts, and hepatic vein thrombosis); skeletal muscular disorders; renal disorders (e.g., renal cell carcinoma, nephritis, polycystic kidney disease); cardiovascular disorders (e.g., atherosclerosis, ischemia reperfusion injury, cardiac hypertrophy, hypertension, coronary artery disease, myocardial infarction, arrhythmia, cardiomyopathies, and congestive heart failure); and/or reproductive disorders (e.g., sterility).
MANGO 003 polypeptides, nucleic acids, or modulators thereof, can be used to treat hepatic (liver) disorders, such as jaundice, hepatic failure, hereditary hyperbiliruinemias (e.g., Gilbert's syndrome, Crigler-Naijar syndromes and Dubin- Johnson and Rotor's syndromes), hepatic circulatory disorders (e.g., hepatic vein thrombosis and portal vein obstruction and thrombosis) hepatitis (e.g., chronic active hepatitis, acute viral hepatitis, and toxic and drug-induced hepatitis) cirrhosis (e.g., alcoholic cirrhosis, biliary cirrhosis, and hemochromatosis), or malignant tumors (e.g., primary carcinoma, hepatoblastoma, and angiosarcoma).
In another example, MANGO 003 polypeptides, nucleic acids, or modulators thereof, can be used to treat disorders of skeletal muscle, such as muscular dystrophy (e.g., Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, Emery-Dreifuss Muscular Dystrophy, Limb-Girdle Muscular Dystrophy, Facioscapulohumeral Muscular Dystrophy, Myotonic Dystrophy, Oculopharyngeal Muscular Dystrophy, Distal Muscular Dystrophy, and Congenital Muscular Dystrophy), motor neuron diseases (e.g., Amyotrophic Lateral Sclerosis, Infantile Progressive Spinal Muscular Atrophy, Intermediate Spinal Muscular Atrophy, Spinal Bulbar Muscular Atrophy, and Adult Spinal Muscular Atrophy), myopathies (e.g., inflammatory myopathies (e.g., Dermatomyositis and Polymyositis), Myotonia Congenita, Paramyotonia Congenita, Central Core Disease, Nemaline Myopathy, Myotubular Myopathy, and Periodic Paralysis), and metabolic diseases of muscle (e.g., Phosphorylase Deficiency, Acid Maltase Deficiency, Phosphofructokinase Deficiency, Debrancher Enzyme Deficiency, Mitochondrial Myopathy, Carnitine Deficiency, Carnitine Palmityl Transferase Deficiency, Phosphoglycerate Kinase Deficiency, Phosphoglycerate Mutase Deficiency, Lactate Dehydrogenase Deficiency, and Myoadenylate Deaminase Deficiency).
In another example, MANGO 003 polypeptides, nucleic acids, or modulators thereof, can be used to treat renal disorders, such as glomerular diseases (e.g., acute and chronic glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, focal proliferative glomerulonephritis, glomerular lesions associated with systemic disease, such as systemic lupus erythematosus, Goodpasture's syndrome, multiple myeloma, diabetes, neoplasia, sickle cell disease, and chronic inflammatory diseases), tubular diseases (e.g., acute tubular necrosis and acute renal failure, polycystic renal diseasemedullary sponge kidney, medullary cystic disease, nephrogenic diabetes, and renal tubular acidosis), tubulointerstitial diseases (e.g., pyelonephritis, drug and toxin induced tubulointerstitial nephritis, hypercalcemic nephropathy, and hypokalemic nephropathy) acute and rapidly progressive renal failure, chronic renal failure, nephrolithiasis, vascular diseases (e.g., hypertension and nephrosclerosis, microangiopathic hemolytic anemia, atheroembolic renal disease, diffuse cortical necrosis, and renal infarcts), or tumors (e.g., renal cell carcinoma and nephroblastoma).
Further, in light of MANGO 003 's pattern of expression in mice, MANGO 003 expression can be utilized as a marker for specific tissues (e.g., liver, skeletal muscle, kidney) and/or cells (e.g., hepatic, skeletal muscle, renal) in which MANGO 003 is expressed. MANGO 003 nucleic acids can also be utilized for chromosomal mapping.
MANGO 347
A cDNA encoding human MANGO 347 was identified by analyzing the sequences of clones present in a human brain cDNA library.
This analysis led to the identification of a clone, jlhbad295gl2, encoding full-length human MANGO 347. The cDNA of this clone is 1423 nucleotides long (Figure 10; SEQ ID NOT0). The 414 nucleotide open reading frame of this cDNA, nucleotides 31 to 444 of SEQ ID NOTO (SEQ ID NO: 12), encodes a 138 amino acid protein (Figure 10; SEQ ID NO: 11).
The signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human MANGO 347 includes a 35 amino acid signal peptide at amino acid 1 to about amino acid 35 of SEQ ED NOT 1 (SEQ ID NOT10) preceding the mature human MANGO 347 protein which corresponds to about amino acid 36 to amino acid 138 of SEQ ID NOT1 (SEQ ED NOT11).
Human MANGO 347 that has not been post-translationally modified is predicted to have a molecular weight of 15.4 kDa prior to cleavage of its signal peptide and a molecular weight of 11.3 kDa subsequent to cleavage of its signal peptide.
10 Human MANGO 347 includes a CUB domain at amino acids 40-136 of SEQ ID NOT 1 (SEQ ID NO:45). An alignment of the CUB domain of human MANGO 347 with a consensus hidden Markov model CUB domain amino acid sequence derived from a hidden Markov model (SEQ ID NO:44) is shown in Figure 12.
Casein kinase II phosphorylation sites are present at amino acids 67-70, and 108-111
15 of SEQ ID NOT 1. N-myristylation sites are present at amino acids 19-24, 31-36, 64-69, and 113-118 of SEQ ID NOT 1.
Clone jlhbad295gl2, which encodes human MANGO 347, was deposited as a composite deposit having a designation EpM347 with the American Type Culture Collection (ATCC® 10801 University Boulevard, Manassas, VA 20110-2209) on June 18, .
20 1999 and assigned Accession Number PTA-250. A description of the deposit conditions used in set forth below. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
25 Figure 11 depicts a hydropathy plot of human MANGO 347. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The hydropathy plot of Figure 11 indicates that human MANGO 347 has a signal peptide at its amino terminus, suggesting that human MANGO
30 347 is a secreted protein.
Use of MANGO 347 Nucleic Acids. Polypeptides. and Modulators Thereof
MANGO 347 includes a CUB domain. Proteins having such a domain play a role in mediating cell interactions during development, and thus can influence a wide variety of
35 developmental processes, including morphogenesis, cellular migration, adhesion, proliferation, differentiation, and/or survival. MANGO 347 polypeptides are expressed in neural (e.g., brain cells). Because MANGO 347 includes a CUB domain and is expressed in neural cells, MANGO 347 polypeptides, nucleic acids, and modulators thereof can be used to treat disorders involving, e.g., cellular migration, proliferation, and differentiation of a cell, e.g., a neural cell (e.g., a CNS disorder, including Alzheimer's disease, Pick's disease, Parkinson's and other Lewy diffuse body diseases, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease; psychiatric disorders, e.g., depression, schizophrenic disorders, Korsakoffs psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss; and neurological disorders, e.g., migraine).
Further, in light of MANGO 347's presence in a human brain cDNA library, MANGO 347 expression can be utilized as a marker for specific tissues (e.g., brain) and/or cells (e.g., brain) in which MANGO 347 is expressed. MANGO 347 nucleic acids can also be utilized for chromosomal mapping.
TANGO 272
A cDNA encoding human TANGO 272 was identified by analyzing the sequences of clones present in a human microvascular endothehal cell library (HMVEC) cDNA library.
This analysis led to the identification of a clone, jthda089h03, encoding full-length human TANGO 272. The cDNA of this clone is 5036 nucleotides long (Figures 13A-13D; SEQ ID NO: 13). The 3149 nucleotide open reading frame of this cDNA, nucleotides 230- 3379 of SEQ ID NO: 13 (SEQ ID NO: 15), encodes a 1050 amino acid protein (Figures 13A- 13D; SEQ ID NO: 14).
Northern blot analysis using the human clone jthda089h03 revealed strong expression of the human TANGO 272 gene in the heart. Moderate expression was detected in the placenta, lung, and liver, and lower levels of expression were detected in the brain, skeletal muscle, kidney, and pancreas.
The signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human TANGO 272 includes an 20 amino acid signal peptide at amino acid 1 to about amino acid 20 of SEQ ID NO: 14 (SEQ ED NO: 112) preceding the mature human TANGO 272 protein which corresponds to about amino acid 21 to amino acid 1050 of SEQ ID NOT4 (SEQ ID NOT13).
Human TANGO 272 that has not been post-translationally modified is predicted to have a molecular weight of 112 kDa prior to cleavage of its signal peptide and a molecular weight of 110 kDa subsequent to cleavage of its signal peptide.
Human TANGO 272 is a transmembrane protein having an extracellular domain which extends from about amino acid 21 to about amino acid 767 of SEQ ID NO: 14 (SEQ ED NOT 14), a transmembrane domain which extends from about amino acid 768 to about amino acid 791 of SEQ ID NOT4 (SEQ ID NOT 15), and a cytoplasmic domain which extends from about amino acid 792 to amino acid 1050 of SEQ ID NOT4 (SEQ ED NO: 116).
Alternatively, in another embodiment, a human TANGO 272 protein contains an extracellular domain which extends from about amino acid 792 to amino acid 1050 of SEQ ID NO 4 (SEQ ED NOT 16), a transmembrane domain which extends from about amino acid 768 to about amino acid 791 of SEQ ID NO: 14 (SEQ ID NO: 115), and a cytoplasmic domain which extends from about amino acid 21 to about amino acid 767 of SEQ ID NO:14 (SEQ ID NO:114).
Human TANGO 272 includes fourteen EGF-like domains at amino acids 151-181 of SEQ ID NO: 14 (SEQ ID NO:49); amino acids 200-229 of SEQ ID NO: 14 (SEQ ID NO:50); amino acids 242-272 of SEQ ID NO: 14 (SEQ ID NO:51); amino acids 285-315 of SEQ ID NO: 14 (SEQ ID NO:52); amino acids 328-358 of SEQ ID NO: 14 (SEQ ED NO:53); amino acids 378-404 of SEQ ID NO: 14 (SEQ ID NO:54); amino acids 417-447 of SEQ ID NO: 14 (SEQ ID NO:55); amino acids 460-490 of SEQ ID NO: 14 (SEQ ID
NO:56); amino acids 503-533 of SEQ ID NO: 14 (SEQ ED NO:57); amino acids 546-576 of SEQ ID NOT4 (SEQ ID NO:58); amino acids 589-619 of SEQ ID NOT4 (SEQ ID NO:59); amino acids 632-661 of SEQ ID NO: 14 (SEQ ID NO:60); amino acids 674-704 of SEQ ID NOT4 (SEQ ID NO:61); and amino acids 717-747 of SEQ ID NOT4 (SEQ ID NO:62). Figures 15A-15C depict alignments of each of the EGF-like domains of TANGO 272 with consensus hidden Markov model EGF-like domains (SEQ ID NO: 46). Human TANGO 272 further includes a delta serrate ligand domain from amino acids 518 to 576 of SEQ ID NO: 14 (SEQ ID NO:63). An alignment of the delta serrate ligand domain of human TANGO 272 with a consensus hidden Markov model of this domain (SEQ ID NO:47) is also depicted (Figure 15B).
An RGD cell attachment site is present at amino acids 177-179 of SEQ ID NO: 14. N-glycosylation sites are present at amino acids 284-287, 405-408, 459-462, 489-492, 504- 507, 588-591, 639-642, 647-650, 716-719, and 873-876 of SEQ ID NOT4. An a idation site is present at amino acids 628-631 of SEQ D NO.T 4. Protein kinase C phosphorylation sites are present at amino acids 38-40, 70-72, 107-109, 359-361, 461-463, 594-596, 809- 811, 896-898, 940-942, 977-979, and 1022-1024 of SEQ ED NO: 14. Casein kinase II phosphorylation sites are present at amino acids 30-33, 38-41, 473-476, 548-551, 579-582, 657-660, 897-900, 921-924, 940-943, and 955-958 of SEQ ID NOT4. A tyrosine kinase phosphorylation site is present at amino acids 361-368 of SEQ ID NO: 14. N-myristylation sites are present at amino acids 14-19, 103-108, 269-274, 302-307, 325-330, 345-350, 401- 406, 427-432, 434-439, 457-462, 520-525, 586-591, 606-611, 648-653, 707-712, 714-719, 769-774, 866-871, 926-931, and 1014-1019 of SEQ ID NOT4.
Clone jthda089h03, which encodes human TANGO 272, was deposited as a composite deposit having a designation EpT272 with the American Type Culture Collection (ATCC® 10801 University Boulevard, Manassas, VA 20110-2236) June 18, 1999 and assigned Accession Number PTA-250. A description of the deposit conditions used is set forth in the section entitled "Deposit of Clones" below. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
Figure 14 depicts a hydropathy plot of human TANGO 272. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The hydropathy plot of Figure 16 indicates the presence of a hydrophobic domain within human TANGO 272, suggesting that human TANGO 272 is a transmembrane protein.
A cDNA encoding mouse TANGO 272 was identified by analyzing the sequences of clones present in a mouse testis cDNA library.
This analysis led to the identification of a clone, jtmzb062c04, encoding partial mouse TANGO 272. The cDNA of this clone is 2569 nucleotides long (Figures 16A-16B; SEQ ID NO: 16). The 1492 nucleotide open reading frame of this cDNA, nucleotides 1- 1492 of SEQ ID NO: 16 (SEQ ID NO: 18), encodes a 497 amino acid protein (Figures 16A- 16B; SEQ ID NO: 17).
Mouse TANGO 272 that has not been post-translationally modified is predicted to have a molecular weight of 53.5 kDa.
Mouse TANGO 272 is a transmembrane protein having an extracellular domain which extends from about amino acid 1 to about amino acid 216 of SEQ ID NO: 17 (SEQ ID NOT 18), a transmembrane domain which extends from about amino acid 217 to about amino acid 240 of SEQ ID NO: 17 (SEQ ID NOT 19), and a cytoplasmic domain which extends from about amino acid 241 to amino acid 497 of SEQ ID NOT7 (SEQ ID NOT20).
Alternatively, in another embodiment, a mouse TANGO 272 protein contains an extracellular domain which extends from about amino acid 241 to amino acid 497 of SEQ ID NO: 17 (SEQ ID NO.T 20), a transmembrane domain which extends from about amino acid 217 to about amino acid 240 of SEQ ID NOT7 (SEQ ED NOT19), and a cytoplasmic domain which extends from about amino acid 1 to about amino acid 216 of SEQ ID NO: 17 (SEQ ID NO: 118). Mouse TANGO 272 includes four EGF-like domains at about amino acids 37-67 of SEQ ID NO: 17 (SEQ ID NO:64); amino acids 80-110 of SEQ ID NO: 17 (SEQ ID NO:65); amino acids 123-153 of SEQ ID NOT7 (SEQ ED NO:66); and amino acids 166-196 of SEQ ID NO: 17 (SEQ ID NO:67). Mouse TANGO 272 further includes four laminin-EGF-like domains at about amino acids 3-37 of SEQ ID NO: 17 (SEQ ID NO:68); amino acids 41-80 of SEQ ID NOT7 (SEQ ID NO:69); amino acids 83-123 of SEQ ID NOT7 (SEQ ID NO:70); and amino acids 127-172 of SEQ ED NOT7 (SEQ ID NO:71). Figures 39A-39B depict alignments of each of the EGF-like- and laminin-EGF-like domains of TANGO 272 with consensus hidden Markov model EGF-like domains (SEQ ID NOs:46 and 48, respectively).
Mouse TANGO 272 further includes a delta serrate ligand domain from amino acids 10 to 67 of SEQ ID NOT7 (SEQ ID NO:72). An alignment of the delta serrate ligand domain of mouse TANGO 272 with a consensus hidden Markov model of this domain (SEQ ID NO:47) is also depicted in Figures 39A-39B.
Based on the Prosite analysis, EGF-like domain cysteine pattern signature are present at amino acids 13-24, 56-67, 99-110, 142-153, and 185-196 of SEQ ID NOT7.
N-glycosylation sites are present at amino acids 36-39, 88-91, 165-168, and 323-326 of SEQ ID NO: 17. An amidation site is present at amino acids 76-79 of SEQ ID NO: 17. Protein kinase C phosphorylation sites are present at amino acids 42-44, 258-260, 354-356, 388-390, 469-471, and 492-494 of SEQ ID NOT7. Casein kinase II phosphorylation sites are present at amino acids 106-109, 192-195, 343-346, 388-391, and 446-449 of SEQ ID NO: 17. N-myristylation sites are present at amino acids 11-16, 34-39, 47-52, 54-59, 97- 102, 120-125, 140-145, 163-168, 199-204, 218-223, 372-377, and 461-466 of SEQ ID NOTE
Figure 17 depicts a hydropathy plot of mouse TANGO 272. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The hydropathy plot of Figure 17 indicates the presence of a hydrophobic domain within mouse TANGO 272, suggesting that mouse TANGO 272 is a transmembrane protein.
A cDNA encoding rat TANGO 272 was identified by analyzing the sequences of clones present in a rat neonatal sciatic nerve cDNA library.
This analysis led to the identification of a clone, atrxa6b6, encoding partial rat TANGO 272. The cDNA of this clone is 3567 nucleotides long (Figures 33A-33C; SΕQ ID NO: 19). The 1908 nucleotide open reading frame of this cDNA, nucleotides 925-2832 of SΕQ ID NO: 19 (SΕQ ID NO:21), encodes a 636 amino acid protein (Figures 33A-33C; SΕQ ID NO:20). Rat TANGO 272 that has not been post-translationally modified is predicted to have a molecular weight of 67.4 kDa.
Rat TANGO 272 is a transmembrane protein having an extracellular domain which extends from about amino acid 1 to about amino acid 500 of SEQ ED NO:20 (SEQ ID- NO: 122), a transmembrane domain which extends from about amino acid 501 to about amino acid 524 of SEQ ID NO:20 (SEQ ID NO: 123), and a cytoplasmic domain which extends from about amino acid 525 to amino acid 636 of SEQ ID NO:20 (SEQ ID NO: 124).
Alternatively, in another embodiment, a rat TANGO 272 protein contains an extracellular domain which extends from about amino acid 525 to amino acid 636 of SEQ ID NO:20 (SEQ ID NO: 124), a transmembrane domain which extends from about amino acid 501 to about amino acid 524 of SEQ ID NO:20 (SEQ ED NO: 123), and a cytoplasmic domain which extends from about amino acid 1 to about amino acid 500 of SEQ ID NO:20 (SEQ ID NO: 122).
Rat TANGO 272 includes eleven EGF-like domains at about amino acids 18-48 of SEQ ID NO:20 (SEQ ID NO:73); amino acids 61-91 of SEQ ID NO:20 (SEQ ID NO:74); amino acids 105-137 of SEQ ID NO:20 (SEQ ID NO:75); amino acids 150-180 of SEQ ED NO:20 (SEQ ID NO:76); amino acids 193-223 of SEQ ID NO:20 (SEQ ID NO:77); amino acids 236-266 of SEQ ID NO:20 (SEQ ID NO:78); amino acids 279-309 of SEQ ID NO:20 (SEQ ID NO:79); amino acids 322-352 of SEQ ID NO:20 (SEQ ID NO:80); amino acids 365-394 of SEQ ID NO:20 (SEQ ID NO:81); amino acids 407-437 of SEQ ID NO:20 (SEQ ID NO:82); and amino acids 450-480 of SEQ ID NO:20 (SEQ ID NO:83). Figures 41 A- 41D depict alignments of each of the EGF-like-domains of rat TANGO 272 with consensus hidden Markov model EGF-like domains (SEQ ID NO:46).
Rat TANGO 272 further includes eleven laminin/EGF-like domains at about amino acids 22-61 of SEQ ID NO:20 (SEQ ID NO:84); arnino acids 65-105 of SEQ ID NO:20 (SEQ ID NO:85); amino acids 109-150 of SEQ ID NO:20 (SEQ ID NO:86); amino acids 154-193 of SEQ ID NO:20 (SEQ ID NO:87); amino acids 197-236 of SEQ ID NO:20 (SEQ ID NO:88); amino acids 240-279 of SEQ ID NO:20 (SEQ ID NO:89); amino acids 283-322 of SEQ ID NO:20 (SEQ ID NO:90); amino acids 326-365 of SEQ ID NO:20 (SEQ ID NO:91); amino acids 368-407 of SEQ ID NO:20 (SEQ ID NO:92); amino acids 411-450; and amino acids 454-489 of SEQ ID NO:20 (SEQ ID NO:93). Figures 41A-41D depict alignments of each of the laminin/EGF-like-domains of rat TANGO 272 with consensus hidden Markov model EGF-like domains (SEQ ID NO:48).
Rat TANGO 272 further includes a delta serrate ligand domain from amino acids
246 to 309 of SEQ ID NO:20 (SEQ ID NO:95). An alignment of the delta serrate ligand domain of rat TANGO 272 with a consensus hidden Markov model of this domain (SEQ ID
NO:47) is also depicted in Figures 41A-41D. Based on the Prosite analysis, EGF-like domain cysteine pattern signature are present at amino acids 37-48, 80-91, 126-137, 169-180, 255-266, 298-309, 341-352, 383- 394, 426-437, and 469-480 of SEQ ID NO:20.
N-glycosylation sites are present at amino acids 17-20, 138-141, 192-195, 222-225, 237-240, 321-324, 372-375, 436-439, and 449-452 of SEQ ID NO:20. A cAMP/cGMP- dependent protein kinase phosphorylation site is present at amino acids 618-621 of SEQ ID NO:20. An amidation site is present at amino acids 361-364 of SEQ ID NO:20. Protein kinase C phosphorylation sites are present at amino acids 92-94, 327-329, 542-544, and 596-598 of SEQ ID NO:20. Casein kinase II phosphorylation sites are present at amino acids 104-107, 206-209, 281-284, and 390-393 of SEQ ID NO:20. A tyrosine kinase
10 phosphorylation site is present at amino acids 94-101 of SEQ ID NO:20. N-myristylation sites are present at amino acids 2-7, 35-40, 58-63, 78-83, 134-139, 160-165, 167-172, 190- 195, 210-215, 253-258, 319-324, 339-344, 381-386, 404-409, 424-429, 447-452, 483-488, and 502-507 of SEQ ID NO:20.
Figure 40 depicts a hydropathy plot of rat TANGO 272. Relatively hydrophobic
15 regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The hydropathy plot of Figure 40 indicates the presence of a hydrophobic domain within rat TANGO 272, suggesting that rat TANGO 272 is a transmembrane protein.
20 A global alignment between the nucleotide sequence of the open reading frame (ORF) of human TANGO 272 (SEQ ID NO: 15) and the nucleotide sequence of the open reading frame of mouse TANGO 272 (SEQ ID NO: 18) revealed a 39.1% identity (Figures 30A-30E). The global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of
25 -79; Myers and Miller, 1989, CABIOS 4:11-1).
A local alignment between the nucleotide sequence of human TANGO 272 (SEQ ID NOT3) and the nucleotide sequence of mouse TANGO 272 (SEQ ID NOT6) revealed 67.6 % identity over nucleotides 1890-4610 of the human TANGO 272 sequence (nucleotides 10-2560 of mouse TANGO 272) (Figures 31A-31D). The local alignment was performed
30 using the L-ALIGN program version 2.0u54 July 1996 (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a score of 8462; Huang and Miller, 1991, Adv. Appl. Math. 12:373- 81).
A global alignment between the amino acid sequence of human TANGO 272 (SEQ ID NO.T 4) and the amino acid sequence of mouse TANGO 272 (SEQ ID NO.T 7) revealed a
35 38.2% identity (Figures 32A-32B). The global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of -19; Myers and Miller, 1989, CABIOS 4:11-1).
A global alignment between the nucleotide sequence of human TANGO 272 (SEQ TD NO: 13) and the nucleotide sequence of rat TANGO 272 (SEQ ID NO: 19) revealed a 55.7%) identity (Figures 34A-34H). The global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of 8635; Myers and Miller, 1989, CABIOS 4:11-7).
A global alignment between the nucleotide sequence of mouse TANGO 272 (SEQ ID NO.T 6) and the nucleotide sequence of rat TANGO 272 (SEQ ID NO.T9) revealed a 43.7% identity (Figures 35A-35F). The global alignment was performed using the ALIGN program version 2.0u (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of 2827; Myers and Miller, 1989, CABIOS 4:11-1).
Use of TANGO 272 Nucleic Acids. Polypeptides. and Modulators Thereof
TANGO 272 includes fourteen EGF-like domains. Proteins having such domains play a role in mediating protein-protein interactions, and thus can influence a wide variety of biological processes, including cell surface recognition; modulation of cell-cell contact; modulation of cell fate determination; and modulation of wound healing and tissue repair.
TANGO 272 further includes an RGD cell attachment site. Proteins having such domains are typically extracellular matrix proteins such as collagens, laminin and fibronectin, among others (reviewed in Ruoslahti, 1996, Annu. Rev. Cell Dev. Biol. 12:691- 715). An RGD cell attachment site typically interacts (e.g., binds to) a cell surface receptor, such as an integrin receptor, and thus mediates a variety of biological processes, including cellular adhesion, migration, among others.
Because TANGO 272 includes EGF-like domains and an RGD cell attachment site, TANGO 272 polypeptides, nucleic acids, and modulators thereof can be used to treat disorders involving, e.g., cellular migration, proliferation, and differentiation of a cell. For example, TANGO 272 polypeptides, nucleic acids, and modulators thereof can be used to treat neoplastic disorders, e.g., cancer, tumor metastasis.
TANGO 272 polypeptides, nucleic acids, and modulators thereof can be used to modulate function, survival, morphology, migration, proliferation, tissue repair and/or differentiation of cells in the tissues in which it is expressed (e.g., micro vascular endothehal cells). For example, TANGO 272 polypeptides, nucleic acids, and modulators thereof can be used to modulate cardiovascular function, and/or to promote wound healing and tissue repair (e.g., of the skin, cornea and mucosal lining). Accordingly, these molecules can be used to treat a variety of cardiovascular diseases including, but not limited to, atherosclerosis, ischemia reperfusion injury, cardiac hypertrophy, hypertension, coronary artery disease, myocardial infarction, an-hythmia, cardiomyopathies, and congestive heart failure.
As TANGO 272 exhibits expression in the heart, TANGO 272 nucleic acids, proteins, and modulators thereof can be used to treat heart disorders, e.g., ischemic heart disease, atherosclerosis, hypertension, angina pectoris, Hypertrophic Cardiomyopathy, and congenital heart disease.
In another example, TANGO 272 polypeptides, nucleic acids, or modulators thereof, can be used to treat placental disorders, such as toxemia of pregnancy (e.g., preeclampsia and eclampsia), placentitis, or spontaneous abortion.
In another example, TANGO 272 polypeptides, nucleic acids, or modulators thereof, can be used to treat pulmonary (lung) disorders, such as atelectasis, cystic fibrosis, rheumatoid lung disease, pulmonary congestion or edema, chronic obstructive airway disease (e.g., emphysema, chronic bronchitis, bronchial asthma, and bronchiectasis), diffuse interstitial diseases (e.g., sarcoidosis, pneumoconiosis, hypersensitivity pneumonitis, Goodpasture's syndrome, idiopathic pulmonary hemosiderosis, pulmonary alveolar proteinosis, desquamative interstitial pneumonitis, chronic interstitial pneumonia, fibrosing alveolitis, hamman-rich syndrome, pulmonary eosinophilia, diffuse interstitial fibrosis, Wegener' s granulomatosis, lymphomatoid granulomatosis, and lipid pneumonia), or tumors (e.g., bronchogenic carcinoma, bronchiolovlveolar carcinoma, bronchial carcinoid, hamartoma, and mesenchymal tumors).
In another example, TANGO 272 polypeptides, nucleic acids, or modulators thereof, can be used to treat hepatic (liver) disorders, such as jaundice, hepatic failure, hereditary hyperbiliruinemias (e.g., Gilbert's syndrome, Crigler-Naijar syndromes and Dubin- Johnson and Rotor's syndromes), hepatic circulatory disorders (e.g., hepatic vein thrombosis and portal vein obstruction and thrombosis) hepatitis (e.g., chronic active hepatitis, acute viral hepatitis, and toxic and drug-induced hepatitis) cirrhosis (e.g., alcoholic cirrhosis, biliary cirrhosis, and hemochromatosis), or malignant tumors (e.g., primary carcinoma, hepatoblastoma, and angiosarcoma).
In another example, TANGO 272 polypeptides, nucleic acids, or modulators thereof, can be used to treat disorders of the brain, such as cerebral edema, hydrocephalus, brain herniations, iatro genie disease (due to, e.g., infection, toxins, or drugs), inflammations (e.g., bacterial and viral meningitis, encephalitis, and cerebral toxoplasmosis), cerebrovascular diseases (e.g., hypoxia, ischemia, and infarction, intracranial hemorrhage and vascular malformations, and hypertensive encephalopathy), and tumors (e.g., neuroglial tumors, neuronal tumors, tumors of pineal cells, meningeal tumors, primary and secondary lymphomas, intracranial tumors, and medulloblastoma), and to treat injury or trauma to the brain. In another example, TANGO 272 polypeptides, nucleic acids, or modulators thereof, can be used to treat disorders of skeletal muscle, such as muscular dystrophy (e.g., Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, Emery-Dreifuss Muscular Dystrophy, Limb-Girdle Muscular Dystrophy, Facioscapulohumeral Muscular Dystrophy, Myotonic Dystrophy, Oculopharyngeal Muscular Dystrophy, Distal Muscular Dystrophy, and Congenital Muscular Dystrophy), motor neuron diseases (e.g. , Amyotrophic Lateral Sclerosis, Infantile Progressive Spinal Muscular Atrophy, Intermediate Spinal Muscular Atrophy, Spinal Bulbar Muscular Atrophy, and Adult Spinal Muscular Atrophy), myopathies (e.g., inflammatory myopathies (e.g., Dermatomyositis and Polymyositis), Myotonia Congenita, Paramyotonia Congenita, Central Core Disease, Nemaline Myopathy,
I Λ
Myotubular Myopathy, and Periodic Paralysis), and metabolic diseases of muscle (e.g., Phosphorylase Deficiency, Acid Maltase Deficiency, Phosphofructokinase Deficiency, Debrancher Enzyme Deficiency, Mitochondrial Myopathy, Carnitine Deficiency, Carnitine Palmityl Transferase Deficiency, Phosphoglycerate Kinase Deficiency, Phosphoglycerate Mutase Deficiency, Lactate Dehydrogenase Deficiency, and Myoadenylate Deaminase Deficiency).
In another example, TANGO 272 polypeptides, nucleic acids, or modulators thereof, can be used to treat renal disorders, such as glomerular diseases (e.g., acute and chronic glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, focal proliferative glomerulonephritis, glomerular lesions associated with systemic disease, such 0 as systemic lupus erythematosus, Goodpasture's syndrome, multiple myeloma, diabetes, neoplasia, sickle cell disease, and chronic inflammatory diseases), tubular diseases (e.g., acute tubular necrosis and acute renal failure, polycystic renal diseasemedullary sponge kidney, medullary cystic disease, nephrogenic diabetes, and renal tubular acidosis), tubulointerstitial diseases (e.g., pyelonephritis, drug and toxin induced tubulointerstitial 5 nephritis, hypercalcemic nephropathy, and hypokalemic nephropathy) acute and rapidly progressive renal failure, chronic renal failure, nephrolithiasis, vascular diseases (e.g., hypertension and nephrosclerosis, microangiopathic hemolytic anemia, atheroembolic renal disease, diffuse cortical necrosis, and renal infarcts), or tumors (e.g., renal cell carcinoma and nephroblastoma). 0 In another example, TANGO 272 polypeptides, nucleic acids, or modulators thereof, can be used to treat pancreatic disorders, such as pancreatitis (e.g., acute hemorrhagic pancreatitis and chronic pancreatitis), pancreatic cysts (e.g., congenital cysts, pseudocysts, and benign or malignant neoplastic cysts), pancreatic tumors (e.g., pancreatic carcinoma and adenoma), diabetes mellitus (e.g., insulin- and non-insulin-dependent types, impaired 5 glucose tolerance, and gestational diabetes), or islet cell tumors (e.g., insulinomas, adenomas, Zollinger-Ellison syndrome, glucagonomas, and somatostatinoma). Further, in light of TANGO 272's pattern of expression in humans, TANGO 272 expression can be utilized as a marker for specific tissues (e.g., cardiovascular) and/or cells (e.g., cardiac) in which TANGO 272 is expressed. TANGO 272 nucleic acids can also be utilized for chromosomal mapping.
TANGO 295
A cDNA encoding human TANGO 295 was identified by analyzing the sequences of clones present in a human mammary epithelium cDNA library.
This analysis led to the identification of a clone, jthvb023d09, encoding full-length human TANGO 295. The cDNA of this clone is 1497 nucleotides long (Figure 18; SEQ ID NO:22). The 468 nucleotide open reading frame of this cDNA, nucleotides 217-684 of SEQ ID NO:22 (SEQ ID NO:34), encodes a 156 amino acid protein (Figure 18; SEQ ID NO:23).
The signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human TANGO 295 includes a 28 amino acid signal peptide at amino acid 1 to about amino acid 28 of SEQ ID NO:23 (SEQ ID NOT25) preceding the mature human TANGO 295 protein which corresponds to about amino acid 29 to amino acid 156 of SEQ ID NO:23 (SEQ ID NO: 126).
Human TANGO 295 that has not been post-translationally modified is predicted to have a molecular weight of 17.5 kDa prior to cleavage of its signal peptide and a molecular weight of 14.6 kDa subsequent to cleavage of its signal peptide.
Secretion assays reveal that human TANGO 295 protein is secreted as a 17 kDa protein. The secretion assays were performed as follows: 8xl05 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/streptomycin) at 37 °C, 5% CO2 overnight. 293T cells were transfected with 2 μg of full-length MANGO 245 inserted in the pMET7 vector/well and 10 μg LipofectAMINE (GIBCO/BRL Cat. # 18324-012) /well according to the protocol for
GIBCO/BRL LipofectAMINE. The transfectant was removed 5 hours later and fresh growth medium was added to allow the cells to recover overnight. The medium was removed and each well was gently washed twice with DMEM without methionine and cysteine (ICN Cat. # 16-424-54). 1 ml DMEM without methionine and cysteine with 50 μCi Trans-35S (ICN Cat. # 51006) was added to each well and the cells were incubated at
37°C5 5% CO2 for the appropriate time period. A 150 μl aliquot of conditioned medium was obtained and 150 μl of 2X SDS sample buffer was added to the aliquot. The sample was heat-inactivated and loaded on a 4-20% SDS-PAGE gel. The gel was fixed and the presence of secreted protein was detected by autoradiography . Human TANGO 295 includes a pancreatic ribonuclease domain at amino acids 32- 156 of SEQ ID NO:23 (SEQ ID NO:97). Figure 20 depicts an alignment of pancreatic ribonuclease domain of human TANGO 295 with a consensus hidden Markov model pancreatic ribonuclease domain (SEQ ID NO:96).
An N-glycosylation site is present at amino acids 127-130 of SEQ ID NO:23. A cAMP/cGMP dependent protein kinase site is present at amino acids 139-142 of SEQ ED NO:23. Protein kinase C phosphorylation sites are present at amino acids 27-29, 62-64, 85- 87, and 113-115 of SEQ ID NO:23. N-myristylation sites are present at amino acids 18-23, and 32-37 of SEQ ID NO:23.
Global alignment of the human TANGO 295 and GenPept AF037081 amino acid
10 sequences revealed 53.2% identity (Matrix file used: pam 120.mat, gap penalties of -12/-4; Myers and Miller, 1989, CABIOS 4:11-1) (Figure 36). A global alignment of the human TANGO 295 and GenPept AF037081 nucleotide sequences revealed a 22.6% identity between these two sequences (Figures 37A-37C) (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of -2718; Myers and Miller, 1989,
15 CABIOS 4:11-1).
Local alignment of the human TANGO 295 and Genbank AF037081 nucleotide sequences revealed 62.7% identity between nucleotides 235-687 of human TANGO 295, and nucleotides 3-453 of AF037081; 43.4%> identity between nucleotides 410-850 of human TANGO 295, and nucleotides 3-450 of AF037081; and 46.5% identity between nucleotides
20 432-700 of human TANGO 295, and nucleotides 5-251 of AF037081 (Matrix file used: pam 120.mat, gap penalties of -12/-4 with a global alignment score of 1214; Huang and Miller, 1991, Adv. Appl. Math. 12:373-81) (Figures 38A-38B).
Clone jthvb023d09, which encodes human TANGO 295, was deposited as a composite deposit having a designation EpT295 with the American Type Culture Collection
25 (ATCC® 10801 University Boulevard, Manassas, NA 20110-2209) on June 18, 1999 and assigned Accession Number PTA-249. Deposit conditions are described below in the section entitled "Deposit of Clones". This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of
30 skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
Figure 19 depicts a hydropathy plot of human TANGO 295. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The hydropathy plot of Figure 19 indicates that human TANGO 295
35 has a signal peptide at its amino terminus, suggesting that human TANGO 295 is a secreted protein. Use of TANGO 295 Nucleic Acids. Polypeptides. and Modulators Thereof
TANGO 295 includes a pancreatic ribonuclease domain. Proteins having such domains have pyrimidine-specific endonuclease activity, and are present at elevated levels in the pancreas of various mammals and few reptiles. TANGO 295 shows some structural similarities to Ribonuclease k6 (RNase k6). RNase k6 is expressed in human monocytes and monophils (but not in eosinophils), suggesting a role for this ribonuclease in regulating host defense. Based on the structural similarities between TANGO 295 and RNase k6,
TANGO 295 may play a role in regulating host defense.
TANGO 295 polypeptides, nucleic acids, and modulators thereof, can be used to modulate the function, morphology, proliferation and/or differentiation of cells in the tissues in which it is expressed (e.g., mammary epithelium). Accordingly, TANGO 295 polypeptides, nucleic acids, and modulators thereof can be used to treat epithelial disorders, e.g., mammary epithelial disorders (e.g., breast cancer).
Further, in light of TANGO 295's presence in a human mamary epithelium cDNA library, TANGO 295 expression can be utilized as a marker for specific tissues (e.g., breast) and/or cells (e.g., mammary) in which TANGO 295 is expressed. TANGO 295 nucleic acids can also be utilized for chromosomal mapping.
TANGO 354
A cDNA encoding human TANGO 354 was identified by analyzing the sequences of clones present in a Mixed Lymphocyte Reaction (MLR) cDNA library.
This analysis led to the identification of a clone, jthLa042a04, encoding full-length human TANGO 354. The cDNA of this clone is 1788 nucleotides long (Figures 21A-21B; SEQ ID NO:25). The 915 nucleotide open reading frame of this cDNA, nucleotides 62-976 of SEQ ID NO:25 (SEQ ID NO:27), encodes a 305 amino acid protein (Figures 21A-21B; SEQ ID NO:26).
Human TANGO 354 that has not been post-translationally modified is predicted to have a molecular weight of 33.8 kDa prior to cleavage of its signal peptide (31.6 kDa after cleavage of its signal peptide).
The signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein
Engineering 10:1-6) predicted that human TANGO 354 includes a 19 amino acid signal peptide at amino acid 1 to about amino acid 19 of SEQ ID NO:26 (SEQ ID NO: 127) preceding the mature human TANGO 354 protein which corresponds to about amino acid
20 to amino acid 305 of SEQ ED NO:26 (SEQ ID NO: 128). Human TANGO 354 is a transmembrane protein having an extracellular domain which extends from about amino acid 20 to about amino acid 169 of SEQ ID NO:26 (SEQ ID NO: 129), a transmembrane domain which extends from about amino acid 170 to about amino acid 193 of SEQ ID NO:26 (SEQ ID NOT30), and a cytoplasmic domain which extends from about amino acid 194 to amino acid 305 of SEQ ID NO:26 (SEQ ID NOT31).
Alternatively, in another embodiment, a human TANGO 354 protein contains an extracellular domain which extends from about amino acid 194 to amino acid 305 of SEQ ID NO:26 (SEQ ID NO: 131), a transmembrane domain which extends from about amino acid 170 to about amino acid 193 of SEQ ID NO:26 (SEQ ID NOT30), and a cytoplasmic domain which extends from about amino acid 20 to about amino acid 169 of SEQ ID NO:26 (SEQ ID NO:129).
Human TANGO 354 includes an immunoglobulin domain at amino acids 33-110 of SEQ ID NO:26 (SEQ ID NO:41). Figure 23 depicts alignments of the immunoglobulin domains of TANGO 354 with consensus hidden Markov model immunoglobulin domains (SEQ ID NO:37).
An N-glycosylation site is present at amino acids 88-91 of SEQ ID NO:26. A cAMP and cGMP-dependent protein kinase phosphorylation site is present at amino acids 233-236 of SEQ ID NO:26. Protein kinase C phosphorylation sites are present at amino acids 81-83, 231-233, and 236-238 of SEQ ID NO:26. Casein kinase II phosphorylation sites are present at amino acids 44-47, 69-72, 81-84, 94-97, 101-104, 113-116, and 146-149 of SEQ ID NO:26. A tyrosine kinase phosphorylation site is present at amino acids 291- 299 of SEQ ID NO:26. N-myristylation sites are present at amino acids 30-35, and 109-114 of SEQ ID NO:26.
Clone jthLa042a04, which encodes human TANGO 354, was deposited as EpT354 with the American Type Culture Collection (ATCC® 10801 University Boulevard, Manassas, VA 20110-2209) on June 18, 1999 and assigned Accession Number PTA-249. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
Figure 22 depicts a hydropathy plot of human TANGO 354. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The hydropathy plot of Figure 22 indicates the presence of a hydrophobic domain within human TANGO 354, suggesting that human TANGO 354 is a transmembrane protein. Use of TANGO 354 Nucleic Acids. Polypeptides. and Modulators Thereof
TANGO 354 includes an immuno globulin-like domain. Proteins having such domains play a role in mediating protein-protein and protein-ligand interactions, and thus can influence a wide variety of biological processes, including modulation of cell surface recognition; modulation of cellular motility, e.g., chemotaxis and chemokinesis; transduction of an extracellular signal (e.g., by interacting with a ligand and or a cell- surface receptor); and/or modulation of a signal transduction pathways.
TANGO 354 polypeptides, nucleic acids, and modulators thereof can be used to modulate function, survival, morphology, migration, proliferation and/or differentiation of cells in the tissues in which it is expressed (e.g., hematopoietic tissues).
Because of the presence of an immunoglobulin domain and the expression of
TANGO 354 in hematopoietic cells, TANGO 354 polypeptides, nucleic acids, and modulators thereof can be used to modulate (e.g., increase or decrease) hematopoietic function, thereby influencing one or more of: (1) regulation of hematopoiesis; (2) modulation of haemostasis; (3) modulation of an inflammatory response; (4) modulation of neoplastic growth, e.g., inhibition of tumor growth; and/or (5) regulation of thrombolysis.
Accordingly, TANGO 354 polypeptides, nucleic acids, and modulators thereof can be used to treat a variety of hematopoietic diseases including, but not limited to, myeloid disorders and/or lymphoid malignancies. Exemplary myeloid diseases that can be treated include acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Naickus, 1991, Crit Rev. in
Oncol/Hemotol. 11 :267-97). Exemplary lymphoid malignancies that can be treated using these molecules include acute lymphoblastic leukemia (ALL) which includes B-lineage
ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia
(PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
Additional forms of malignant lymphomas include non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T- cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF) and Hodgkin's disease.
In one embodiment, TANGO 354 polypeptides, nucleic acids, and modulators thereof can be used to treat a variety of neoplastic diseases, including malignancies of the various organ systems, such as affecting lung, breast, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art recognized and refers to malignant tumors of mesenchymal derivation.
TANGO 354 polypeptides, nucleic acids, and modulators thereof can also be used to treat a variety of non-cancerous diseases or conditions involving, for example, aberrant T cell activity (e.g., aberrant T cell proliferation and/or secretion). Examples of such T cell diseases or conditions include inflammation; allergy, for example, atopic allergy; organ rejection after transplantation (e.g., skin graft, cardiac graft, islet graft); graft- versus-host disease; autoimmune diseases (including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, diabetes, myasthenia gravis, systemic lupus erythematosus, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjδgren's Syndrome, including keratoconjunctivitis sicca secondary to Sjδgren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens- Johnson syndrome, idiopathic sprue, lichen planus, Crohn's disease, Graves ophthalmopathy, sarcoidosis, primary biliary- cirrhosis, uveitis posterior, and interstitial lung fibrosis).
Further, in light of TANGO 345's presence in a Mixed Lymphocyte Reaction cDNA library, TANGO 345 expression can be utilized as a marker for specific tissues (e.g., lymphoid tissues such as the thymus and spleen) and/or cells (e.g., lymphocytes) in which TANGO 345 is expressed. TANGO 345 nucleic acids can also be utilized for chromosomal mapping.
TANGO 378
A cDNA encoding human TANGO 378 was identified by analyzing the sequences of clones present in a human natural killer cell cDNA library. This analysis led to the identification of a clone, jthta028f04, encoding full-length human TANGO 378. The cDNA of this clone is 3258 nucleotides long (Figures 24A-24C; SEQ ID NO:28). The 1584 nucleotide open reading frame of this cDNA, nucleotides 42 to 1625 of SEQ ID NO:28 (SEQ ID NO:30), encodes a 528 amino acid protein (Figure 25; SEQ ID NO:29).
The signal peptide prediction program SIGNALP (Nielsen et al., 1997, Protein Engineering 10:1-6) predicted that human TANGO 378 includes a 21 amino acid signal peptide at amino acid 1 to about amino acid 21 of SEQ ID NO:29 (SEQ ID NO: 132) preceding the mature human MANGO 347 protein which corresponds to about amino acid 22 to amino acid 528 of SEQ ID NO:29 (SEQ ID NO: 133).
Human TANGO 378 that has not been post-translationally modified is predicted to have a molecular weight of 59.0 kDa prior to cleavage of its signal peptide and a molecular weight of 56.7 kDa subsequent to cleavage of its signal peptide.
Human TANGO 378 is a seven transmembrane G-protein coupled receptor (GPCR) protein having an N-terminal extracellular domain which extends from about amino acid 22 to about amino acid 244 of SEQ ID NO:29 (SEQ ID NO: 134); seven transmembrane domains which extend from about amino acids 245 to about amino acid 269 of SEQ ID NO:29 (SEQ ID NO: 135), about amino acids 287 to about amino acid 306 of SEQ ID NO:29 (SEQ ID NOT36), about amino acids 323 to about amino acid 343 of SEQ ID NO:29 (SEQ ID NOT37), about amino acids 358 to about amino acid 376 of SEQ ID NO:29 (SEQ ID NO: 138), about amino acids 414 to about amino acid 438 of SEQ ID NO:29 (SEQ ID NO: 139), about amino acids 457 to about amino acid 477 of SEQ ID NO:29 (SEQ ID NO: 140), and about amino acids 485 to about amino acid 504 of SEQ ID NO:29 (SEQ ID NO: 141); and a C-terminal cytoplasmic domain which extends from about amino acid 505 to amino acid 528 of SEQ ED NO:29 (SEQ ID NO: 142). Figure 26 depicts an alignment of each of the transmembrane domains of TANGO 378 with the consensus hidden Markov model seven transmembrane receptor sequences (SEQ ID NO: 98).
Alternatively, in another embodiment, a human TANGO 378 protein contains an N- terminal extracellular domain which extends from about amino acid 505 to amino acid 528 of SEQ ID NO:29 (SEQ ID NO: 142); seven transmembrane domains which extend from about amino acids 245 to about amino acid 269 of SEQ ID NO:29 (SEQ ID NOT35), about amino acids 287 to about amino acid 306 of SEQ ID NO:29 (SEQ ID NO: 136), about amino acids 323 to about amino acid 343 of SEQ ID NO:29 (SEQ ID NOT37), about amino acids 358 to about amino acid 376 of SEQ ID NO:29 (SEQ ID NOT38), about amino acids 414 to about amino acid 438 of SEQ ED NO:29 (SEQ ID NO: 139), about amino acids 457 to about amino acid 477 of SEQ ID NO:29 (SEQ ID NO: 140), and about amino acids 485 to about amino acid 504 of SEQ ED NO:29 (SEQ ID NO: 141); and a C- terminal cytoplasmic domain which extends from about amino acid 22 to about amino acid 244 of SEQ ED NO:29 (SEQ ID NOT34).
Human TANGO 378 includes three extracellular loops which extend from about amino acid 307 to about amino acid 322 of SEQ ID NO:29 (SEQ ID NO: 143), about amino acid 377 to about amino acid 413 of SEQ ID NO:29 (SEQ ID NO: 144), and about amino acid 478 to about amino acid 484 of SEQ ID NO:29 (SEQ ID NO.T 45).
Human TANGO 378 includes three intracellular loops which extend from about amino acid 270 to about amino acid 286 of SEQ ID NO:29 (SEQ ID NO: 146), about amino acid 344 to about amino acid 357 of SEQ ID NO:29 (SEQ ID NO: 147), and about amino acid 439 to about amino acid 456 of SEQ ID NO:29 (SEQ ID NO: 148).
N-glycosylation sites are present at amino acids 18-21, 58-61, 65-68, 146-149, 173- 176, 179-182, 394-397, and 400-403 of SEQ ID NO:29. A cAMP and cGMP-dependent protein kinase phosphorylation site is present at amino acids 274-277 of SEQ ID NO:29. Protein kinase C phosphorylation sites are present at amino acids 45-47, 93-95, 375-377, 437-439, 449-451, and 505-507 of SEQ ID NO:29. Casein kinase II phosphorylation sites are present at amino acids 23-26, 29-32, and 510-513 of SEQ ID NO:29. N-myristylation sites are present at amino acids 86-91, 101-106, 157-162, 255-260, 311-316, 420-425, and 467-472 of SEQ ID NO:29. A thiol (cysteine) protease histidine site is present at amino acid 410-420 of SEQ ID NO:29.
Clone jthta028f04, which encodes human TANGO 378, was deposited as EpT378 with the American Type Culture Collection (ATCC® 10801 University Boulevard, Manassas, NA 20110-2209) on June 18, 1999 and assigned Accession Number PTA-249. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S. C. §112.
Figure 25 depicts a hydropathy plot of human TANGO 378. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The hydropathy plot of Figure 25 indicates that human TANGO 378 has a signal peptide at its amino terminus and seven hydrophobic domains within human TANGO 378, suggesting that human TANGO 378 is a transmembrane protein.
Use of TANGO 378 Nucleic Acids. Polypeptides. and Modulators Thereof
TANGO 378 includes a seven transmembrane domain which is typically found in G-protein coupled receptors. Proteins having such a domain play a role in transducing an extracellular signal, e.g., by interacting with a ligand and/or a cell-surface receptor, followed by mobilization of intracellular molecules that participate in signal transduction pathways (e.g., adenylate cyclase, or phosphatidylinositol 4,5-bisphosphate (PIP2), inositol 1,4,5-triphosphate (EP3)).
TANGO 378 polypeptides, nucleic acids, and modulators thereof can be used to modulate function, survival, morphology, migration, proliferation and/or differentiation of cells in the tissues in which it is expressed (e.g., natural killer cells). For example, TANGO 354 polypeptides, nucleic acids, and modulators thereof can be used to modulate an immune response in a subject by, for example, (1) modulating immune cytotoxic responses against pathogenic organisms, e.g., viruses, bacteria, and parasites; (2) by modulating organ rejection after transplantation (e.g., skin graft, cardiac graft, islet graft); (3) by modulating immune recognition and lysis of normal and malignant cells; (4) by modulating T cell diseases; and (5) by controlling neoplastic growth, e.g., inhibition of tumor growth.
Accordingly, TANGO 378 polypeptides, nucleic acids, and modulators thereof can be used to treat a variety of diseases involving aberrant immune responses, for example, aberrant T cell activity (e.g., aberrant T cell proliferation and/or secretion). A non-limiting list of diseases involving aberrant T cell activity is provided in the section entitled "TANGO 354" above.
In other embodiments, TANGO 378 polypeptides, nucleic acids, and modulators thereof can be used to treat a variety of neoplastic diseases, including hematopoietic malignancies and including, but not limited to, myeloid disorders, lymphoid malignancies, and/or malignancies of the various organ systems. ). A non-limiting list of such neoplastic diseases is provided in the section entitled "TANGO 354" above.
Further, in light of TANGO 378's presence in a Natral Killer cell cDNA library,
TANGO 378 expression can be utilized as a marker for specific tissues (e.g., lymphoid tissues such as the thymus and spleen) and/or cells (e.g., Natural Killer cells) in which
TANGO 345 is expressed. TANGO 345 nucleic acids can also be utilized for chromosomal mapping.
Tables 1 and 2 below provide summaries of INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, and TANGO 378 sequence information.
TABLE 1 : Summary of Sequence Information for INTERCEPT 340, MANGO 003,
MANGO 347, TANGO 272, TANGO 295, TANGO 354, and TANGO 378
Figure imgf000055_0001
TABLE 2: Summary of Protein Domains of INTERCEPT 340, MANGO 003,
MANGO 347, TANGO 272, TANGO 295, TANGO 354, and TANGO 378
Figure imgf000056_0001
TABLE 2 continued
Figure imgf000057_0001
Various aspects of the invention are described in further detail in the following subsections
I. Isolated Nucleic Acid Molecules
One aspect of the invention pertains to isolated nucleic acid molecules that encode a polypeptide of the invention or a biologically active portion thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single- stranded or double-stranded, but preferably is double-stranded DNA. An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Preferably, an "isolated" nucleic acid molecule is free of sequences (preferably protein encoding sequences) which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. In other embodiments, the "isolated" nucleic acid is free of intron sequences. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, the nucleic acid molecules of the invention comprise a contiguous open reading frame encoding a polypeptide of the invention.
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOsT, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, or a complement thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequences of SEQ ID NOs.T, 3, 4, 6, 1, 9, 10, 12, 13, 15, 16, 18,
19, 21, 22, 24, 25, 27, 28 or 30 as a hybridization probe, nucleic acid molecules of the invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, 2nd ed.,1989, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY).
A nucleic acid molecule of the invention can be amplified using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard
PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to all or a portion of a nucleic acid molecule of the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence of
SEQ ID NOsT, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, or a portion thereof. A nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence thereby forming a stable duplex.
Moreover, a nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence encoding a full length polypeptide of the invention for example, a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a polypeptide of the invention. The nucleotide sequence determined from the cloning one gene allows for the generation of probes and primers designed for use in identifying and/or cloning homologues in other cell types, e.g., from other tissues, as well as homologues from other mammals. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide
10 sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides of the sense or anti-sense sequence of SEQ ID NOsT, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, or of a naturally occurring mutant of SEQ ID NOsT, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30.
15 Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts or genomic sequences encoding the same protein molecule encoded by a selected nucleic acid molecule. The probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which
20 mis-express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted.
A nucleic acid fragment encoding a biologically active portion of a polypeptide of the invention can be prepared by isolating a portion of any of SEQ ID NOs: 1 , 3, 4, 6, 7, 9,
25 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, expressing the encoded portion of the polypeptide protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the polypeptide.
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of SEQ ID NOsT, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24,
30 25, 27, 28 or 30, due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequence SEQ ID NOsT, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30.
In addition to the nucleotide sequences of SEQ ID NOsT, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, it will be appreciated by those skilled in the art
35 that DNA sequence polymorphisms that lead to changes in the amino acid sequence may exist within a population (e.g., the human population). Such genetic polymorphisms may exist among individuals within a population due to natural allehc variation. An allele is one of a group of genes which occur alternatively at a given genetic locus. As used herein, the phrase "allelic variant" refers to a nucleotide sequence which occurs at a given locus or to a polypeptide encoded by the nucleotide sequence. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene. Alternative alleles can be identified by 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 or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding proteins of the invention from other species (homologues), which have a nucleotide sequence which differs from that of the human protein described herein are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of a cDNA of the invention can be isolated based on their identity to the human nucleic acid molecule disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
For example, a cDNA encoding a soluble form of a membrane-bound protein of the invention isolated based on its hybridization to a nucleic acid molecule encoding all or part of the membrane-bound form. Likewise, a cDNA encoding a membrane-bound form can be isolated based on its hybridization to a nucleic acid molecule encoding all or part of the soluble form.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900,
1000, or 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600,
3800, 4000, or 4200) nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence, preferably the coding sequence, of SEQ ID NOsT, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, or a complement thereof.
As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 10%, preferably 75%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, 1989, John Wiley & Sons, NY, sections
6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 C, followed by one or more washes in 0.2 X SSC, 0.1 % SDS at 50-65 C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NOsT, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, or a complement thereof, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In addition to naturally-occurring allelic variants of a nucleic acid molecule of the invention sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation thereby leading to changes in the amino acid sequence of the encoded protein, without altering the biological activity of the protein. For example, one can make nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are not conserved or only semi-conserved among homologues of various species may be non-essential for activity and thus would be likely targets for alteration. Alternatively, amino acid residues that are conserved among the homologues of various species (e.g., murine and human) may be essential for activity and thus would not be likely targets for alteration.
Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding a polypeptide of the invention that contain changes in amino acid residues that are not essential for activity. Such polypeptides differ in amino acid sequence from SEQ ID
NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule includes a nucleotide sequence encoding a protein that includes an amino acid sequence that is at least about 45% identical, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29.
An isolated nucleic acid molecule encoding a variant protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, 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. Briefly, PCR primers are designed that delete the trinucleotide codon of the amino acid to be changed and replace it with the trinucleotide codon of the amino acid to be included. This primer is used in the PCR amplification of DNA encoding the protein of interest. This fragment is then isolated and inserted into the full length cDNA encoding the protein of interest and expressed recombinantly. The resulting protein now includes the amino acid replacement.
Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are can be divided into four families: (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In similar fashion, the amino acid repertoire can be grouped as (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine histidine, (3) aliphatic = glycine, alanine, valine, leucine, isoleucine, serine, threonine, with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic = phenylalanine, tyrosine, tryptophan; (5) amide = asparagine, glutamine; and (6) sulfur - containing = cysteine and methionine. (See, for example, Biochemistry, 4th ed., Ed. by L. Stryer, WH Freeman and Co.: 1995).
Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
In a preferred embodiment, a mutant polypeptide that is a variant of a polypeptide of the invention can be assayed for: (1) the ability to form protein-protein interactions with proteins in a signaling pathway of the polypeptide of the invention; (2) the ability to bind a ligand of the polypeptide of the invention; or (3) the ability to bind to an intracellular target protein of the polypeptide of the invention. In yet another preferred embodiment, the mutant polypeptide can be assayed for the ability to modulate cellular proliferation, cellular migration or chemotaxis, or cellular differentiation.
The present invention encompasses antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid encoding a polypeptide of the invention, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can be antisense to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding a polypeptide of the invention. The non-coding regions ("5' and 3' untranslated regions") are the 5' and 3' sequences which flank the coding region and are not translated into amino acids.
An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40,
45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenme, 2-methylguanme, 3-methylcytosme, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxy acetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-
3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a selected polypeptide of the invention to thereby inhibit expression, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
An antisense nucleic acid molecule of the invention can be an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al., 1987, Nucleic Acids Res. 15:6625-41). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al.,1987, Nucleic Acids Res. 15:6131-48) or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-30).
The invention also encompasses ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes; described in Haselhoff and Gerlach, 1988, Nature 334:585- 91) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. A ribozyme having specificity for a nucleic acid molecule encoding a polypeptide of the invention can be designed based upon the nucleotide sequence of a cDNA disclosed herein. For example, a derivative of a Tetrahymena L-l 9 INS RΝA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742. Alternatively, an mRNA encoding a polypeptide of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak, 1993, Science 261:1411-8.
The invention also encompasses nucleic acid molecules which form triple helical structures. For example, expression of a polypeptide of the invention can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g., the promoter and/or enhancer) to form triple helical structures that prevent transcription of the gene in target cells. See generally Helene, 1991, Anticancer Drug Des. 6(6):569-84; Helene, 1992, Ann. N.Y. Acad. Sci. 660:21-36; and Maher, 1992, Bioassays 14(12):807-15.
In various embodiments, the nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al., 1996, Bioorganic & Medicinal Chemistry 4(1): 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al., 1996, supra; Perry-O'Keefe et al., 1996, Proc. Natl Acad. Sci. USA 93:14670-5.
PNAs can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup, 1996, supra); or as probes or primers for DNA sequence and hybridization (Hyrup, 1996, supra; Perry-O'Keefe et al., 1996, Proc. Natl Acad. Sci. USA 93:14670-675).
In another embodiment, PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup, 1996, supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996, supra) and Finn et al. (1996, Nucleic Acids Res. 24(17):3357-63). For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs. Compounds such as 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite can be used as a link between the PNA and the 5' end of DNA (Mag et al., 1989, Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al., 1996, Nucleic Acids Res. 24(17):3357-63). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser et al., 1975, Bioorganic Med. Chem. Lett. 5:1119-1124). In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6; Lemaitre et al, 1987, Proc. Natl. Acad. Sci. USA 84:648-52; PCT Publication No. W0 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W0 89/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988, Bio/Techniques 6:958-16) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-49). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
II. Isolated Proteins and Antibodies
One aspect of the invention pertains to isolated proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise antibodies directed against a polypeptide of the invention. In one embodiment, the native polypeptide can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, polypeptides of the invention are produced by recombinant DNA techniques. Alternative to recombinant expression, a polypeptide of the invention can be synthesized chemically using standard peptide synthesis techniques.
An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein"). When the protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest. The term "pure" or "isolated" as used herein preferably has the same numerical limits as "purified" or "isolated" immediately above. "Isolated" and "purified" do not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure (e.g., lacking contaminating proteins, or chromatography reagents such as denaturing agents and polymers, e.g., acrylamide or agarose) substances or solutions. In preferred embodiments, purified or isolated preparations will lack any contaminating proteins from the same animal from which the protein is normally produced, as can be accomplished by recombinant expression of, for example, a human protein in a non-human cell.
Biologically active portions of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the protein (e.g., the amino acid sequence shown in any of SEQ ID NOs:2, 5, 8, 11, 14, or 17), which include fewer amino acids than the full length protein, and exhibit at least one activity of the corresponding full-length protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the corresponding protein. A biologically active portion of a protein of the invention can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the invention.
Preferred polypeptides have the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14,
17, 20, 23, 26, or 29. Other useful proteins are substantially identical (e.g., at least about
45%, preferably 55%, 65%, 75%, 85%, 95%, or 99%) to any of SEQ ID NOs:2, 5, 8, 11, 14,
17, 20, 23, 26, or 29 and retain the functional activity of the protein of the corresponding naturally-occurring protein yet differ in amino acid sequence due to natural allelic variation or mutagenesis.
To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison 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 percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions (e.g., overlapping positions) x 100). In one embodiment the two sequences are the same length. The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-8), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-7). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990, J Mol. Biol. 215:403-10). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389- 402). Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988, CABIOS 4:11-7). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
The invention also provides chimeric or fusion proteins. As used herein, a "chimeric protein" or "fusion protein" comprises all or part (preferably biologically active) of a polypeptide of the invention operably linked to a heterologous polypeptide (i.e., a polypeptide other than the same polypeptide of the invention). Within the fusion protein, the tenn "operably linked" is intended to indicate that the polypeptide of the invention and the heterologous polypeptide are fused in-frame to each other. The heterologous polypeptide can be fused to the N-terminus or C-terminus of the polypeptide of the invention.
One useful fusion protein is a GST fusion protein in which the polypeptide of the invention is fused to the C-terminus of GST sequences. Such fusion proteins can facilitate the purification of a recombinant polypeptide of the invention. In another embodiment, the fusion protein contains a heterologous signal peptide at its N-terminus. For example, the native signal peptide of a polypeptide of the invention can be removed and replaced with a signal peptide from another protein. For example, the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal peptide (Current Protocols in Molecular Biology, 1992, Ausubel et al., eds., John Wiley & Sons). Other examples of eukaryotic heterologous signal peptides include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California). In yet another example, useful prokaryotic heterologous signal peptides include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).
In yet another embodiment, the fusion protein is an immunoglobulin fusion protein in which all or part of a polypeptide of the invention is fused to sequences derived from a member of the immunoglobulin protein family. The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand (soluble or membrane-bound) and a protein on the surface of a cell (receptor), to thereby suppress signal transduction in vivo. The immunoglobulin fusion protein can be used to affect the bioavailability of a cognate ligand of a polypeptide of the invention. Inhibition of ligand receptor interaction may be useful therapeutically, both for treating proliferative and differentiative disorders and for modulating (e.g., promoting or inhibiting) cell survival. Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies directed against a polypeptide of the invention in a subject, to purify ligands and in screening assays to identify molecules which inhibit the interaction of receptors with ligands.
Chimeric and fusion proteins of the invention can be produced by standard recombinant DNA techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel et al., supra). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide of the invention.
A signal peptide of a polypeptide of the invention (SEQ ID NOsTOl, 110, 112, 125, 127, or 132) can be used to facilitate secretion and isolation of the secreted protein or other proteins of interest. Signal peptides are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal peptide from the mature proteins as they pass through the secretory pathway. Thus, the invention pertains to the described polypeptides having a signal peptide, as well as to the signal peptide itself and to the polypeptide in the absence of the signal peptide (i.e., the cleavage products). In one embodiment, a nucleic acid sequence encoding a signal peptide of the invention can be operably linked in an expression vector to a protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate. The signal peptide directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal peptide is subsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by art recognized methods. Alternatively, the signal peptide can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.
In another embodiment, the signal peptides of the present invention can be used to identify regulatory sequences, e.g., promoters, enhancers, repressors. Since signal peptides are the most amino-terminal sequences of a peptide, it is expected that the nucleic acids which flank the signal peptide on its amino-terminal side will be regulatory sequences which affect transcription. Thus, a nucleotide sequence which encodes all or a portion of a signal peptide can be used as a probe to identify and isolate signal peptides and their flanking regions, and these flanking regions can be studied to identify regulatory elements therein. 0 The present i •nventi •on also pertai •ns to vari ■ants of the polypeptides of the invention.
Such variants have an altered amino acid sequence which can function as either agonists
(mimetics) or as antagonists. Nariants can be generated by mutagenesis, e.g., discrete point mutation or truncation. An agonist can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the protein. An antagonist of a 5 protei ■n can i ■nhi ■bit one or more of the activities of the naturally occurring form of the protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring 0 form of the protein can have fewer side effects in a subject relative to treatment with the naturally occurring form of the protein.
Modification of the structure of the subject polypeptides can be for such purposes as enhancing therapeutic or prophylactic efficacy, stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo), or post-translational modifications (e.g., to
J alter phosphorylation pattern of protein). Such modified peptides, when designed to retain at least one activity of the naturally-occurring form of the protein, or to produce specific antagonists thereof, are considered functional equivalents of the polypeptides described in more detail herein. Such modified peptides can be produced, for instance, by amino acid substitution, deletion, or addition.
For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e. isosteric and/or isoelectric mutations) will not have a major effect on the biological activity of the resulting molecule.
Whether a change in the amino acid sequence of a peptide results in a functional homolog (e.g., functional in the sense that the resulting polypeptide mimics or antagonizes the wild-type form) can be readily determined by assessing the ability of the variant peptide to produce a response in cells in a fashion similar to the wild-type protein, or competitively inhibit such a response. Polypeptides in which more than one replacement has taken place can readily be tested in the same manner.
Nariants of a protein of the invention which function as either agonists (mimetics) or as antagonists can be identified by screening combinatorial libraries of mutants, e.g. , truncation mutants, of the protein of the invention for agonist or antagonist activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display). There are a variety of methods which can be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known m the art (see, e.g., Νarang, 1983, Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev.
Biochem. 53:323; Itakura et al., 1984, Science 198:1056; Ike et al., 1983, Nucleic Acid
Res.11:477).
In addition, libraries of fragments of the coding sequence of a polypeptide of the invention can be used to generate a variegated population of polypeptides for screening and subsequent selection of variants. For example, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DΝA, renaturing the DΝA to form double stranded DΝA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention (Arkin and Yourvan, 1992, Proc. Natl. Acad. Sci. USA 89:1811-5; Delgrave et al., 1993, Protein Engineering 6(3):327-31).
An isolated polypeptide of the invention, or a fragment thereof, can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. The full-length polypeptide or protein can be used or, alternatively, the invention provides antigenic peptide fragments for use as immunogens. The antigenic peptide of a protein of the invention comprises at least 8 (preferably 10, 15, 20, or 30) amino acid residues of the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23,
20 26, or 29, and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.
Preferred epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, e.g., hydrophilic regions. Hydropathy plots or similar analyses can be used to identify hydrophilic regions.
25 An immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal). An appropriate immunogenic preparation can contain, for example, recombinantly expressed or chemically synthesized polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
30 Accordingly, another aspect of the invention pertains to antibodies directed against a polypeptide of the invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention, e.g., an epitope of a polypeptide of the invention. A molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope.
Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide of the invention as an immunogen. Preferred polyclonal antibody compositions are ones that have been selected for antibodies directed against a polypeptide or polypeptides of the invention. Particularly preferred polyclonal antibody preparations are ones that contain only antibodies directed against a polypeptide or polypeptides of the invention. Particularly preferred immunogen compositions are those that contain no other human proteins such as, for example, immunogen compositions made using a non-human host cell for recombinant expression of a polypeptide of the invention. In such a manner, the only human epitope or epitopes recognized by the resulting antibody compositions raised against this immunogen will be present as part of a polypeptide or polypeptides of the invention.
The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. Alternatively, antibodies specific for a protein or polypeptide of the invention can be selected for (e.g., partially purified) or purified by, e.g., affinity chromatography. For example, a recombinantly expressed and purified (or partially purified) protein of the invention is produced as described herein, and covalently or non-covalently coupled to a solid support such as, for example, a chromatography column. The column can then be used to affinity purify antibodies specific for the proteins of the invention from a sample containing antibodies directed against a large number of different epitopes, thereby generating a substantially purified antibody composition, i.e., one that is substantially free of contaminating antibodies. By a substantially purified antibody composition is meant, in this context, that the antibody sample contains at most only 30% (by dry weight) of contaminating antibodies directed against epitopes other than those on the desired protein or polypeptide of the invention, and preferably at most 20%>, yet more preferably at most 10%, and most preferably at most 5% (by dry weight) of the sample is contaminating antibodies. A purified antibody composition means that at least 99% of the antibodies in the composition are directed against the desired protein or polypeptide of the invention.
At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique (Kohler and Milstein, 1975, Nature 256:495-1), 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., pgs. 77-96) or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology, 1994, Coligan et al.,eds., John Wiley & Sons, Inc., New York, NY). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide of interest. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAPJ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al., 1991, Bio/Technology 9:1370-2; Hay et al., 1992, Hum. Antibod. Hybridomas 3:81-5; Huse et al., 1989, Science 246:1275-81; Griffiths et al., 1993, EMBO J. 12:725-34.
Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; and Boss et al., U.S. Patent No. 4,816,397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Patent No. 4,816,567; European Patent Application 125,023; Better et al, 1988, Science 240:1041-3; Liu et al, 1987, Proc. Natl. Acad. Sci. USA 84:3439-43; Liu et al., 1987, J. Immunol. 139:3521-6; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-8; Nishimura et al., 1987, Cane. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-9; and Shaw et al, 1988, J Natl. Cancer Inst. 80:1553-9; Morrison, 1985, Science 229:1202-7; Oi et al, 1986, Bio/Techniques 4:214; U.S. Patent 5,225,539; Jones et al., 1986, Nature 321:522-5; Verhoeyan et al, 1988, Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053- 60.
Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced , for example, using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Patent 5,625,126; U.S. Patent 5,633,425; U.S. Patent 5,569,825; U.S. Patent 5,661,016; and U.S. Patent 5,545,806. In addition, companies such as Abgenix, Inc. (Freemont, CA), can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., 1994, Bio/technology 12:899-903).
Further, an antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxcrubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (I) (IDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example , a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2
("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-
CSF"), granulocyte colony stimulating factor ("G-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.
Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies for Immunotargeting of Drugs in Cancer
Therapy," in Monoclonal Antibodies and Cancer Therapy, 1985, Reisfeld et al., eds., pgs.
243-56; Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery 2nd
Ed., 1987, Robinson et al., eds.; Thorpe, "Antibody Carriers of Cytotoxic Agents in Cancer
Therapy: A Review," in Monoclonal Antibodies '84 Biological and Clinical Applications,
1985, Pinchera et al, eds, pgs. 475-506; "Analysis, Results, and Future Prospective of the
Therapeutic Use of Radiolabeled Antibody in Cancer Therapy," in Monoclonal Anitbodies or Cancer Detection and Therapy, 1985, Baldwin et al., eds., pgs. 303-16; and Thorpe et al.,1982, Immunol. Rev., 62:119-58. Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent
No. 4,676,980.
An antibody directed against a polypeptide of the invention (e.g., monoclonal antibody) can be used to isolate the polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, such an antibody can be used to detect the protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide. The antibodies can also be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, 8-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 1251, 1311, 35S or 3H.
Further, an antibody (or fragment thereof) can be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorabicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents
(e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorabicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti- mitotic agents (e.g., vincristine and vinblastine).
The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α- interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.
Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., A non et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, 1985, Reisfeld et al. (eds.), pgs. 243-56, Alan R. Liss, Inc.; Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), 1987, Robinson et al. (eds.), pgs. 623-53, Marcel Dekker, Inc.; Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, 1985, Pinchera et al. (eds.), pgs. 475-506; "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, 1985, Baldwin et al. (eds.), pgs. 303-16, Academic Press, and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 1982, 62:119-58.
Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980. Accordingly, in one aspect, the invention provides substantially purified antibodies or fragment thereof, and human or non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of any one of SEQ
ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29; or an amino acid sequence encoded by the cDNA of a clone deposited as ATCC® Accession Number 207178, ATCC® Accession
Number PTA-249, or ATCC® Accession Number PTA-250; a fragment of at least 15 amino acid residues of the amino acid sequence of any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20,
23, 26, or 29; an amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4; and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of any one of SEQ ID NOsT, 3, 4, 6, 7, 9, 10, 12, 13, 15,
16, 18, 19, 21, 22, 24, 25, 27, 28, or 30, or the cDNA of a clone deposited as ATCC®
Accession Number 207178, ATCC® Accession Number PTA-249, or ATCC® Accession
Number PTA-250, or a complement thereof, under conditions of hybridization of 6X SSC at
45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C. In various embodiments, the substantially purified antibodies of the invention, or fragments thereof, can be human, non- human, chimeric and/or humanized antibodies. In another aspect, the invention provides human or non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, or an amino acid sequence encoded by the cDNA of a clone deposited as ATCC® Accession Number 207178, ATCC® Accession Number PTA-249, or ATCC® Accession Number PTA-250; a fragment of at least 15 amino acid residues of the amino acid sequence of any one of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, an amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4; and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid nolecule consisting of any one of SEQ ID NOsT, 3, 4, 6, 1, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, or 30, or the cDNA of a clone deposited as ATCC® Accession Number 207178, ATCC® Accession Number PTA-249, or ATCC® Accession Number PTA-250, or a complement thereof, under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C. Such non-human antibodies can be goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies. Alternatively, the non-human antibodies of the invention can be chimeric and/or humanized antibodies. In addition, the human or non-human antibodies of the invention can be polyclonal antibodies or monoclonal antibodies.
In still a further aspect, the invention provides monoclonal antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, or an amino acid sequence encoded by the cDNA of a clone deposited as ATCC® Accession Number 207178, ATCC®
Accession Number PTA-249, or ATCC® Accession Number PTA-250; a fragment of at least 15 amino acid residues of the amino acid sequence of any one of SEQ ID NOs:2, 5, 8,
11, 14, 17, 20, 23, 26, or 29, an amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4; and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of any one of SEQ ID NOs: 1, 3, 4, 6, 7,
9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, or 30, or the cDNA of a clone deposited as any of ATCC® Accession Number 207178, ATCC® Accession Number PTA-249, or
ATCC® Accession Number PTA-250, or a complement thereof, under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C. The monoclonal antibodies can be human, humanized, chimeric and/or non-human antibodies.
The substantially purified antibodies or fragments thereof specifically bind to a signal peptide, a secreted sequence, an extracellular domain, a transmembrane or a cytoplasmic domain cytoplasmic membrane of a polypeptide of the invention. In a particularly preferred embodiment, the substantially purified antibodies or fragments thereof, the human or non-human antibodies or fragments thereof, and/or the monoclonal antibodies or fragments thereof, of the invention specifically bind to a secreted sequence or an extracellular domain of the amino acid sequence of SEQ ID NOs:103, 107, 114, 118, 122, 129, or 134. Preferably, the secreted sequence or extracellular domain to which the
1 A antibody, or fragment thereof, binds comprises from about amino acids 25-374 of SEQ ID NO:5 (SEQ ID NO: 103), from amino acids 1-73 of SEQ ID NO:8 (SEQ ID NO: 107), from amino acids 21-767 of SEQ ID NOT4 (SEQ ID NOT 14), from amino acids 1-216 of SEQ ID NOT7 (SEQ ID NOT 18), from amino acids 1-500 of SEQ ID NO:20 (SEQ ID NO: 122) from amino acids 20-169 of SEQ ID NO:26 (SEQ ID NO: 129), and from amino acids 22- 15 244 of SEQ ID NO:29 (SEQ ID NO:134).
Any of the antibodies of the invention can be conjugated to a therapeutic moiety or to a detectable substance. Non-limiting examples of detectable substances that can be conjugated to the antibodies of the invention are an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, and a radioactive 0 material.
The invention also provides a kit containing an antibody of the invention conjugated to a detectable substance, and instructions for use. Still another aspect of the invention is a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier. In preferred embodiments, the pharmaceutical 5 composition contains an antibody of the invention, a therapeutic moiety, and a pharmaceutically acceptable carrier.
Still another aspect of the invention is a method of making an antibody that specifically recognizes INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272,
TANGO 295, TANGO 354, and TANGO 378, the method comprising immunizing a 0 mammal with a polypeptide. The polypeptide used as an immunogen comprises an amino acid sequence selected from the group consisting of: the amino acid sequence of any one of
SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, or an amino acid sequence encoded by the cDNA of a clone deposited as ATCC® Accession Number 207178, ATCC® Accession
Number PTA-249, or ATCC® Accession Number PTA-250; a fragment of at least 15 amino 5 acid residues of the amino acid sequence of any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20,
23, 26, or 29, an amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4; and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of any one of SEQ ID NOs: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, or 30, or the cDNA of a clone deposited as ATCC® Accession Number 207178, ATCC® Accession Number PTA-249, or ATCC® Accession Number PTA-250, or a complement thereof, under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C. After immunization, a sample is collected from the mammal that contains an antibody that specifically recognizes GPVI. Preferably, the polypeptide is recombinantly produced using a non-human host cell. Optionally, the antibodies can be further purified from the sample using techniques well known to those of skill in the art. The method can further comprise producing a monoclonal antibody-producing cell from the cells of the mammal. Optionally, antibodies are collected from the antibody-producing cell.
III. Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide of the invention (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors, expression vectors, are capable of directing the expression of genes to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retrovirases, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell. This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, 1990, Academic Press, San Diego, CA. Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
The recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells (using baculovirus expression vectors), yeast cells or mammalian cells). Suitable host cells are discussed further in Goeddel, supra. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three 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. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988,
Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia,
Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al.,1988, Gene 69:301-15) and pET lid (Studier et al., Gene Expression Technology: Methods in Enzymology, 1990, Academic Press, San Diego, CA pgs. 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 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.
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, Gene Expression Technology: Methods in Enzymology, 1990, Academic Press, San Diego, CA pgs. 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., 1992, Nucleic Acids Res. 20:2111-8). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerivisae include pYepSecl (Baldari et al., 1987, EMBOJ. 6:229-34), pMFa (Kurjan and Herskowitz, 1982, Cell 30:933-43), pJRY88 20 (Schultz et al, 1987, Gene 54:113-23), pYES2 (Invitrogen Corporation, San Diego, CA), and pPicZ (Invitrogen Corp, San Diego, CA).
Alternatively, the expression vector is a baculoviras expression vector. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., 1983, Mol. Cell Biol. 3:2156-65) and the pVL series (Lucklow 25 and Summers, 1989, Virology 170:31-9).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987, Nature 329:840) and pMT2PC (Kaufinan et al., 1987, EMBO J. 6:187-95). When used in mammalian cells, the expression vector's
30 control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian
Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al., supra.
In another embodiment, the recombinant mammalian expression vector is capable of τ _τ directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue- specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., 1987, Genes Dev. 1:268-77), lymphoid-specific promoters (Calame and Eaton, 1988, Adv. Immunol. 43:235- 75), in particular promoters of T cell receptors (Winoto and Baltimore, 1989, EMBO J. 8:729-33) and immunoglobulins (Banerji et al., 1983, Cell 33:729-40; Queen and Baltimore, 1983, Cell 33:741-8), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989, Proc. Natl. Acad. Sci. USA 86:5473-7), pancreas- specific promoters (Edlund et al., 1985, Science 230:912-6), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Grass, 1990, Science 249:314-9) and the α- fetoprotein promoter (Campes and Tilghman, 1989, Genes Dev. 3:537-46).
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to the mRNA encoding a polypeptide of the invention. Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al. (1985, Reviews - Trends in Genetics l(l):22-5).
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell (e.g., insect cells, yeast or mammalian cells).
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drags, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
In another embodiment, the expression characteristics of an endogenous (e.g., INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, and TANGO 378) nucleic acid within a cell, cell line or microorganism may be modified by inserting a DNA regulatory element heterologous to the endogenous gene of interest into the genome of a cell, stable cell line or cloned microorganism such that the inserted regulatory element is operatively linked with the endogenous gene (e.g., INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, and TANGO 378) and controls, modulates or activates the endogenous gene. For example, endogenous INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, and TANGO 378 which are normally "transcriptionally silent", i.e., INTERCEPT 340,
MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, andTANGO 378 genes which are normally not expressed, or are expressed only at very low levels in a cell line or microorganism, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell line or microorganism. Alternatively, transcriptionally silent, endogenous INTERCEPT 340,
MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, andTANGO 378 genes may be activated by insertion of a promiscuous regulatory element that works across cell types.
A heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with and activates expression of endogenous INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295,
TANGO 354, and TANGO 378 genes, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, and described e.g., in Chappel, U.S. Patent No. 5,272,071 ; PCT publication No. WO 91/06667, published May 16, 1991.
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce a polypeptide of the invention. Accordingly, the invention further provides methods for producing a polypeptide of the invention using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell.
The host cells of the invention can also be used to produce nonhuman transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a sequences encoding a polypeptide of the invention have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous sequences encoding a polypeptide of the invention have been introduced into their genome or homologous recombinant animals in which endogenous encoding a polypeptide of the invention sequences have been altered. Such animals are useful for studying the function and/or activity of the polypeptide and for identifying and/or evaluating modulators of polypeptide activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, an "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
A transgenic animal of the invention can be created by introducing nucleic acid encoding a polypeptide of the invention (or a homologue thereof) into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the polypeptide of the invention to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent NOs. 4,736,866; 4,870,009; 4,873,191 and in Hogan (Manipulating the Mouse Embryo, 1986, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of mRNA encoding the transgene in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying the transgene can further be bred to other transgenic animals carrying other transgenes.
To create an homologous recombinant animal, a vector is prepared which contains at least a portion of a gene encoding a polypeptide of the invention into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the gene. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous protein). In the homologous recombination vector, the altered portion of the gene is flanked at its 5' and 3' ends by additional nucleic acid of the gene to allow for homologous recombination to occur between the exogenous gene carried by the vector and an endogenous gene in an embryonic stem cell. The additional flanking nucleic acid sequences are of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see, e.g., Thomas and Capecchi, 1987, Cell 51:503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by elecfroporation) and cells in which the introduced gene has homologously recombined with the endogenous gene are selected (see, e.g., Li et al., 1992, Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see, e.g., Bradley in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, 1987, . Robertson, ed., IRL, Oxford pgs. 113-52). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991, Current Opinion in Bio/Technology 2:823-9 and in PCT Publication NOs. WO 90/11354, WO 91/01140, WO 92/0968 and WO 93/04169.
In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al., 1991, Science 251:1351-5). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al., 1997, Nature 385:810-3 and PCT Publication NOs. WO 97/07668 and WO 97/07669.
IN. Pharmaceutical Compositions
The nucleic acid molecules, polypeptides, and antibodies (also referred to herein as "active compounds") of the invention 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. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and 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.
The invention includes methods for preparing pharmaceutical compositions for modulating the expression or activity of a polypeptide or nucleic acid of the invention. Such methods comprise formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of the invention. Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of the invention and one or more additional active compounds. The agent which modulates expression or activity may, for example, be a small molecule. For example, such small molecules include peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. It is understood that appropriate doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention. Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g. about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein.
When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drag combination, and the degree of expression or activity to be modulated.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal
(topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELJ (BASF; Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged 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 polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, 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.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza 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. Patent No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. (1997, J Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).
As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments. In a preferred example, a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein. The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Patent 5,328,470) or by stereotactic injection (see, e.g., Chen et al., 1994, Proc. Natl. Acad. Sci. USA 91:3054-7). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
V. Uses and Methods of the Invention
The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) detection assays (e.g., chromosomal mapping, tissue typing, forensic biology); c) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenomics); and d) methods of treatment (e.g., therapeutic and prophylactic). For example, the INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295,
TANGO 354, and TANGO 378 polypeptides of the invention can to used to modulate cellular function, survival, morphology, proliferation, and/or differentiation of the cells in which they are expressed. For example, the polypeptides of the invention can be used to treat diseases such as neoplastic disorders (e.g., cancer, tumors), hematopoietic disorders
(e.g., T cell disorders), among others. The isolated nucleic acid molecules of the invention can be used to express proteins (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect mRNA (e.g., in a biological sample) or a genetic lesion, and to modulate activity of a polypeptide of the invention. In addition, the polypeptides of the invention can be used to screen drags or compounds which modulate activity or expression of a polypeptide of the invention as well as to treat disorders characterized by insufficient or excessive production of a protein of the invention or production of a form of a protein of the invention which has decreased or aberrant activity compared to the wild type protein. In addition, the antibodies of the invention can be used to detect and isolate a protein of the invention and modulate activity of a protein of the invention.
This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein. A. Screening Assays
The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drags) which bind to polypeptide of the invention or have a stimulatory or inhibitory effect on, for example, expression or activity of a polypeptide of the invention.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a polypeptide of the invention or biologically active portion thereof. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non- peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12:145).
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., 1993, Proc. Natl. Acad. Sci. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al, 1994, J. Med. Chem. 37:2678; Cho et al., 1993, Science 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al., 1994, J. Med. Chem. 37:1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992, Bio/Techniques 13:412-21), or on beads (Lam, 1991, Nature 354:82-4), chips (Fodor, 1993, Nature 364:555-6), bacteria (U.S. Patent No. 5,223,409), spores (U.S. Patent NOs. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al, 1992, Proc. Natl. Acad. Sci USA 89:1865-9) or phage (Scott and Smith, 1990, Scz'ence 249:386-90; Devlin, 1990, Science 249:404-6; Cwirla et al, 1990, Proc. Natl. Acad. Sci. USA 87:6378-82; and Felici, 1991, J. Mol. Biol. 222:301-10).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to the polypeptide determined. The cell, for example, can be a yeast cell or a cell of mammalian origin. Determining the ability of the test compound to bind to the polypeptide can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the polypeptide or biologically active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 1251, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In a preferred embodiment, the assay comprises contacting a cell which expresses a membrane- bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or a biologically active portion thereof as compared to the known compound.
In another embodiment, the assay involves assessment of an activity characteristic of the polypeptide, wherein binding of the test compound with the polypeptide or a biologically active portion thereof alters (e.g., increases or decreases) the activity of the polypeptide.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the polypeptide or biologically active portion thereof. Detennining the ability of the test compound to modulate the activity of the polypeptide or a biologically active portion thereof can be accomplished, for example, by determining the ability of the polypeptide protein to bind to or interact with a target molecule or to transport molecules across the cytoplasmic membrane.
Determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by one of the methods described above for determining direct binding. As used herein, a "target molecule" is a molecule with which a selected polypeptide (e.g., a polypeptide of the invention binds or interacts with in nature, for example, a molecule on the surface of a cell which expresses the selected protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. A target molecule can be a polypeptide of the invention or some other polypeptide or protein. For example, a target molecule can be a component of a signal transduction pathway which facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a compound to a polypeptide of the invention) through the cell membrane and into the cell or a second intercellular protein which has catalytic activity or a protein which facilitates the association of downstream signaling molecules with a polypeptide of the invention. Determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g. luciferase), or detecting a cellular response, for example, cellular differentiation, or cell proliferation. hi yet another embodiment, an assay of the present invention is a cell-free assay comprising contacting a polypeptide of the invention or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to the polypeptide or biologically active portion thereof. Binding of the test compound to the polypeptide can be determined either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the polypeptide of the invention or biologically active portion thereof with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or biologically active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-free assay comprising contacting a polypeptide of the invention or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the polypeptide or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished, for example, by determining the ability of the polypeptide to bind to a target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished by determining the ability of the polypeptide of the invention to further modulate the target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as previously described. In yet another embodiment, the cell-free assay comprises contacting a polypeptide of the invention or biologically active portion thereof with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the polypeptide to preferentially bind to or modulate the activity of a target molecule.
The cell-free assays of the present invention are amenable to use of both a soluble form or the membrane-bound form of a polypeptide of the invention. In the case of cell-free assays comprising the membrane-bound form of the polypeptide, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of the polypeptide is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-octyhnaltoside, octanoyl-N- methylglucamide, decanoyl-N-methylglucamide, Triton X-100, Triton X-l 14, Thesit, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-l- propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-l- propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-l -propane sulfonate.
In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either the polypeptide of the invention or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to the polypeptide, or interaction of the polypeptide with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or A polypeptide of the invention, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of the polypeptide of the invention can be determined using standard techniques. Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the polypeptide of the invention or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated polypeptide of the invention or target molecules can be prepared from biotin- NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with the polypeptide of the invention or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the invention trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST- immobilized complexes, include immunodetection of complexes using antibodies reactive with the polypeptide of the invention or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the polypeptide of the invention or target molecule.
In another embodiment, modulators of expression of a polypeptide of the invention are identified in a method in which a cell is contacted with a candidate compound and the expression of the selected mRNA or protein (i.e., the mRNA or protein corresponding to a polypeptide or nucleic acid of the invention) in the cell is determined. The level of expression of the selected mRNA or protein in the presence of the candidate compound is compared to the level of expression of the selected mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of expression of the polypeptide of the invention based on this comparison. For example, when expression of the selected mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of the selected mRNA or protein expression.
Alternatively, when expression of the selected mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of the selected mRNA or protein expression. The level of the selected mRNA or protein expression in the cells can be determined by methods described herein.
In yet another aspect of the invention, a polypeptide of the inventions can be used as
"bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No.
5,283,317; Zervos et al., 1993, Cell 72:223-32; Madura et al., 1993, J. Biol. Chem.
268:12046-54; Bartel et al, 1993, Bio/Techniques 14:920-4; Iwabuchi et al., 1993, Oncogene 8:1693-6; and PCT Publication No. WO 94/10300), to identify other proteins, which bind to or interact with the polypeptide of the invention and modulate activity of the polypeptide of the invention. Such binding proteins are also likely to be involved in the propagation of signals by the polypeptide of the inventions as, for example, upstream or downstream elements of a signaling pathway involving the polypeptide of the invention.
This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
B. Detection Assays
Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.
1. Chromosome Mapping
Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. Accordingly, nucleic acid molecules described herein or fragments thereof, can be used to map the location of the corresponding genes on a chromosome. The mapping of the sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
Briefly, genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the sequence of a gene of the invention. Computer analysis of the sequence of a gene of the invention can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the gene sequences will yield an amplified fragment. For a review of this technique, see D'Eustachio et al. (1983, Science 220:919-24).
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the nucleic acid sequences of the invention to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a gene to its chromosome include in situ hybridization (described in Fan et al., 1990, Proc. Natl. Acad. Sci. USA 87:6223-7), pre-screening with labeled flow-sorted chromosomes (CITE), and pre-selection by hybridization to chromosome specific cDNA libraries. Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques, 1988, Pergamon Press, NY.
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.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland et al., 1987, Nature 325:783-7.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with a gene of the invention can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
Furthermore, the nucleic acid sequences disclosed herein can be used to perform searches against "mapping databases", e.g., BLAST-type search, such that the chromosome position of the gene is identified by sequence homology or identity with known sequence fragments which have been mapped to chromosomes.
A polypeptide and fragments and sequences thereof and antibodies specific thereto can be used to map the location of the gene encoding the polypeptide on a chromosome. This mapping can be carried out by specifically detecting the presence of the polypeptide in members of a panel of somatic cell hybrids between cells of a first species of animal from which the protein originates and cells from a second species of animal and then determining which somatic cell hybrid(s) expresses the polypeptide and noting the chromosome(s) from the first species of animal that it contains. For examples of this technique, see Pajunen et al, 1988, Cytogenet. Cell Genet. 47:37-41 and Van Keuren et al., 1986, Hum. Genet. 74:34-40. Alternatively, the presence of the polypeptide in the somatic cell hybrids can be determined by assaying an activity or property of the polypeptide, for example, enzymatic activity, as described in Bordelon-Riser et al., 1979, Somatic Cell Genetics 5:597-613 and Owerbach et al., 1978, Proc. Natl. Acad. Sci. USA 75:5640-5644.
2. Tissue Typing
The nucleic acid sequences of the present invention can also be used to identify individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult. The c sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
Furthermore, the sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the nucleic acid sequences described herein can 0 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 5 be used to obtain such identification sequences from individuals and from tissue. The nucleic acid sequences of the invention uniquely represent portions of the human genome.
Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Each of the 0 sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NOsT, 4, 7, 10, 13, 16, 19,
22, 25, and 28 can comfortably provide positive individual identification with a panel of 5 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 NOs:3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
If a panel of reagents from the nucleic acid sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.
3. Use of Partial Gene Sequences in Forensic Biology
DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique. Examples of polynucleotide reagents include the nucleic acid sequences of the invention or portions thereof, e.g., fragments derived from noncoding regions having a length of at least 20 or 30 bases.
The nucleic acid sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such probes can be used to identify tissue by species and/or by organ type.
C. Predictive Medicine:
The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials 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 INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 protein and/or nucleic acid expression as well as INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 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 or unwanted INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 gene expression or activity. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 protein or nucleic acid expression or activity. For example, mutations in a 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 protein or nucleic acid expression or activity.
As an alternative to making determinations based on the absolute expression level of selected genes, determinations may be based on the normalized expression levels of these genes. Expression levels are normalized by correcting the absolute expression level of a INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 gene by comparing its expression to the expression of a gene that is not a INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, e.g., a non-disease sample, or between samples from different sources.
Alternatively, the expression level can be provided as a relative expression level. To determine a relative expression level of a gene, the level of expression of the gene is determined for 10 or more samples of different cell isolates, preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the gene(s) in question. The expression level of the gene determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that gene. This provides a relative expression level and aids in identifying extreme cases of disease.
Preferably, the samples used in the baseline determination will be from diseased or from non-diseased cells of tissue. The choice of the cell source is dependent on the use of the relative expression level. Using expression found in normal tissues as a mean expression score aids in validating whether the INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 gene assayed is diseased cell-type specific (versus normal cells). Such a use is particularly important in identifying whether a INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 gene can serve as a target gene. In addition, as more data is accumulated, the mean expression value can be revised, providing improved relative expression values based on accumulated data. Expression data from cells provide a means for grading the severity of the disease state.
Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of INTERCEPT 340, MANGO 003, MANGO 347, TANGO 272, TANGO 295, TANGO 354, or TANGO 378 genes in clinical trials.
These and other agents are described in further detail in the following sections.
1. Diagnostic Assays
An exemplary method for detecting the presence or absence of a polypeptide or nucleic acid of the invention in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of the invention such that the presence of a polypeptide or nucleic acid of the invention is detected in the biological sample. A preferred agent for detecting mRNA or genomic DNA encoding a polypeptide of the invention is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA encoding a polypeptide of the invention. The nucleic acid probe can be, for example, a full-length cDNA, such as the nucleic acid of SEQ ID NOs , 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28 or 30, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a polypeptide of the invention. Other suitable probes for use in the diagnostic assays of the invention are described herein.
A preferred agent for detecting a polypeptide of the invention is an antibody capable of binding to a polypeptide of the invention, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g. , Fab or F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of a polypeptide of the invention include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of a polypeptide of the invention include introducing into a subject a labeled antibody directed against the polypeptide. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting a polypeptide of the invention or mRNA or genomic DNA encoding a polypeptide of the invention, such that the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide is detected in the biological sample, and comparing the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide in the control sample with the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide in the test sample.
The invention also encompasses kits for detecting the presence of a polypeptide or nucleic acid of the invention in a biological sample (a test sample). Such kits can be used to determine if a subject is suffering from or is at increased risk of developing a disorder associated with aberrant expression of a polypeptide of the invention (e.g., a proliferative disorder, e.g., psoriasis or cancer). For example, the kit can comprise a labeled compound or agent capable of detecting the polypeptide or mRNA encoding the polypeptide in a biological sample and means for determining the amount of the polypeptide or mRNA in the sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide). Kits can also include instractions for observing that the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of the polypeptide if the amount of the polypeptide or mRNA encoding the polypeptide is above or below a normal level.
For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.
For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule encoding a polypeptide of the invention. The kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit can also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit is usually enclosed within an individual container and all of the various containers are within a single package along with instractions for observing whether the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of the polypeptide.
Prognostic Assays
The methods described herein can furthermore be utilized as diagnostic or prognostic assays to identify subjects having or at risk of developing a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with aberrant expression or activity of a polypeptide of the invention.
Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing such a disease or disorder. Thus, the present invention provides a method in which a test sample is obtained from a subject and a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of the invention is detected, wherein the presence of the polypeptide or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant expression or activity of the polypeptide. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest.
For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention. For example, such methods can be used to determine whether a subject can be effectively treated with a specific agent or class of agents (e.g., agents of a type which decrease activity of the polypeptide). Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant expression or activity of a polypeptide of the invention in which a test sample is obtained and the polypeptide or nucleic acid encoding the polypeptide is detected (e.g., wherein the presence of the polypeptide or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant expression or activity of the polypeptide).
The methods of the invention can also be used to detect genetic lesions or mutations in a gene of the invention, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized aberrant expression or activity of a polypeptide of the invention. In preferred embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion or mutation characterized by at least one of an alteration affecting the integrity of a gene encoding the polypeptide of the invention, or the mis-expression of the gene encoding the polypeptide of the invention. For example, such genetic lesions or mutations can be detected by ascertaining the existence of at least one of: 1) a deletion of one or more nucleotides from the gene; 2) an addition of one or more nucleotides to the gene; 3) a substitution of one or more nucleotides of the gene; 4) a chromosomal rearrangement of the gene; 5) an alteration in the level of a messenger RNA transcript of the gene; 6) an aberrant modification of the gene, such as of the methylation pattern of the genomic DNA; 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of the gene; 8) a non- wild type level of a the protein encoded by the gene; 9) an allelic loss of the gene; and 10) an inappropriate post-translational modification of the protein encoded by the gene. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in a gene.
In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent NOs. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., 1988, Science 241:1077-80; and Nakazawa et al., 1994, Proc. Natl. Acad. Sci USA 91 :360-4), the latter of which can be particularly useful for detecting point mutations in a gene (see, e.g., Abravaya et al., 1995, Nucleic Acids Res. 23:675-82). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to the selected gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication (Guatelli et al, 1990, Proc. Natl. Acad. Sci. USA 87:1874-78), transcriptional amplification system (Kwoh, et al, 1989, Proc. Natl. Acad. Sci. USA 86:1173-7), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6:1191), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in a selected gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g.,
U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin et al., 1996, Human Mutation 7:244-55;
Kozal et al., 1996, Nature Medicine 2:753-9). For example, genetic mutations can be identified in two-dimensional arrays containing light-generated DNA probes as described in
Cronin et al., supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the selected gene and detect mutations by comparing the sequence of the sample nucleic acids with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert (1977, Proc. Natl. Acad. Sci. USA 74:560) or Sanger (1977, Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays developed by Naeve et al. (1995, Bio/Techniques 19:448-53), including sequencing by mass spectrometry (see, e.g., PCT Publication No. WO 94/16101; Cohen et al., 1996, Adv. Chromatogr. 36:127-62; and Griffin et al., 1993, Appl. Biochem. Biotechnol. 38:147-59).
Other methods for detecting mutations in a selected gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al., 1985, Science 230:1242). In general, the technique of mismatch cleavage entails providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. RNA/DNA duplexes can be treated with RNase to digest mismatched regions, and DNA DNA hybrids can be treated with SI nuclease to digest mismatched regions.
In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton et al., 1988, Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al., 1992, Methods Enzymol 217:286-95. In a preferred embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called DNA mismatch repair enzymes) in defined systems for detecting and mapping point mutations in cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al., 1994, Carcinogen esis 15:1657-62). According to an exemplary embodiment, a probe based on a selected sequence, e.g., a wild-type sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al, 1989, Proc. Natl. Acad. Sci. USA 86:2766; see also Cotton, 1993,
Mutat. Res. 285:125-44; Hayashi, 1992, Genet. Anal. Tech. Appl. 9:73-9). Single-stranded
DNA fragments of sample and control nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, and the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al.,
10 1991, Trends Genet. 7:5). hi yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al., 1985, Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely 1 c denature, for example by adding a "GC clamp of approximately 40 bp of high-melting GC- rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA
(Rosenbaum and Reissner, 1981, Biophys. Chem. 265:12753).
Examples of other techniques for detecting point mutations include, but are not
20 limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al., 1986, Nature
324:163; Saiki et al., 1989, Proc. Natl Acad. Sci USA 86:6230). Such allele specific
25 oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology which depends on selective
PCR amplification may be used in conjunction with the instant invention. Oligonucleotides
30 used as pπmers 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. 17:2437-48) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent or reduce polymerase extension (Prossner,
1993, Tibtech 11 :238). In addition, it may be desirable to introduce a novel restriction site
35 in the region of the mutation to create cleavage-based detection (Gasparini et al., 1992, Mol.
Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany, 1991, Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is aperfect 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 prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a gene encoding a polypeptide of the invention. Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which the polypeptide of the invention is expressed may be utilized in the prognostic assays described herein.
3. Pharmaco genomics
Agents, or modulators which have a stimulatory or inhibitory effect on activity or expression of a polypeptide of the invention as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders associated with aberrant activity of the polypeptide. In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drag) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drag. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drags) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of a polypeptide of the invention, expression of a nucleic acid of the invention, or mutation content of a gene of the invention in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
Pharmacogenomics deals with clinically significant hereditary variations in the response to drags due to altered drag disposition and abnormal action in affected persons. See, e.g., Linder, 1997, Clin. Chem. 43(2):254-66. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drags 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. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drag metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drag response and serious toxicity after taking the standard and safe dose of a drag. These polymoφhisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, a PM will show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
Thus, the activity of a polypeptide of the invention, expression of a nucleic acid encoding the polypeptide, or mutation content of a gene encoding the polypeptide in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymoφhic alleles encoding drag-metabolizing enzymes to the identification of an individual's drag responsiveness phenotype. This knowledge, when applied to dosing or drag selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a modulator of activity or expression of the polypeptide, such as a modulator identified by one of the exemplary screening assays described herein.
4. Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of a polypeptide of the invention (e.g., the ability to modulate aberrant cell proliferation chemotaxis, and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent, as determined by a screening assay as described herein, to increase gene expression, protein levels or protein activity, can be monitored in clinical trials of subjects exhibiting decreased gene expression, protein levels, or protein activity. Alternatively, the effectiveness of an agent, as determined by a screening assay, to decrease gene expression, protein levels or protein activity, can be monitored in clinical trials of subjects exhibiting increased gene expression, protein levels, or protein activity. In such clinical trials, expression or activity of a polypeptide of the invention and preferably, that of other polypeptide that have been implicated in for example, a cellular proliferation disorder, can be used as a marker of the immune responsiveness of a particular cell.
For example, and not by way of limitation, genes, including those of the invention, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates activity or expression of a polypeptide of the invention (e.g., as identified in a screening assay described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of a gene of the invention and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of a gene of the invention or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of the polypeptide or nucleic acid of the invention in the preadministration sample;
(iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level the of the polypeptide or nucleic acid of the invention in the post-administration samples; (v) comparing the level of the polypeptide or nucleic acid of the invention in the pre-administration sample with the level of the polypeptide or nucleic acid of the invention in the post-administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased admimstration of the agent may be desirable to increase the expression or activity of the polypeptide to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of the polypeptide to lower levels than detected, i.e., to decrease the effectiveness of the agent. C. Methods of Treatment
The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant expression or activity of a polypeptide of the invention, e.g., cardiac infection (e.g., myocarditis or dilated cardiomyopathy), central nervous system infection (e.g., nonspecific febrile illness or meningoencephalitis), pancreatic infection (e.g., acute pancreatitis), respiratory infection (pneumonia), gastrointestinal infection, type I diabetes, cancer, familia hypercholesterolemia, treat hemophilia B, Marfan syndrome, protein S deficiency, allergy, inflammation, and gastroduodenal ulcer. Moreover, the polypeptides of the invention can be used to modulate cellular function, survival, moφhology, proliferation
10 and/or differentiation.
1. Prophylactic Methods In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant expression or activity of a polypeptide of the
15 invention, by administering to the subject an agent which modulates expression or at least one activity of the polypeptide. Subjects at risk for a disease which is caused or contributed to by aberrant expression or activity of a polypeptide of the invention can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms
20 characteristic of the aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of aberrancy, for example, an agonist or antagonist agent can be used for treating the subject.
2. Therapeutic Methods
25 Another aspect of the invention pertains to methods of modulating expression or activity of a polypeptide of the invention for therapeutic p poses. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of the polypeptide. An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of the
30 polypeptide, a peptide, a peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more of the biological activities of the polypeptide. Examples of such stimulatory agents include the active polypeptide of the invention and a nucleic acid molecule encoding the polypeptide of the invention that has been introduced into the cell. In another embodiment, the agent inhibits one or more of the biological activities of the
^5 polypeptide of the invention. Examples of such inhibitory agents include antisense nucleic acid molecules and antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a polypeptide of the invention. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) expression or activity. In another embodiment, the method involves administering a polypeptide of the invention or a nucleic acid molecule of the invention as therapy to compensate for reduced or aberrant expression or activity of the polypeptide.
Stimulation of activity is desirable in situations in which activity or expression is abnormally low or downregulated and/or in which increased activity is likely to have a beneficial effect. Conversely, inhibition of activity is desirable in situations in which activity or expression is abnormally high or upregulated and/or in which decreased activity is likely to have a beneficial effect.
The contents of all references, patents and published patent applications cited throughout this application are hereby incoφorated by reference.
Deposit of Clones
Clones containing cDNA molecules encoding human MANGO 003 were deposited with the American Type Culture Collection (ATCC® 10801 University Boulevard, Manassas, VA 20110-2209) on March 30, 1999 as Accession Number 207178, as part of a composite deposit representing a mixture of three strains, each carrying one recombinant plasmid harboring a particular cDNA clone.
To distinguish the strains and isolate a strain harboring a particular cDNA clone, an aliquot of the mixture can be streaked out to single colonies on nutrient medium (e.g., LB plates) supplemented with 100 g/ml ampicillin, single colonies grown, and then plasmid DNA extracted using a standard minipreparation procedure. Next, a sample of the DNA minipreparation can be digested with a combination of the restriction enzymes Sal I and Not I, and the resultant products resolved on a 0.8% agarose gel using standard DNA electrophoresis conditions. The digest liberates fragments as follows:
human MANGO 003 (clone EpthLaόal): 3.2 kB
The identity of the strains can be inferred from the fragments liberated.
Clones containing cDNA molecules encoding human INTERCEPT 340, MANGO 347, and TANGO 272 were deposited with the American Type Culture Collection (ATCC® 10801 University Boulevard, Manassas, NA 20110-2209) on June 18, 1999 as Accession Number PTA-250, as part of a composite deposit representing a mixture of three strains, each carrying one recombinant plasmid harboring a particular cDNA clone.
To distinguish the strains and isolate a strain harboring a particular cDNA clone, an aliquot of the mixture can be streaked out to single colonies on nutrient medium (e.g., LB plates) supplemented with 100 g/ml ampicillin, single colonies grown, and then plasmid DNA extracted using a standard minipreparation procedure. Next, a sample of the DNA minipreparation can be digested with a combination of the restriction enzymes Sal I and Not I, and the resultant products resolved on a 0.8% agarose gel using standard DNA electrophoresis conditions. The digest liberates fragments as follows:
human INTERCEPT 340 (clone EpI340): 3.3 kB human MANGO 347 (clone EpM347): 1.4 kB human TANGO 272 (clone EpT272): 5.0 kB
The identity of the strains can be inferred from the fragments liberated.
Clones containing cDNA molecules encoding human TANGO 295, TANGO 354, and TANGO 378 were deposited with the American Type Culture Collection (ATCC® 10801 University Boulevard, Manassas, VA 20110-2209) on June 18, 1999 as Accession Number PTA-249, as part of a composite deposit representing a mixture of three strains, each carrying one recombinant plasmid harboring a particular cDNA clone.
To distinguish the strains and isolate a strain harboring a particular cDNA clone, an aliquot of the mixture can be streaked out to single colonies on nutrient medium (e.g., LB plates) supplemented with 100 g/ml ampicillin, single colonies grown, and then plasmid DNA extracted using a standard minipreparation procedure. Next, a sample of the DNA minipreparation can be digested with a combination of the restriction enzymes Sal I and Not I, and the resultant products resolved on a 0.8% agarose gel using standard DNA electrophoresis conditions. The digest liberates fragments as follows:
human TANGO 295 (clone EpT295) 1.5 kB human TANGO 354 (clone EpT354) 1.8 kB human TANGO 378 (clone EpT378) 3.3 kB
The identity of the strains can be inferred from the fragments liberated. All publications, patents and patent applications mentioned in this specification are herein incoφorated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incoφorated herein by reference.
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following Claims.
-1 16.1 -
International Application No: PCT/ /
MICROORGANISMS
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A. IDENTIFICATION OF DEPOSIT '
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American Type Culture Collection
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Accession No. Date of Deposit
PTA-249 June 18, 1999
PTA-250 June 18, 1999

Claims

What is claimed is:
1. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 55% identical to the nucleotide sequence of SEQ ID NOs:l, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30, the cDNA insert of the plasmid deposited with the ATCC® as Accession Number 207178, the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-249, the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-250, or a complement thereof; b) a nucleic acid molecule comprising a fragment of at least 300 nucleotides of the nucleotide sequence of SEQ ID NOs:l, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30, the cDNA insert of the plasmid deposited with the ATCC® as Accession Number 207178, the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-249, the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-250, or a complement thereof; c) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number 207178, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-249, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-250; d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number 207178, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-249, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-250, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number 207178, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-249, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-250; and e) a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20,
- 117 23, 26, 29, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number 207178, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-249, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-250, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NOs.l, 3, 4, 6, 1, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30, or a complement thereof, under stringent conditions.
2. The isolated nucleic acid molecule of Claim 1, which is selected from the group consisting of: a) a nucleic acid comprising the nucleotide sequence of SEQ ID NOs:l, 3, 4, 6, 1, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30, the cDNA insert of the plasmid deposited with the ATCC® as Accession Number 207178, the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-249, the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-250, or a complement thereof; and b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number 207178, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-249, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-250.
3. The nucleic acid molecule of Claim 1 further comprising vector nucleic acid sequences.
4. The nucleic acid molecule of Claim 1 further comprising nucleic acid sequences encoding a heterologous polypeptide.
5. A host cell which contains the nucleic acid molecule of Claim 1.
6. The host cell of Claim 5 which is a mammalian host cell.
7. A non-human mammalian host cell containing the nucleic acid molecule of Claim 1.
8. An isolated polypeptide selected from the group consisting of:
- 118 - a) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number 207178, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-249, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-250, wherein the polypeptide is encoded by a nucleic acid molecule which
10 hybridizes to a nucleic acid molecule comprising SEQ ID NOs: 1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, or a complement thereof under stringent conditions; and c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 55% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NOs:l, 3, 4, 6, 1, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24,
15 25, 27, 28, 30, or a complement thereof.
9. The isolated polypeptide of Claim 8 comprising the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29.
10. The polypeptide of Claim 8 further comprising heterologous amino acid 0 sequences.
11. An antibody which selectively binds to a polypeptide of Claim 8.
12. A method for producing a polypeptide selected from the group consisting of: 5 a) a polypeptide comprising the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number 207178, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-249, or the amino acid sequence encoded by the cDNA insert of the plasmid 0 deposited with the ATCC® as Accession Number PTA-250; b) a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number 207178, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-249, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-250, wherein the
- 119 - fragment comprises at least 15 contiguous amino acids of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number 207178, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA- 249, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-250; and c) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number 207178, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-249, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC® as Accession Number PTA-250, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NOs:l, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30, or a complement thereof under stringent conditions; comprising culruring the host cell of Claim 5 under conditions in which the nucleic acid molecule is expressed.
13. A method for detecting the presence of a polypeptide of Claim 8 in a sample, comprising: a) contacting the sample with a compound which selectively binds to a polypeptide of Claim 8; and b) determining whether the compound binds to the polypeptide in the sample.
14. The method of Claim 13, wherein the compound which binds to the polypeptide is an antibody.
15. A kit comprising a compound which selectively binds to a polypeptide of Claim 8 and instructions for use.
16. A method for detecting the presence of a nucleic acid molecule of Claim 1 in a sample, comprising the steps of: a) contacting the sample with a nucleic acid probe or primer which selectively hybridizes to the nucleic acid molecule; and b) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample.
120 -
17. The method of Claim 16, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.
18. A kit comprising a compound which selectively hybridizes to a nucleic acid molecule of Claim 1 and instructions for use.
19. A method for identifying a compound which binds to a polypeptide of Claim 8 comprising the steps of: a) contacting a polypeptide, or a cell expressing a polypeptide of Claim 8 with a test compound; and b) determining whether the polypeptide binds to the test compound.
20. The method of Claim 19, wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of: a) detection of binding by direct detecting of test compound/polypeptide binding; b) detection of binding using a competition binding assay; c) detection of binding using an assay for INTERCEPT 340-, MANGO 003-, MANGO 347-, TANGO 272-, TANGO 295-, TANGO 354-, or TANGO 378-mediated signal transduction.
21. A method for modulating the activity of a polypeptide of Claim 8 comprising contacting a polypeptide or a cell expressing a polypeptide of Claim 8 with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide.
22. A method for identifying a compound which modulates the activity of a polypeptide of Claim 8, comprising: a) contacting a polypeptide of Claim 8 with a test compound; and b) determining the effect of the test compound on the activity of the polypeptide to thereby identify a compound which modulates the activity of the polypeptide.
121 -
PCT/US2000/018198 1999-06-30 2000-06-29 Membrane-associated and secreted proteins and uses thereof WO2001000673A1 (en)

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Publication number Priority date Publication date Assignee Title
US7083791B2 (en) 1999-03-25 2006-08-01 Genesis Research & Development Corporation Limited Methods for enhancing immune responses by fibroblast growth factor receptor 5 polypeptides
US6242419B1 (en) * 1999-03-25 2001-06-05 Genesis Research & Development Corporation Ltd. Compositions isolated from stromal cells and methods for their use
CA2398251A1 (en) * 2000-01-25 2001-08-02 Hyseq, Inc. Methods and materials relating to leukocyte immunoglobulin receptor-like (lir-like) polypeptides and polynucleotides
US20050208498A1 (en) * 2000-01-25 2005-09-22 Nuvelo, Inc. Methods of therapy and diagnosis using targeting of cells that express killer cell immunoglobulin-like receptor-like protein
US20040005558A1 (en) * 2001-01-18 2004-01-08 Anderson David W. Proteins, polynucleotides ecoding them and methods of using the same
US7049096B2 (en) 2001-04-11 2006-05-23 Bristol-Meyers Squibb Company Polynucleotides encoding a novel human G-protein coupled receptor splice variant HGPRBMY29sv1
CN108997498A (en) 2008-12-09 2018-12-14 霍夫曼-拉罗奇有限公司 Anti- PD-L1 antibody and they be used to enhance the purposes of T cell function
CN114028549A (en) * 2016-02-19 2022-02-11 伊玛提克斯生物技术有限公司 Novel peptides and peptide compositions for immunotherapy of NHL and other cancers
WO2022101193A1 (en) * 2020-11-10 2022-05-19 Fundació Hospital Universitari Vall D'hebron - Institut De Recerca Cell-penetrating peptides

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